Oligonucleotide compositions and methods of use thereof

Abstract

Among other things, the present disclosure provides designed oligonucleotides, compositions, and methods of use thereof. In some embodiments, the present disclosure provides technologies useful for reducing levels of transcripts. In some embodiments, the present disclosure provides technologies useful for modulating transcript splicing. In some embodiments, provided technologies can alter splicing of a dystrophin (DMD) transcript. In some embodiments, the present disclosure provides methods for treating diseases, such as Duchenne muscular dystrophy, Becker's muscular dystrophy, etc.

Claims

1. A method for preparing an oligonucleotide, wherein the oligonucleotide comprises one or more phosphoramidate internucleotidic linkages, comprising providing a phosphoramidite compound having the structure of: ##STR01165## or a salt thereof, wherein a base is contacted with an oligonucleotide having an internucleotidic linkage having the structure of formula III (the formula III oligonucleotide): ##STR01166## wherein the contact is performed substantially absent of water and the contact is after the length of the oligonucleotide to be prepared is achieved before deprotection and cleavage of the formula III oligonucleotide; and wherein: P.sup.N is P(N-L-R.sup.5), ##STR01167## R.sup.5s is independently R or OR; each BA is independently an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.5-30 heteroaryl having 1-10 heteroatoms, C.sub.3-30 heterocyclyl having 1-10 heteroatoms, a natural nucleobase moiety, and a modified nucleobase moiety; each R.sup.s is independently H, halogen, CN, N.sub.3, NO, NO.sub.2, -L-R, -L-Si(R).sub.3, -L-OR, -L-SR, -L-N(R).sub.2, O-L-R, O-L-Si(R).sub.3, O-L-OR, O-L-SR, or O-L-N(R).sub.2; each s is independently 0-20; each L is independently C(R.sup.5s).sub.2, or L; Q.sup. is an anion; L.sup.b is L or N(R.sup.1); g is 0-18; each of X, Y and Z is independently O, S, N(-L-R.sup.5), or L; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; Ring A.sup.L is an optionally substituted 3-20 monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the two depicted nitrogen atoms; each of G.sup.1, G.sup.2, G.sup.3, G.sup.4, and G.sup.5is independently R.sup.1; each of R.sup.1 and R.sup.5 is independently H, -L-R, halogen, CN, NO.sub.2, -L-Si(R).sub.3, OR, SR, or N(R).sub.2; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms; X-L-R.sup.1 is ##STR01168## and wherein G.sup.2 comprises an electron-withdrawing group.

2. The method of claim 1, wherein G.sup.5 and one of G.sup.3 and G.sup.4 are taken together to form an optionally substituted 3-8 membered saturated ring having 0-3 heteroatoms in addition to the nitrogen of NG.sup.5-.

3. The method of claim 1, wherein P.sup.N is P(=NLR.sup.5), wherein L is S(O).sub.2and R.sup.5 is an optionally substituted group selected from phenyl and C.sub.1-30 aliphatic.

4. The method of claim 1, wherein G.sup.2 is -LS(O).sub.2R, wherein L is optionally substituted CH.sub.2.

5. The method of claim 4, wherein R is optionally substituted C.sub.1-6 aliphatic.

6. The method of claim 4, wherein R is t-butyl.

7. The method of claim 4, wherein R is optionally substituted phenyl.

8. The method of claim 4, wherein R is phenyl.

9. The method of claim 1, comprising one or more cycles, each of which independently comprises or consisting of: 1) Deblocking; 2) Coupling; 3) Optionally a first capping; 4) Modifying; and 5) Optionally a second capping.

10. The method of claim 1, wherein the base is an amine base having the structure of NR.sub.3.

11. The method of claim 10, wherein the base is N,N-diethylamine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an example of multiple exon skipping.

(2) FIG. 2 shows a cartoon of a method for detecting multiple exon skipping.

(3) FIG. 3 illustrates various strategies for multiple exon skipping.

DEFINITIONS

(4) As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001.

(5) Aliphatic: The term aliphatic or aliphatic group, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic or polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as carbocycle cycloaliphatic or cycloalkyl), or combinations thereof. In some embodiments, aliphatic groups contain 1-100 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphatic carbon atoms. In some embodiments, cycloaliphatic (or carbocycle or cycloalkyl) refers to a monocyclic or bicyclic or polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic. In some embodiments, cycloaliphatic (or carbocycle or cycloalkyl) refers to a monocyclic C.sub.3-C.sub.6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

(6) Alkenyl: As used herein, the term alkenyl refers to an aliphatic group, as defined herein, having one or more double bonds.

(7) Alkyl: As used herein, the term alkyl is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C.sub.1-C.sub.20 for straight chain, C.sub.2-C.sub.20 for branched chain), and alternatively, about 1-10. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure where such rings are monocyclic, bicyclic, or polycyclic, and alternatively about 5, 6 or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, wherein a lower alkyl group comprises 1-4 carbon atoms (e.g., C.sub.1-C.sub.4 for straight chain lower alkyls).

(8) Alkynyl: As used herein, the term alkynyl refers to an aliphatic group, as defined herein, having one or more triple bonds.

(9) Animal: As used herein, the term animal refers to any member of the animal kingdom. In some embodiments, animal refers to humans, at any stage of development. In some embodiments, animal refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In some embodiments, an animal may be a transgenic animal, a genetically-engineered animal, and/or a clone.

(10) Approximately: As used herein, the terms approximately or about in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). In some embodiments, use of the term about in reference to dosages means5 mg/kg/day.

(11) Aryl: The term aryl, as used herein, used alone or as part of a larger moiety as in aralkyl, aralkoxy, or aryloxyalkyl, refers to monocyclic, bicyclic or polycyclic ring systems having a total of, e.g., five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, an aryl group is a biaryl group. The term aryl may be used interchangeably with the term aryl ring. In certain embodiments of the present disclosure, aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term aryl, as it is used herein, is an aromatic ring fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

(12) Characteristic sequence: A characteristic sequence is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.

(13) Comparable: The term comparable is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In some embodiments, comparable sets of conditions or circumstances are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of conditions are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under the different sets of conditions or circumstances are caused by or indicative of the variation in those features that are varied.

(14) Cycloaliphatic: The term cycloaliphatic, carbocycle, carbocyclyl, carbocyclic radical, and carbocyclic ring, are used interchangeably, and as used herein, refer to saturated or partially unsaturated, but non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having, unless otherwise specified, from 3 to 30 ring members. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a cycloaliphatic group has 3-6 carbons. In some embodiments, a cycloaliphatic group is saturated and is cycloalkyl. The term cycloaliphatic may also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or 1,2,3,4-tetrahydronaphth-1-yl. In some embodiments, a cycloaliphatic group is bicyclic. In some embodiments, a cycloaliphatic group is tricyclic. In some embodiments, a cycloaliphatic group is polycyclic. In some embodiments, cycloaliphatic refers to C.sub.3-C.sub.6 monocyclic hydrocarbon, or C.sub.8-C.sub.10 bicyclic or polycyclic hydrocarbon, that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, or a C.sub.9-C.sub.16 polycyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic.

(15) Dosing regimen: As used herein, a dosing regimen or therapeutic regimen refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regime comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.

(16) Heteroaliphatic: The term heteroaliphatic refers to an aliphatic group wherein one or more units selected from C, CH, CH.sub.2, and CH.sub.3 are independently replaced by one or more heteroatoms. In some embodiments, a heteroaliphatic group is heteroalkyl. In some embodiments, a heteroaliphatic group is heteroalkenyl.

(17) Heteroaryl: The terms heteroaryl and heteroar-, as used herein, used alone or as part of a larger moiety, e.g., heteroaralkyl, or heteroaralkoxy, refer to monocyclic, bicyclic or polycyclic ring systems having a total of, e.g., five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroaryl group is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, a heteroaryl group has 6, 10, or 14 electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl and the like. The terms heteroaryl and heteroar-, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be monocyclic, bicyclic or polycyclic. The term heteroaryl may be used interchangeably with the terms heteroaryl ring, heteroaryl group, or heteroaromatic, any of which terms include rings that are optionally substituted. The term heteroaralkyl refers to an alkyl group substituted by a heteroaryl group, wherein the alkyl and heteroaryl portions independently are optionally substituted.

(18) Heteroatom: The term heteroatom means an atom that is not carbon or hydrogen. In some embodiments, a heteroatom is oxygen, sulfur, nitrogen, phosphorus, boron or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring (for example, N as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR.sup.+ (as in N-substituted pyrrolidinyl); etc.). In some embodiments, a heteroatom is boron, nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, silicon, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, a heteroatom is nitrogen, oxygen or sulfur.

(19) Heterocycle: As used herein, the terms heterocycle, heterocyclyl, heterocyclic radical, and heterocyclic ring, as used herein, are used interchangeably and refer to a monocyclic, bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term nitrogen includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur and nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or .sup.+NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms heterocycle, heterocyclyl, heterocyclyl ring, heterocyclic group, heterocyclic moiety, and heterocyclic radical, are used interchangeably herein, and also include heterocyclyl rings fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic, bicyclic or polycyclic. The term heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

(20) Intraperitoneal: The phrases intraperitoneal administration and administered intraperitonealy as used herein have their art-understood meaning referring to administration of a compound or composition into the peritoneum of a subject.

(21) In vitro: As used herein, the term in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant, and/or microbe).

(22) In vivo: As used herein, the term in vivo refers to events that occur within an organism (e.g., animal, plant, and/or microbe).

(23) Lower alkyl: The term lower alkyl refers to a C.sub.1-4 straight or branched alkyl group. Example lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

(24) Lower haloalkyl: The term lower haloalkyl refers to a C.sub.1-4 straight or branched alkyl group that is substituted with one or more halogen atoms.

(25) Optionally substituted: As described herein, compounds of the disclosure, e.g., oligonucleotides, lipids, carbohydrates, etc., may contain optionally substituted moieties. In general, the term substituted, whether preceded by the term optionally or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an optionally substituted group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term stable, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

(26) Suitable monovalent substituents are halogen; (CH.sub.2).sub.0-4R.sup.o; (CH.sub.2).sub.0-4OR.sup.o; O(CH.sub.2).sub.0-4R.sup.o, O(CH.sub.2).sub.0-4C(O)OR.sup.o; (CH.sub.2).sub.0-4CH(OR.sup.o).sub.2; (CH.sub.2).sub.0-4Ph, which may be substituted with R.sup.o; (CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1Ph which may be substituted with R.sup.o; CHCHPh, which may be substituted with R.sup.o; (CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1 pyridyl which may be substituted with R.sup.o; NO.sub.2; CN; N.sub.3; (CH.sub.2).sub.0-4N(R.sup.o).sub.2; (CH.sub.2).sub.0-4N(R.sup.o)C(O)R.sup.o; N(R.sup.o)C(S)R.sup.o; (CH.sub.2).sub.0-4N(R.sup.o)C(O)N(R.sup.o).sub.2; N(R.sup.o)C(S)N(R.sup.o).sub.2; (CH.sub.2).sub.0-4N(R.sup.o)C(O)OR.sup.o; N(R.sup.o)N(R.sup.o)C(O)R.sup.o; N(R.sup.o)N(R.sup.o)C(O)N(R.sup.o).sub.2; N(R.sup.o)N(R.sup.o)C(O)OR.sup.o; (CH.sub.2).sub.0-4C(O)R.sup.o; C(S)R.sup.o; (CH.sub.2).sub.0-4C(O)OR.sup.o; (CH.sub.2).sub.0-4C(O)SR.sup.o; (CH.sub.2).sub.0-4C(O)OSi(R.sup.o).sub.3; (CH.sub.2).sub.0-4OC(O)R.sup.o; OC(O)(CH.sub.2).sub.0-4SR.sup.o, SC(S)SR.sup.o; (CH.sub.2).sub.0-4SC(O)R.sup.o; (CH.sub.2).sub.0-4C(O)N(R.sup.o).sub.2; C(S)N(R.sup.o).sub.2; C(S)SR.sup.o; SC(S)SR.sup.o, (CH.sub.2).sub.0-4OC(O)N(R.sup.o).sub.2; C(O)N(OR.sup.o)R.sup.o; C(O)C(O)R.sup.o; C(O)CH.sub.2C(O)R.sup.o; C(NOR.sup.o)R.sup.o; (CH.sub.2).sub.0-4SSR.sup.o; (CH.sub.2).sub.0-4S(O).sub.2R.sup.o; (CH.sub.2).sub.0-4S(O).sub.2OR.sup.o; (CH.sub.2).sub.0-4OS(O).sub.2R.sup.o; S(O).sub.2N(R.sup.o).sub.2; (CH.sub.2).sub.0-4S(O)R.sup.o; N(R.sup.oS(O).sub.2N(R.sup.o).sub.2; N(R.sup.oS(O).sub.2R.sup.o; N(OR.sup.o)R.sup.o; C(NH)N(R.sup.o).sub.2; Si(R.sup.o).sub.3; OSi(R.sup.o).sub.3; P(R.sup.o).sub.2; P(OR.sup.o).sub.2; P(R.sup.o) (OR.sup.o); OP(R.sup.o).sub.2; OP(OR.sup.o).sub.2; OP(R.sup.o)(OR.sup.o); P[N(R.sup.o).sub.2].sub.2; P(R.sup.o)[N(R.sup.o).sub.2]; P(OR.sup.o)[N(R.sup.o).sub.2]; OP[N(R.sup.o).sub.2].sub.2; OP(R.sup.o)[N(R.sup.o).sub.2]; OP(OR.sup.o)[N(R.sup.o).sub.2]; N(R.sup.o)P(R.sup.o).sub.2; N(R.sup.o)P(OR.sup.o).sub.2; N(R.sup.o)P(R.sup.o)(OR.sup.o); N(R.sup.o)P[N(R.sup.o).sub.2].sub.2; N(R.sup.o)P(R.sup.o)[N(R.sup.o).sub.2]; N(R.sup.o)P(OR.sup.o)[N(R.sup.o).sub.2]; B(R.sup.o).sub.2; B(R.sup.o)(OR.sup.o); B(OR.sup.o).sub.2; OB(R.sup.o).sub.2; OB(R.sup.o)(OR.sup.o); OB(OR.sup.o).sub.2; P(O)(R.sup.o).sub.2; P(O)(R.sup.o)(OR.sup.o); P(O)(R.sup.o)(SR.sup.o); P(O)(R.sup.o)[N(R.sup.o).sub.2]; P(O)(OR.sup.o).sub.2; P(O)(SR.sup.o).sub.2; P(O)(OR.sup.o)[N(R.sup.o).sub.2]; P(O)(SR.sup.o)[N(R.sup.o).sub.2]; P(O)(OR.sup.o)(SR.sup.o); P(O)[N(R.sup.o).sub.2].sub.2; OP(O)(R.sup.o).sub.2; OP(O)(R.sup.o)(OR.sup.o); OP(O)(R.sup.o)(SR.sup.o); OP(O)(R.sup.o)[N(R.sup.o).sub.2]; OP(O)(OR.sup.o).sub.2; OP(O)(SR.sup.o).sub.2; OP(O)(OR.sup.o)[N(R.sup.o).sub.2]; OP(O)(SR.sup.o)[N(R.sup.o).sub.2]; OP(O)(OR.sup.o)(SR.sup.o); OP(O)[N(R.sup.o).sub.2].sub.2; SP(O)(R.sup.o).sub.2; SP(O)(R.sup.o)(OR.sup.o); SP(O)(R.sup.o)(SR.sup.o); SP(O)(R.sup.o)[N(R.sup.o).sub.2]; SP(O)(OR.sup.o).sub.2; SP(O)(SR.sup.o).sub.2; SP(O)(OR.sup.o)[N(R.sup.o).sub.2]; SP(O)(SR.sup.o)[N(R.sup.o).sub.2]; SP(O)(OR.sup.o)(SR.sup.o); SP(O)[N(R.sup.o).sub.2].sub.2; N(R.sup.o)P(O)(R.sup.o).sub.2; N(R.sup.o)P(O)(R.sup.o)(OR.sup.o); N(R.sup.o)P(O)(R.sup.o)(SR.sup.o); N(R.sup.o)P(O)(R.sup.o)[N(R.sup.o).sub.2]; N(R.sup.o)P(O)(OR.sup.o).sub.2; N(R.sup.o)P(O)(SR.sup.o).sub.2; N(R.sup.o)P(O)(OR.sup.o)[N(R.sup.o).sub.2]; N(R.sup.o)P(O)(SR.sup.o)[N(R.sup.o).sub.2]; N(R.sup.o)P(O)(OR.sup.o)(SR.sup.o); N(R.sup.o)P(O)[N(R.sup.o).sub.2].sub.2; P(R.sup.o).sub.2[B(R.sup.o).sub.3]; P(OR.sup.o).sub.2[B(R.sup.o).sub.3]; P(NR.sup.o).sub.2[B(R.sup.o).sub.3]; P(R.sup.o)(OR.sup.o)[B(R.sup.o).sub.3]; P(R.sup.o)[N(R.sup.o).sub.2][B(R.sup.o).sub.3]; P(OR.sup.o)[N(R.sup.o).sub.2][B(R.sup.o).sub.3]; OP(R.sup.o).sub.2[B(R.sup.o).sub.3]; OP(OR.sup.o).sub.2[B(R.sup.o).sub.3]; OP(NR.sup.o).sub.2[B(R.sup.o).sub.3]; OP(R.sup.o)(OR.sup.o)[B(R.sup.o).sub.3]; OP(R.sup.o)[N(R.sup.o).sub.2][B(R.sup.o).sub.3]; OP(OR.sup.o)[N(R.sup.o).sub.2][B(R.sup.o).sub.3]; N(R.sup.o)P(R.sup.o).sub.2[B(R.sup.o).sub.3]; N(R.sup.o)P(OR.sup.o).sub.2[B(R.sup.o).sub.3]; N(R.sup.o)P(NR.sup.o).sub.2[B(R.sup.o).sub.3]; N(R.sup.o)P(R.sup.o)(OR.sup.o)[B(R.sup.o).sub.3]; N(R.sup.o)P(R.sup.o)[N(R.sup.o).sub.2][B(R.sup.o).sub.3]; N(R.sup.o)P(OR.sup.o)[N(R.sup.o).sub.2][B(R.sup.o).sub.3]; P(OR)[B(R).sub.3]; (C.sub.1-4 straight or branched alkylene)ON(R.sup.o).sub.2; or (C.sub.1-4 straight or branched alkylene)C(O)ON(R.sup.o).sub.2, wherein each R.sup.o may be substituted as defined below and is independently hydrogen, C.sub.1-20 aliphatic, C.sub.1-20 heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, CH.sub.2(C.sub.6-20 aryl), O(CH.sub.2).sub.0-1 (C.sub.6-20 aryl), CH.sub.2-(5-20 membered heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus), a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R.sup.o, taken together with their intervening atom(s), form a 3-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

(27) Suitable monovalent substituents on R.sup.o (or the ring formed by taking two independent occurrences of R.sup.o together with their intervening atoms), are independently halogen, (CH.sub.2).sub.0-2R.sup..circle-solid., -(haloR.sup..circle-solid.), (CH.sub.2).sub.0-2OH, (CH.sub.2).sub.0-2OR.sup..circle-solid., (CH.sub.2).sub.0-2CH(OR.sup..circle-solid.).sub.2; O(haloR.sup..circle-solid.), CN, N.sub.3, (CH.sub.2).sub.0-2C(O)R.sup..circle-solid., (CH.sub.2).sub.0-2C(O)OH, (CH.sub.2).sub.0-2C(O)OR.sup..circle-solid., (CH.sub.2).sub.0-2SR.sup..circle-solid., (CH.sub.2).sub.0-2SH, (CH.sub.2).sub.0-2NH.sub.2, (CH.sub.2).sub.0-2NHR.sup..circle-solid., (CH.sub.2).sub.0-2NR.sup..circle-solid..sub.2, NO.sub.2, SiR.sup..circle-solid..sub.3, OSiR.sup..circle-solid..sub.3, C(O)SR.sup..circle-solid., (C.sub.1-4 straight or branched alkylene)C(O)OR.sup..circle-solid., or SSR.sup..circle-solid. wherein each R.sup..circle-solid. is unsubstituted or where preceded by halo is substituted only with one or more halogens, and is independently selected from C.sub.1-4 aliphatic, CH.sub.2Ph, O(CH.sub.2).sub.0-1Ph, and a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R.sup.o include O and S.

(28) Suitable divalent substituents, e.g., on a suitable carbon atom, nitrogen atom, are independently the following: O, S, CR.sup.*.sub.2, NNR.sup.*.sub.2, NNHC(O)R*, NNHC(O)OR*, NNHS(O).sub.2R*, NR*, NOR*, O(C(R.sup.*.sub.2)).sub.2-3O, or S(C(R.sup.*.sub.2)).sub.2-3S, wherein each R* may be substituted as defined below and is independently hydrogen, C.sub.1-20 aliphatic, C.sub.1-20 heteroaliphatic having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, CH.sub.2(C.sub.6-20 aryl), O(CH.sub.2).sub.0-1 (C.sub.6-20 aryl), CH.sub.2-(5-20 membered heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus), a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the definition above, two independent occurrences of R*, taken together with their intervening atom(s), form a 3-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below. Suitable divalent substituents that are bound to vicinal substitutable atoms of an optionally substituted group include: O(CR.sup.*.sub.2).sub.2-3O.

(29) Suitable monovalent substituents on R* (or the ring formed by taking two independent occurrences of R* together with their intervening atoms), are independently halogen, (CH.sub.2).sub.0-2R.sup..circle-solid., -(haloR.sup..circle-solid.), (CH.sub.2).sub.0-2OH, (CH.sub.2).sub.0-2OR.sup..circle-solid., (CH.sub.2).sub.0-2CH(OR.sup..circle-solid.).sub.2; O(haloR.sup..circle-solid.), CN, N.sub.3, (CH.sub.2).sub.0-2C(O)R.sup..circle-solid., (CH.sub.2).sub.0-2C(O)OH, (CH.sub.2).sub.0-2C(O)OR.sup..circle-solid., (CH.sub.2).sub.0-2SR.sup..circle-solid., (CH.sub.2).sub.0-2SH, (CH.sub.2).sub.0-2NH.sub.2, (CH.sub.2).sub.0-2NHR.sup..circle-solid., (CH.sub.2).sub.0-2NR.sup..circle-solid..sub.2, NO.sub.2, SiR.sup..circle-solid..sub.3, OSiR.sup..circle-solid..sub.3, C(O)SR.sup..circle-solid., (C.sub.1-4 straight or branched alkylene)C(O)OR.sup..circle-solid., or SSR.sup..circle-solid. wherein each R.sup..circle-solid. is unsubstituted or where preceded by halo is substituted only with one or more halogens, and is independently selected from C.sub.1-4 aliphatic, CH.sub.2Ph, O(CH.sub.2).sub.0-1Ph, and a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R* include O and S.

(30) In some embodiments, suitable substituents on a substitutable nitrogen of an optionally substituted group include R.sup., NR.sup..sub.2, C(O)R.sup., C(O)OR.sup., C(O)C(O)R.sup., C(O)CH.sub.2C(O)R.sup., S(O).sub.2R.sup., S(O).sub.2NR.sup..sub.2, C(S)NR.sup..sub.2, C(NH)NR.sup..sub.2, or N(R.sup.)S(O).sub.2R.sup.; wherein each R.sup. is independently hydrogen, C.sub.1-6 aliphatic which may be substituted as defined below, unsubstituted OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R.sup., taken together with their intervening atom(s) form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

(31) In some embodiments, suitable substituents on the aliphatic group of R.sup. are independently halogen, R.sup..circle-solid., -(haloR.sup..circle-solid.), OH, OR.sup..circle-solid., O(haloR.sup..circle-solid.), CN, C(O)OH, C(O)OR.sup..circle-solid., NH.sub.2, NHR.sup..circle-solid., NR.sup..circle-solid..sub.2, or NO.sub.2, wherein each R.sup..circle-solid. is unsubstituted or where preceded by halo is substituted only with one or more halogens, and is independently C.sub.1-4 aliphatic, CH.sub.2Ph, O(CH.sub.2).sub.0-1Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

(32) Oral: The phrases oral administration and administered orally as used herein have their art-understood meaning referring to administration by mouth of a compound or composition.

(33) Parenteral: The phrases parenteral administration and administered parenterally as used herein have their art-understood meaning referring to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

(34) Partially unsaturated: As used herein, the term partially unsaturated refers to a ring moiety that includes at least one double or triple bond. The term partially unsaturated is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

(35) Pharmaceutical composition: As used herein, the term pharmaceutical composition refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a controlled therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

(36) Pharmaceutically acceptable: As used herein, the phrase pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

(37) Pharmaceutically acceptable carrier: As used herein, the term pharmaceutically acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

(38) Pharmaceutically acceptable salt: The term pharmaceutically acceptable salt, as used herein, refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a provided compound comprises one or more acidic groups, e.g., an oligonucleotide, and a pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium (e.g., an ammonium salt of N(R).sub.3, wherein each R is independently as defined and described in the present disclosure) salt. Representative alkali or alkaline earth metal salts include salts of sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, a pharmaceutically acceptable salt is a potassium salt. In some embodiments, a pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. In some embodiments, a provided compound comprises more than one acid groups, for example, a provided oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate linkages and/or modified internucleotidic linkages). In some embodiments, a pharmaceutically acceptable salt, or generally a salt, of such a compound comprises two or more cations, which can be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or generally, a salt), each acidic group having sufficient acidity independently exists as its salt form (e.g., in an oligonucleotide comprising natural phosphate linkages and phosphorothioate internucleotidic linkages, each of the natural phosphate linkages and phosphorothioate internucleotidic linkages independently exists as its salt form). In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide. In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide, wherein each acidic linkage, e.g., each natural phosphate linkage and phosphorothioate internucleotidic linkage, exists as a sodium salt form (all sodium salt).

(39) Protecting group: The term protecting group, as used herein, is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Also included are those protecting groups specially adapted for nucleoside and nucleotide chemistry, e.g., those described in Current Protocols in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. June 2012, the entirety of Chapter 2 is incorporated herein by reference. Suitable amino-protecting groups include methyl 1 carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo) fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2- and 4-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N-p-toluenesulfonylaminocarbonyl derivative, N-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido) propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N(N,N-dimethylaminomethylene)amine, N,N-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, 0-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, -trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), -trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4,8-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

(40) Suitably protected carboxylic acids further include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

(41) Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, -naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4-bromophenacyloxyphenyl)diphenylmethyl, 4,4,4-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4,4-tris(levulinoyloxyphenyl)methyl, 4,4,4-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4,4-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), -butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio) pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy) butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl) phenoxyacetate, 2,4-bis(1,1-dimethylpropyl) phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, -naphthoate, nitrate, alkyl N,N,N,N-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho -methoxybenzylidene ester, ortho ester, 1-(N,N-dimethylamino)ethylidene derivative, -(N,N-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho di-t-butylsilylene ester, group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

(42) In some embodiments, a hydroxyl protecting group is acetyl, t-butyl, tbutoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl (trityl), 4,4-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4-dimethoxytrityl, (DMTr) and 4,4,4-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4,4-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is, independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and 4,4-dimethoxytrityl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of trityl, monomethoxytrityl and 4,4-dimethoxytrityl group.

(43) In some embodiments, a phosphorous protecting group is a group attached to the internucleotide phosphorous linkage throughout oligonucleotide synthesis. In some embodiments, the phosphorous protecting group is attached to the sulfur atom of the internucleotide phosphorothioate linkage. In some embodiments, the phosphorous protecting group is attached to the oxygen atom of the internucleotide phosphorothioate linkage. In some embodiments, the phosphorous protecting group is attached to the oxygen atom of the internucleotide phosphate linkage. In some embodiments the phosphorous protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

(44) Protein: As used herein, the term protein refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). In some embodiments, proteins include only naturally-occurring amino acids. In some embodiments, proteins include one or more non-naturally-occurring amino acids (e.g., moieties that form one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues in a protein chain contain a non-amino-acid moiety (e.g., a glycan, etc). In some embodiments, a protein includes more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. In some embodiments, proteins contain L-amino acids, D-amino acids, or both; in some embodiments, proteins contain one or more amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. The term peptide is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

(45) Subject: As used herein, the term subject or test subject refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be suffering from, and/or susceptible to a disease, disorder, and/or condition.

(46) Substantially: As used herein, the term substantially refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term substantially is therefore used herein to capture the potential lack of completeness inherent in many biological and/or chemical phenomena.

(47) Suffering from: An individual who is suffering from a disease, disorder, and/or condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition.

(48) Susceptible to: An individual who is susceptible to a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

(49) Systemic: The phrases systemic administration, administered systemically, peripheral administration, and administered peripherally as used herein have their art-understood meaning referring to administration of a compound or composition such that it enters the recipient's system.

(50) Tautomeric forms: The phrase tautomeric forms, as used herein and generally understood in the art, is used to describe different isomeric forms of organic compounds that are capable of facile interconversion. Tautomers may be characterized by the formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and adjacent double bond. In some embodiments, tautomers may result from prototropic tautomerism (i.e., the relocation of a proton). In some embodiments, tautomers may result from valence tautomerism (i.e., the rapid reorganization of bonding electrons). All such tautomeric forms are intended to be included within the scope of the present disclosure. In some embodiments, tautomeric forms of a compound exist in mobile equilibrium with each other, so that attempts to prepare the separate substances results in the formation of a mixture. In some embodiments, tautomeric forms of a compound are separable and isolatable compounds. In some embodiments of the disclosure, chemical compositions may be provided that are or include pure preparations of a single tautomeric form of a compound. In some embodiments of the disclosure, chemical compositions may be provided as mixtures of two or more tautomeric forms of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomeric forms; in certain embodiments, such mixtures contain different amounts of at least two different tautomeric forms of a compound. In some embodiments of the disclosure, chemical compositions may contain all tautomeric forms of a compound. In some embodiments of the disclosure, chemical compositions may contain less than all tautomeric forms of a compound. In some embodiments of the disclosure, chemical compositions may contain one or more tautomeric forms of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the disclosure, the tautomerism is keto-enol tautomerism. One of skill in the chemical arts would recognize that a keto-enol tautomer can be trapped (i.e., chemically modified such that it remains in the enol form) using any suitable reagent known in the chemical arts in to provide an enol derivative that may subsequently be isolated using one or more suitable techniques known in the art. Unless otherwise indicated, the present disclosure encompasses all tautomeric forms of relevant compounds, whether in pure form or in admixture with one another.

(51) Therapeutic agent: As used herein, the phrase therapeutic agent refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.

(52) Therapeutically effective amount: As used herein, the term therapeutically effective amount means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

(53) Treat: As used herein, the term treat, treatment, or treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

(54) Unit dose: The expression unit dose as used herein refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical composition. In many embodiments, a unit dose contains a predetermined quantity of an active agent. In some embodiments, a unit dose contains an entire single dose of the agent. In some embodiments, more than one unit dose is administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect. A unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc. It will be appreciated that a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s). For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc., may be included as described infra. It will be appreciated by those skilled in the art, in many embodiments, a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment. In some embodiments, the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.

(55) Unsaturated: The term unsaturated, as used herein, means that a moiety has one or more units of unsaturation.

(56) Wild-type: As used herein, the term wild-type has its art-understood meaning that refers to an entity having a structure and/or activity as found in nature in a normal (as contrasted with mutant, diseased, altered, etc) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

(57) Nucleic acid: The term nucleic acid includes any nucleotides, analogs thereof, and polymers thereof. The term polynucleotide as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or analogs thereof. These terms refer to the primary structure of the molecules and include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom bridges (also referred to herein as internucleotidic linkages). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, natural natural phosphate internucleotidic linkages or non-natural internucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly-refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo-refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

(58) Nucleotide: The term nucleotide as used herein refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups or phosphorus-containing internucleotidic linkages. Naturally occurring bases, (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included. Naturally occurring sugars include the pentose (five-carbon sugar) deoxyribose (which is found in natural DNA) or ribose (which is found in natural RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included, such as sugars with 2-modifications, sugars in locked nucleic acid (LNA) and phosphorodiamidate morpholino oligomer (PMO). Nucleotides are linked via internucleotidic linkages to form nucleic acids, or polynucleotides. Many internucleotidic linkages are known in the art (such as, though not limited to, natural phosphate linkage, phosphorothioate linkages, boranophosphate linkages and the like). Artificial nucleic acids include PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates and other variants of the phosphate backbone of native nucleic acids, etc. In some embodiments, a nucleotide is a natural nucleotide comprising a naturally occurring nucleobase, a natural occurring sugar and the natural phosphate linkage. In some embodiments, a nucleotide is a modified nucleotide or a nucleotide analog, which is a structural analog that can be used in lieu of a natural nucleotide.

(59) Modified nucleotide: The term modified nucleotide includes any chemical moiety which differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, a modified nucleotide comprises a modification at a sugar, base and/or internucleotidic linkage. In some embodiments, a modified nucleotide comprises a modified sugar, modified nucleobase and/or modified internucleotidic linkage. In some embodiments, a modified nucleotide is capable of at least one function of a nucleotide, e.g., forming a subunit in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

(60) Analog: The term analog includes any chemical moiety which differs structurally from a reference chemical moiety or class of moieties, but which is capable of performing at least one function of such a reference chemical moiety or class of moieties. As non-limiting examples, a nucleotide analog differs structurally from a nucleotide but performs at least one function of a nucleotide; a nucleobase analog differs structurally from a nucleobase but performs at least one function of a nucleobase; a sugar analog differs structurally from a nucleobase but performs at least one function of a sugar, etc.

(61) Nucleoside: The term nucleoside refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or modified sugar.

(62) Modified nucleoside: The term modified nucleoside refers to a chemical moiety which is chemically distinct from a natural nucleoside, but which is capable of performing at least one function of a nucleoside. In some embodiments, a modified nucleoside is derived from or chemically similar to a natural nucleoside, but which comprises a chemical modification which differentiates it from a natural nucleoside. Non-limiting examples of modified nucleosides include those which comprise a modification at the base and/or the sugar. Non-limiting examples of modified nucleosides include those with a 2-modification at a sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a nucleobase). In some embodiments, a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases.

(63) Nucleoside analog: The term nucleoside analog refers to a chemical moiety which is chemically distinct from a natural nucleoside, but which is capable of performing at least one function of a nucleoside. In some embodiments, a nucleoside analog comprises an analog of a sugar and/or an analog of a nucleobase. In some embodiments, a modified nucleoside is capable of at least one function of a nucleoside, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising a complementary sequence of bases.

(64) Sugar: The term sugar refers to a monosaccharide or polysaccharide in closed and/or open form. In some embodiments, sugars are monosaccharides. In some embodiments, sugars are polysaccharides. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term sugar also encompasses structural analogs used in lieu of conventional sugar molecules, such as glycol, polymer of which forms the backbone of the nucleic acid analog, glycol nucleic acid (GNA), etc. As used herein, the term sugar also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified sugars and nucleotide sugars. In some embodiments, a sugar is D-2-deoxyribose. In some embodiments, a sugar is beta-D-deoxyribofuranose. In some embodiments, a sugar moiety is a beta-D-deoxyribofuranose moiety. In some embodiments, a sugar is D-ribose. In some embodiments, a sugar is beta-D-ribofuranose. In some embodiments, a sugar moiety is a beta-D-ribofuranose moiety. In some embodiments, a sugar is optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose. In some embodiments, a sugar moiety is an optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose moiety. In some embodiments, a sugar moiety/unit in an oligonucleotide, nucleic acid, etc. is a sugar which comprises one or more carbon atoms each independently connected to an internucleotidic linkage, e.g., optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose whose 5-C and/or 3-C are each independently connected to an internucleotidic linkage (e.g., a natural phosphate linkage, a modified internucleotidic linkage, a chirally controlled internucleotidic linkage, etc.).

(65) Modified sugar: The term modified sugar refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. In some embodiments, a modified sugar is substituted beta-D-deoxyribofuranose or beta-D-ribofuranose. In some embodiments, a modified sugar comprises a 2-modification. In some embodiments, a modified sugar comprises a linker (e.g., optionally substituted bivalent heteroaliphatic) connecting two sugar carbon atoms (e.g., C2 and C4), e.g., as found in LNA. In some embodiments, a linker is OCH(R), wherein R is as described in the present disclosure. In some embodiments, a linker is OCH(R), wherein O is connected to C2, and CH(R) is connected to C4 of a sugar, and R is as described in the present disclosure. In some embodiments, R is methyl. In some embodiments, R is H. In some embodiments, CH(R) is of S configuration. In some embodiments, CH(R) is of R configuration.

(66) Nucleobase: The term nucleobase refers to the parts of nucleic acids that are involved in the hydrogen-bonding that binds one nucleic acid strand to another complementary strand in a sequence specific manner. The most common naturally-occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is a substituted nucleobase which nucleobase is selected from A, T, C, G, U, and tautomers thereof. In some embodiments, the naturally-occurring nucleobases are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally-occurring nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase is a modified nucleobase, e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobases are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobase mimics the spatial arrangement, electronic properties, or some other physicochemical property of the nucleobase and retains the property of hydrogen-bonding that binds one nucleic acid strand to another in a sequence specific manner. In some embodiments, a modified nucleobase can pair with all of the five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide duplex. As used herein, the term nucleobase also encompasses structural analogs used in lieu of natural or naturally-occurring nucleotides, such as modified nucleobases and nucleobase analogs. In some embodiments, a nucleobase is an optionally substituted A, T, C, G, or U, or a substituted nucleobase which nucleobase is selected from A, T, C, G, U, and tautomers thereof.

(67) Modified nucleobase: The terms modified nucleobase, modified base and the like refer to a chemical moiety which is chemically distinct from a nucleobase, but which is capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase which comprises a modification. In some embodiments, a modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases. In some embodiments, a modified nucleobase is a substituted nucleobase which nucleobase is selected from A, T, C, G, U, and tautomers thereof.

(68) Chiral ligand: The term chiral ligand or chiral auxiliary refers to a moiety that is chiral and can be incorporated into a reaction so that the reaction can be carried out with certain stereoselectivity. In some embodiments, the term may also refer to a compound that comprises such a moiety.

(69) Blocking group: The term blocking group refers to a group that masks the reactivity of a functional group. The functional group can be subsequently unmasked by removal of the blocking group. In some embodiments, a blocking group is a protecting group.

(70) Moiety: The term moiety refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule. In some embodiments, a moiety of a compound is a monovalent, bivalent, or polyvalent group formed from the compound by removing one or more H and/or equivalents thereof from a compound. In some embodiments, depending on its context, moiety may also refer to a compound or entity from which the moiety is derived from.

(71) Solid support: The term solid support when used in the context of preparation of nucleic acids, oligonucleotides, or other compounds refers to any support which enables synthesis of nucleic acids, oligonucleotides or other compounds. In some embodiments, the term refers to a glass or a polymer, that is insoluble in the media employed in the reaction steps performed to synthesize nucleic acids, and is derivatized to comprise reactive groups. In some embodiments, the solid support is Highly Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). In some embodiments, the solid support is Controlled Pore Glass (CPG). In some embodiments, the solid support is hybrid support of Controlled Pore Glass (CPG) and Highly Cross-linked Polystyrene (HCP).

(72) Reading frame: The term reading frame refers to one of the six possible reading frames, three in each direction, of a double stranded DNA molecule. The reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA molecule.

(73) Antisense: As used herein, an antisense nucleic acid molecule comprises a nucleotide sequence which is complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid molecule can associate via hydrogen bonds to a sense nucleic acid molecule. In some embodiments, transcripts may be generated from both strands. In some embodiments, transcripts may or may not encode protein products. In some embodiments, when directed or targeted to a particular nucleic acid sequence, a antisense sequence may refer to a sequence that is complementary to the particular nucleic acid sequence.

(74) Oligonucleotide: the term oligonucleotide refers to a polymer or oligomer of nucleotide monomers, containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, natural phosphate linkages, or non-natural internucleotidic linkages.

(75) Oligonucleotides can be single-stranded or double-stranded. As used herein, the term oligonucleotide strand encompasses a single-stranded oligonucleotide. A single-stranded oligonucleotide can have double-stranded regions and a double-stranded oligonucleotide can have single-stranded regions. Example oligonucleotides include, but are not limited to structural genes, genes including control and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double-stranded siRNAs and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immuno-stimulatory oligonucleotides, and decoy oligonucleotides.

(76) Double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference may also be referred to as siRNA, RNAi agent, or iRNA agent. In some embodiments, these RNA interference inducing oligonucleotides associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). In many embodiments, single-stranded and double-stranded RNAi agents are sufficiently long that they can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller oligonucleotides that can enter the RISC machinery and participate in RISC mediated cleavage of a target sequence, e.g. a target mRNA.

(77) Oligonucleosides of the present disclosure can be of various lengths. In particular embodiments, oligonucleosides can range from about 2 to about 200 nucleosides in length. In various related embodiments, oligonucleosides, single-stranded, double-stranded, and triple-stranded, can range in length from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides in length. In some embodiments, the oligonucleoside is from about 9 to about 39 nucleosides in length. In some embodiments, the oligonucleoside is at least 15 nucleosides in length. In some embodiments, the oligonucleoside is at least 20 nucleosides in length. In some embodiments, the oligonucleoside is at least 25 nucleosides in length. In some embodiments, the oligonucleoside is at least 30 nucleosides in length. In some embodiments, the oligonucleoside is a duplex of complementary strands of at least 18 nucleosides in length. In some embodiments, the oligonucleoside is a duplex of complementary strands of at least 21 nucleosides in length. In some embodiments, for the purpose of oligonucleotide lengths, each nucleoside counted independently comprises an optionally substituted nucleobase selected from A, T, C, G, U and their tautomers.

(78) Internucleotidic linkage: As used herein, the phrase internucleotidic linkage refers generally to a linkage, typically a phosphorus-containing linkage, between nucleotide units of a nucleic acid or an oligonucleotide, and is interchangeable with inter-sugar linkage, internucleosidic linkage, and phosphorus atom bridge, as used above and herein. As appreciated by those skilled in the art, natural DNA and RNA contain natural phosphate linkages. In some embodiments, an internucleotidic linkage is a natural phosphate linkage (OP(O)(OH)O, typically existing as its anionic form OP(O)(O.sup.)O at pH e.g., 7.4), as found in naturally occurring DNA and RNA molecules. In some embodiments, an internucleotidic linkage is a modified internucleotidic linkage (or non-natural internucleotidic linkage), which is structurally different from a natural phosphate linkage but may be utilized in place of a natural phosphate linkage, e.g., phosphorothioate internucleotidic linkage, PMO linkages, etc. In some embodiments, an internucleotidic linkage is a modified internucleotidic linkage wherein one or more oxygen atoms of a natural phosphodiester linkage are independently replaced by one or more organic or inorganic moieties. In some embodiments, such an organic or inorganic moiety is selected from but not limited to S, Se, NR, SR, SeR, N(R).sub.2, B(R).sub.3, S, Se, and N(R), wherein each R is independently as defined and described below. In some embodiments, an internucleotidic linkage is a phosphotriester linkage. In some embodiments, an internucleotidic linkage is a phosphorothioate diester linkage (phosphorothioate internucleotidic linkage,

(79) ##STR00011##
typically existing as its anionic form OP(O)(S)O at pH e.g., 7.4). It is understood by a person of ordinary skill in the art that an internucleotidic linkage may exist as an anion or cation at a given pH due to the existence of acid or base moieties in the linkage. In some embodiments, an internucleotidic linkage is a non-negatively charged internucleotidic linkage at a given pH. In some embodiments, an internucleotidic linkage is a neutral internucleotidic linkage at a given pH. In some embodiments, a given pH is pH 7.4. In some embodiments, a given pH is in the range of pH about 0, 1, 2, 3, 4, 5, 6 or 7 to pH about 7, 8, 9, 10, 11, 12, 13 or 14. In some embodiments, a given pH is in the range of pH 5-9. In some embodiments, a given pH is in the range of pH 6-8. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., as described in the present disclosure. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., as described in the present disclosure. In some embodiments, an internucleotidic linkage is one of, e.g., PNA (peptide nucleic acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. In some embodiments, an internucleotidic linkage comprises a chiral linkage phosphorus. In some embodiments, an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, an internucleotidic linkage is selected from: s (phosphorothioate), s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 or s18, wherein each of s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 and s18 is independently as described in WO 2017/062862.

(80) Unless otherwise specified, the Rp/Sp designations preceding an oligonucleotide sequence describe the configurations of linkage phosphorus in chirally controlled internucleotidic linkages sequentially from 5 to 3 of the oligonucleotide sequence. For instance, in (Rp, Sp)-ATsCs1GA, the phosphorus in the s linkage between T and C has Rp configuration and the phosphorus in s1 linkage between C and G has Sp configuration. In some embodiments, All-(Rp) or All-(Sp) is used to indicate that all chiral linkage phosphorus atoms in chirally controlled internucleotidic linkages have the same Rp or Sp configuration, respectively. For instance, All-(Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC(SEQ ID NO: 60) indicates that all the chiral linkage phosphorus atoms in the oligonucleotide have Rp configuration; All-(Sp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC(SEQ ID NO: 60) indicates that all the chiral linkage phosphorus atoms in the oligonucleotide have Sp configuration.

(81) Oligonucleotide type: As used herein, the phrase oligonucleotide type is used to define oligonucleotides that have a particular base sequence, pattern of backbone linkages (i.e., pattern of internucleotidic linkage types, for example, natural phosphate linkages, phosphorothioate internucleotidic linkages, negatively charged internucleotidic linkages, neutral internucleotidic linkages etc), pattern of backbone chiral centers (i.e. pattern of linkage phosphorus stereochemistry (Rp/Sp)), and pattern of backbone phosphorus modifications (e.g., pattern of X-L-R.sup.1 groups in formula I). In some embodiments, oligonucleotides of a common designated type are structurally identical to one another.

(82) One of skill in the art will appreciate that synthetic methods of the present disclosure provide for a degree of control during the synthesis of an oligonucleotide strand such that each nucleotide unit of the oligonucleotide strand can be designed and/or selected in advance to have a particular stereochemistry at the linkage phosphorus and/or a particular modification at the linkage phosphorus, and/or a particular base, and/or a particular sugar. In some embodiments, an oligonucleotide strand is designed and/or selected in advance to have a particular combination of stereocenters at the linkage phosphorus. In some embodiments, an oligonucleotide strand is designed and/or determined to have a particular combination of modifications at the linkage phosphorus. In some embodiments, an oligonucleotide strand is designed and/or selected to have a particular combination of bases. In some embodiments, an oligonucleotide strand is designed and/or selected to have a particular combination of one or more of the above structural characteristics. The present disclosure provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chirally controlled oligonucleotide compositions). In some embodiments, all such molecules are of the same type. In some embodiments, all such molecules are structurally identical to one another. In some embodiments, provided compositions comprise a plurality of oligonucleotides of different types, typically in pre-determined (non-random) relative amounts.

(83) Chiral control: As used herein, chiral control refers to control of the stereochemical designation of a chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide. In some embodiments, a control is achieved through a chiral element that is absent from the sugar and base moieties of an oligonucleotide, for example, in some embodiments, a control is achieved through use of one or more chiral auxiliaries during oligonucleotide preparation as exemplified in the present disclosure, which chiral auxiliaries often are part of chiral phosphoramidites used during oligonucleotide preparation. In contrast to chiral control, a person having ordinary skill in the art appreciates that conventional oligonucleotide synthesis which does not use chiral auxiliaries cannot control stereochemistry at a chiral internucleotidic linkage if such conventional oligonucleotide synthesis is used to form the chiral internucleotidic linkage. In some embodiments, the stereochemical designation of each chiral linkage phosphorus in a chiral internucleotidic linkage within an oligonucleotide is controlled.

(84) Chirally controlled oligonucleotide composition: The terms chirally controlled (stereocontrolled or stereodefined) oligonucleotide composition, chirally controlled (stereocontrolled or stereodefined) nucleic acid composition, and the like, as used herein, refers to a composition that comprises a plurality of oligonucleotides (or nucleic acids, chirally controlled oligonucleotides or chirally controlled nucleic acids) which share 1) a common base sequence, 2) a common pattern of backbone linkages; 3) a common pattern of backbone chiral centers, and 4) a common pattern of backbone phosphorus modifications (oligonucleotides of a particular type), wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotidic linkages (chirally controlled internucleotidic linkages, whose chiral linkage phosphorus is Rp or Sp, not a random Rp and Sp mixture as non-chirally controlled internucleotidic linkages). Level of the plurality of oligonucleotides (or nucleic acids) in a chirally controlled oligonucleotide composition is non-random (pre-determined, controlled). Chirally controlled oligonucleotide compositions are typically prepared through chirally controlled oligonucleotide preparation to stereoselectively form one or more chiral internucleotidic linkages (e.g., using chiral auxiliaries as exemplified in the present disclosure, compared to non-chirally controlled (stereorandom, non-stereoselective, racemic) oligonucleotide synthesis such as traditional phosphoramidite-based oligonucleotide synthesis using no chiral auxiliaries or chiral catalysts to purposefully control stereoselectivity). A chirally controlled oligonucleotide composition is enriched, relative to a substantially racemic preparation of oligonucleotides having the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications, for oligonucleotides of the plurality. In some embodiments, a chirally controlled oligonucleotide composition comprises a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications, wherein it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type. As one having ordinary skill in the art readily appreciates, such enrichment can be characterized in that compared to a substantially racemic preparation, at each chirally controlled internucleotidic linkage, a higher level of the linkage phosphorus has the desired configuration. In some embodiments, each chirally controlled internucleotidic linkage independently has a diastereopurity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to its chiral linkage phosphorus. In some embodiments, each independently has a diastereopurity of at least 90%. In some embodiments, each independently has a diastereopurity of at least 95%. In some embodiments, each independently has a diastereopurity of at least 97%. In some embodiments, each independently has a diastereopurity of at least 98%. In some embodiments, oligonucleotides of a plurality have the same constitution. In some embodiments, oligonucleotides of a plurality have the same constitution and stereochemistry, and are structurally identical.

(85) In some embodiments, the plurality of oligonucleotides in a chirally controlled oligonucleotide composition share the same base sequence, the same, if any, nucleobase, sugar, and internucleotidic linkage modifications, and the same stereochemistry (Rp or Sp) independently at linkage phosphorus chiral centers of one or more chirally controlled internucleotidic linkages, though stereochemistry of certain linkage phosphorus chiral centers may differ. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition that share the common base sequence, the common pattern of backbone linkages, and the common pattern of backbone phosphorus modifications are oligonucleotides of the plurality. In some embodiments, about 0.1%-100%, (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a chirally controlled oligonucleotide composition, or of all oligonucleotides in a composition that share a common base sequence (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share a common base sequence, a common patter of base modifications, a common pattern of sugar modifications, a common pattern of internucleotidic linkage types, and/or a common pattern of internucleotidic linkage modifications (e.g., of a plurality of oligonucleotide or an oligonucleotide type), or of all oligonucleotides in a composition that share the same constitution, are oligonucleotides of the plurality. In some embodiments, a percentage is at least (DP).sup.NCI, wherein DP is a percentage selected from 85%-100%, and NCI is the number of chirally controlled internucleotidic linkage. In some embodiments, DP is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, DP is at least 85%. In some embodiments, DP is at least 90%. In some embodiments, DP is at least 95%. In some embodiments, DP is at least 96%. In some embodiments, DP is at least 97%. In some embodiments, DP is at least 98%. In some embodiments, DP is at least 99%. In some embodiments, DP reflects diastereopurity of linkage phosphorus chiral centers chirally controlled internucleotidic linkages. In some embodiments, diastereopurity of a linkage phosphorus chiral center of an internucleotidic linkage may be typically assessed using an appropriate dimer comprising such an internucleotidic linkage and the two nucleoside units being linked by the internucleotidic linkage. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotidic linkages. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotidic linkages. In some embodiments, each chiral internucleotidic linkage is a chiral controlled internucleotidic linkage, and the composition is a completely chirally controlled oligonucleotide composition. In some embodiments, not all chiral internucleotidic linkages are chiral controlled internucleotidic linkages, and the composition is a partially chirally controlled oligonucleotide composition. In some embodiments, a chirally controlled oligonucleotide composition comprises predetermined levels of individual oligonucleotide or nucleic acids types. For instance, in some embodiments a chirally controlled oligonucleotide composition comprises one oligonucleotide type at a predetermined level (e.g., as described above). In some embodiments, a chirally controlled oligonucleotide composition comprises more than one oligonucleotide type, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition comprises multiple oligonucleotide types, each independently at a predetermined level. In some embodiments, a chirally controlled oligonucleotide composition is a composition of oligonucleotides of an oligonucleotide type, which composition comprises a predetermined level of a plurality of oligonucleotides of the oligonucleotide type.

(86) Chirally pure: as used herein, the phrase chirally pure is used to describe an oligonucleotide or compositions thereof, in which all or nearly all (the rest are impurities) of the oligonucleotide molecules exist in a single diastereomeric form with respect to the linkage phosphorus atoms. In many embodiments, as appreciated by those skilled in the art, a chirally pure oligonucleotide composition is substantially pure in that substantially all of the oligonucleotides in the composition are structurally identical (being the same stereoisomer).

(87) Linkage phosphorus: as defined herein, the phrase linkage phosphorus is used to indicate that the particular phosphorus atom being referred to is the phosphorus atom present in an internucleotidic linkage, which phosphorus atom corresponds to the phosphorus atom of a natural phosphate linkage as occurs in naturally occurring DNA and RNA. In some embodiments, a linkage phosphorus atom is in a modified internucleotidic linkage. In some embodiments, a linkage phosphorus atom is the P of P.sup.L of formula I. In some embodiments, a linkage phosphorus atom is chiral.

(88) P-modification: as used herein, the term P-modification refers to any modification at the linkage phosphorus other than a stereochemical modification. In some embodiments, a P-modification comprises addition, substitution, or removal of a pendant moiety covalently attached to a linkage phosphorus. In some embodiments, the P-modification is W, Y, Z, or X-L-R.sup.1 of formula I.

(89) Blockmer: the term blockmer, as used herein, refers to an oligonucleotide whose pattern of structural features characterizing each individual nucleotide unit is characterized by the presence of at least two consecutive nucleotide units sharing a common structural feature at the nucleobase, sugar and/or internucleotidic linkage. By common structural feature is meant common chemistry and/or stereochemistry, e.g., common modifications at nucleobases, sugars, and/or internucleotidic linkages and common stereochemistry at linkage phosphorus chiral centers. In some embodiments, the at least two consecutive nucleotide units sharing a common structural feature are referred to as a block.

(90) In some embodiments, a blockmer is a stereoblockmer, e.g., at least two consecutive nucleotide units have the same stereochemistry at the linkage phosphorus. Such at least two consecutive nucleotide units form a stereoblock. For instance, (Sp, Sp)-ATsCs1GA is a stereoblockmer because at least two consecutive nucleotide units, the Ts and the Cs1, have the same stereochemistry at the linkage phosphorus (both Sp). In the same oligonucleotide (Sp, Sp)-ATsCs1GA, TsCs1 forms a block, and it is a stereoblock.

(91) In some embodiments, a blockmer is a P-modification blockmer, e.g., at least two consecutive nucleotide units have the same modification at the linkage phosphorus. Such at least two consecutive nucleotide units form a P-modification block. For instance, (Rp, Sp)-ATsCsGA is a P-modification blockmer because at least two consecutive nucleotide units, the Ts and the Cs, have the same P-modification (i.e., both are a phosphorothioate diester). In the same oligonucleotide of (Rp, Sp)-ATsCsGA, TsCs forms a block, and it is a P-modification block.

(92) In some embodiments, a blockmer is a linkage blockmer, e.g., at least two consecutive nucleotide units have identical stereochemistry and identical modifications at the linkage phosphorus. At least two consecutive nucleotide units form a linkage block. For instance, (Rp, Rp)-ATsCsGA is a linkage blockmer because at least two consecutive nucleotide units, the Ts and the Cs, have the same stereochemistry (both Rp) and P-modification (both phosphorothioate). In the same oligonucleotide of (Rp, Rp)-ATsCsGA, TsCs forms a block, and it is a linkage block.

(93) In some embodiments, a blockmer is a sugar modification blockmer, e.g., at least two consecutive nucleotide units have identical sugar modifications. In some embodiments, a sugar modification blockmer is a 2-F blockmer wherein at least two consecutive nucleotide units have 2-F modification at their sugars. In some embodiments, a sugar modification blockmer is a 2-OR blockmer wherein at lead two consecutive nucleotide units independently have 2-OR modification at their sugars, wherein each R is independent as described in the present disclosure. In some embodiments, a sugar modification blockmer is a 2-OMe blockmer wherein at least two consecutive nucleotide units have 2-OMe modification at their sugars. In some embodiments, a sugar modification blockmer is a 2-MOE blockmer wherein at lead two consecutive nucleotide units have 2-MOE modification at their sugars. In some embodiments, a sugar modification blockmer is a LNA blockmer wherein at least two consecutive nucleotide units have LNA sugars.

(94) In some embodiments, a blockmer comprises one or more blocks independently selected from a sugar modification block, a stereoblock, a P-modification block and a linkage block. In some embodiments, a blockmer is a stereoblockmer with respect to one block, and/or a P-modification blockmer with respect to another block, and/or a linkage blockmer with respect to yet another block.

(95) Altmer: the term altmer, as used herein, refers to an oligonucleotide whose pattern of structural features characterizing each individual nucleotide unit is characterized in that no two consecutive nucleotide units of the oligonucleotide strand share a particular structural feature at the nucleobase, sugar, and/or the internucleotidic phosphorus linkage. In some embodiments, an altmer is designed such that it comprises a repeating pattern. In some embodiments, an altmer is designed such that it does not comprise a repeating pattern.

(96) In some embodiments, an altmer is a stereoaltmer, e.g., no two consecutive nucleotide units have the same stereochemistry at the linkage phosphorus. For instance, (Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp Rp, Sp, Rp, Sp, Rp, Sp, Rp, Sp, Rp)-GsCsCsTsCsAsGsTsCsTsGsCsTsTsCsGsCsAsCsC(SEQ ID NO: 60).

(97) Gapmer: as used herein, the term gapmer refers to an oligonucleotide characterized in that one or more nucleotide units (gap) do not have the structural features (e.g., nucleobase modifications, sugar modifications, internucleotidic linkage modifications, linkage phosphours stereochemistry, etc.) contained by nucleotide units flanking such one or more nucleotide units at both ends. In some embodiments, a gapmer comprises a gap of one or more natural phosphate linkages, independently flanked at both ends by non-natural internucleotidic linkages. In some embodiments, a gapmer is a sugar modification gapmer, wherein the gapmer comprises a gap of one or more nucleotide units comprising no sugar modifications which the flanking nucleotide at both ends contain. In some embodiments, a gapmer comprises a gap, wherein each nucleotide unit in the gap region contains no 2-modification that is contained in nucleotide units flanking the gap at both ends. In some embodiments, a provided oligonucleotide comprising a gap, wherein each nucleotide unit in the gap region contains no 2-OR modification, while nucleotide units flanking the gap at each end independently comprise a 2-OR modification. In some embodiments, a provided oligonucleotide comprising a gap, wherein each nucleotide unit in the gap region contains no 2-F modification, while nucleotide units flanking the gap at each end independently comprise a 2-F modification.

(98) Skipmer: as used herein, the term skipmer refers to a type of gapmer in which every other internucleotidic phosphorus linkage of the oligonucleotide strand is a phosphate diester linkage (a natural phosphate linkage), for example such as those found in naturally occurring DNA or RNA, and every other internucleotidic phosphorus linkage of the oligonucleotide strand is a modified internucleotidic linkage (a non-natural internucleotidic linkage).

(99) For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

(100) Unless otherwise specified, salts, such as pharmaceutically acceptable acid or base addition salts, stereoisomeric forms, and tautomeric forms, of compounds (e.g., oligonucleotides, agents, etc.) are included. Unless otherwise specified, singular forms a, an, and the include the plural reference unless the context clearly indicates otherwise (and vice versa). Thus, for example, a reference to a compound may include a plurality of such compounds.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

(101) Synthetic oligonucleotides provide useful molecular tools in a wide variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research, and new nanomaterials applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endo- and exo-nucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings. These include synthetic oligonucleotides that contain chemical modification, e.g., base modifications, sugar modifications, backbone modifications, etc., which, among other things, render these molecules less susceptible to degradation and improve other properties of oligonucleotides. Chemical modifications may also lead to certain undesired effects, such as increased toxicities, etc. From a structural point of view, modifications to natural phosphate linkages can introduce chirality, and certain properties of oligonucleotides may be affected by the configurations of the phosphorus atoms that form the backbone of the oligonucleotides.

(102) In some embodiments, an oligonucleotide or oligonucleotide composition is: a DMD oligonucleotide or oligonucleotide composition; an oligonucleotide or oligonucleotide composition comprising a non-negatively charged internucleotidic linkage; or a DMD oligonucleotide comprising a non-negatively charged internucleotidic linkage.

(103) In some embodiments, the chirality of the backbone (e.g., the configurations of the phosphorus atoms) or inclusion of natural phosphate linkages or non-natural internucleotidic linkages in the backbone and/or modifications of a sugar and/or nucleobase, and/or the addition of chemical moieties can affect properties and activities of oligonucleotides, e.g., the ability of a DMD oligonucleotide (e.g., an oligonucleotide antisense to a Dystrophin (DMD) transcript sequence) to skip one or more exons, and/or other properties of a DMD oligonucleotide, including but not limited to, increased stability, improved pharmacokinetics, and/or decreased immunogenicity, etc. Suitable assays for assessing properties and/or activities of provided compounds, e.g., oligonucleotides, and compositions thereof are widely known in the art and can be utilized in accordance with the present disclosure. For example, to test immunogenicity, various DMD oligonucleotides were tested in mouse serum in vivo and demonstrated minimal activation of cytokines, and various DMD oligonucleotides were tested ex vivo in human PBMC (peripheral blood mononuclear cells) for cytokine activity (e.g., IL-12p40, IL-12p70, IL-1alpha, IL-1beta, IL-6, MCP-1, MIP-1alpha, MIP-1beta, and TNF-alpha).

(104) In some embodiments, technologies (e.g., oligonucleotides, compositions, and methods of use thereof) of the present disclosure can be utilized to target various nucleic acids (e.g., by hybridizing to a target sequence of a target nucleic acid, and/or providing level reduction, degradation, splicing modulation, transcription suppression, etc. of the target nucleic acid, etc.) In some embodiments, provided technologies are particularly useful for modulating splicing of transcripts, e.g., to increase levels of desired splicing products and/or to reduce levels of undesired splicing products. In some embodiments, provided technologies are particularly useful for reducing levels of transcripts, e.g., pre-mRNA, RNA, etc., and in many instances, reducing levels of products arising from or encoded by such transcripts such as mRNA, proteins, etc.

(105) In some embodiments, a transcript is pre-mRNA. In some embodiments, a splicing product is mature RNA. In some embodiments, a splicing product is mRNA. In some embodiments, splicing modulation or alteration comprises skipping one or more exons. In some embodiments, splicing of a transcript is improved in that exon skipping increases levels of mRNA and proteins that have improved beneficial activities compared with absence of exon skipping. In some embodiments, an exon causing frameshift is skipped. In some embodiments, an exon comprising an undesired mutation is skipped. In some embodiments, an exon comprising a premature termination codon is skipped. An undesired mutation can be a mutation causing changes in protein sequences; it can also be a silent mutation. In some embodiments, a transcript is a transcript of Dystrophin (DMD).

(106) In some embodiments, splicing of a transcript is improved in that exon skipping lowers levels of mRNA and proteins that have undesired activities compared with absence of exon skipping. In some embodiments, a target is knocked down through exon skipping which, by skipping one or more exons, causes premature stop codon and/or frameshift mutations. In some embodiments, provided oligonucleotides in provided compositions, e.g., oligonucleotides of a plurality, comprise base modifications, sugar modifications, and/or internucleotidic linkage modifications. In some embodiments, provided oligonucleotides comprise base modifications and sugar modifications. In some embodiments, provided oligonucleotides comprise base modifications and internucleotidic linkage modifications. In some embodiments, provided oligonucleotides comprise sugar modifications and internucleotidic modifications. In some embodiments, provided compositions comprise base modifications, sugar modifications, and internucleotidic linkage modifications. Example chemical modifications, such as base modifications, sugar modifications, internucleotidic linkage modifications, etc. are widely known in the art including but not limited to those described in this disclosure. In some embodiments, a modified base is substituted A, T, C, G or U. In some embodiments, a sugar modification is 2-modification. In some embodiments, a 2-modification is 2-F modification. In some embodiments, a 2-modification is 2-OR.sup.1, wherein R.sup.1 is not hydrogen. In some embodiments, a 2-modification is 2-OR.sup.1, wherein R.sup.1 is optionally substituted alkyl. In some embodiments, a 2-modification is 2-OMe. In some embodiments, a 2-modification is 2-MOE. In some embodiments, a modified sugar moiety is a bridged bicyclic or polycyclic ring. In some embodiments, a modified sugar moiety is a bridged bicyclic or polycyclic ring having 5-20 ring atoms wherein one or more ring atoms are optionally and independently heteroatoms. Example ring structures are widely known in the art, such as those found in BNA, LNA, etc. In some embodiments, provided oligonucleotides comprise both one or more modified internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, oligonucleotides comprising both modified internucleotidic linkage and natural phosphate linkage and compositions thereof provide improved properties, e.g., activities and toxicities, etc. In some embodiments, a modified internucleotidic linkage is a chiral internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a modified internucleotidic linkage is a substituted phosphorothioate linkage.

(107) In some embodiments, provided oligonucleotides comprise one or more non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is a positively charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a modified internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage) comprises optionally substituted triazolyl. In some embodiments, a modified internucleotidic linkage (e.g., a non-negatively charged internucleotidic linkage) comprises optionally substituted alkynyl. In some embodiments, a modified internucleotidic linkage comprises a triazole or alkyne moiety. In some embodiments, a triazole moiety, e.g., a triazolyl group, is optionally substituted. In some embodiments, a triazole moiety, e.g., a triazolyl group) is substituted. In some embodiments, a triazole moiety is unsubstituted. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted guanidine moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted cyclic guanidine moiety and has the structure of:

(108) ##STR00012##
wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, a non-negatively charged internucleotidic linkage is stereochemically controlled.

(109) In some embodiments, an internucleotidic linkage comprising an optionally substituted guanidine moiety is an internucleotidic linkage of formula I-n-2, I-n-3, I-n-4, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein. In some embodiments, an internucleotidic linkage comprising an optionally substituted cyclic guanidine moiety is an internucleotidic linkage of formula II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2.

(110) Among other things, the present disclosure encompasses the recognition that stereorandom oligonucleotide preparations contain a plurality of distinct chemical entities that differ from one another, e.g., in the stereochemical structure of individual backbone linkage phosphorus chiral centers within the oligonucleotide chain. Without control of stereochemistry of backbone chiral centers, stereorandom oligonucleotide preparations provide uncontrolled compositions comprising undetermined levels of oligonucleotide stereoisomers with respect to the uncontrolled chiral centers, e.g., chiral linkage phosphorus. Even though these stereoisomers may have the same base sequence, they are different chemical entities at least due to their different backbone stereochemistry, and they can have, as demonstrated herein, different properties, e.g., activities, toxicities, etc. Among other things, the present disclosure provides new oligonucleotide compositions wherein stereochemistry of one or more linkage phosphorus chiral centers are independently controlled (e.g., in chirally controlled internucleotidic linkages). In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions which are or contain particular stereoisomers of oligonucleotides of interest.

(111) In some embodiments, provided oligonucleotides contain increased levels of one or more isotopes. In some embodiments, provided oligonucleotides are labeled, e.g., by one or more isotopes of one or more elements, e.g., hydrogen, carbon, nitrogen, etc. In some embodiments, provided oligonucleotides in provided compositions, e.g., oligonucleotides of a plurality, comprise base modifications, sugar modifications, and/or internucleotidic linkage modifications, wherein the oligonucleotides contain an enriched level of deuterium. In some embodiments, provided oligonucleotides are labeled with deuterium (replacing .sup.1H with .sup.2H) at one or more positions. In some embodiments, one or more .sup.1H of an oligonucleotide or any moiety conjugated to the oligonucleotide (e.g., a targeting moiety, lipid, etc.) is substituted with .sup.2H. Such oligonucleotides can be used in any composition or method described herein.

(112) In some embodiments, in an oligonucleotide, a pattern of backbone chiral centers can provide improved activity(s) or characteristic(s), including but not limited to: improved skipping of one or more exons, increased stability, increased activity, increased stability and activity, low toxicity, low immune response, improved protein binding profile, increased binding to certain proteins, and/or enhanced delivery.

(113) In some embodiments, a pattern of backbone chiral centers is or comprises S, SS, SSS, SSSS, SSSSS, SSSSSS, SSSSSSS, SOS, SSOSS, SSSOSSS, SSSSOSSSS, SSSSSOSSSSS, SSSSSSOSSSSSS, SSSSSSSOSSSSSSS, SSSSSSSSOSSSSSSSS, SSSSSSSSSOSSSSSSSSS, SOSOSOSOS, SSOSOSOSOSS, SSSOSOSOSOSSS, SSSSOSOSOSOSSSS, SSSSSOSOSOSOSSSSS, SSSSSSOSOSOSOSSSSSS, SOSOSSOOS, SSOSOSSOOSS, SSSOSOSSOOSSS, SSSSSSOSOSSOOSSSSSS, SOSOOSOOS, SSSSOSOSSOOSSSS, SSSSSOSOSSOOSSSSS, ssoSOOSOOSS, SSSOSOOSOOSSS, SSSSOSOOSOOSSSS, SSSSSOSOOSOOSSSSS, SSSSSSOSOOSOOSSSSSS, SOSOSSOOS, SSOSOSSOOSO, SSSOSOSSOOSOS, SSSSOSOSSOOSOSS, SSSSSOSOSSOOSOSSS, SSSSSSOSOSSOOSOSSSS, soSOOSOOSO, SSOSOOSOOSOS, SSSOSOOSOOSOS, SSSSOSOOSOOSOSS, SSSSSOSOOSOOSOSSS, SSSSSSOSOOSOOSOSSSS, SSOSOSSOO, SSSOSOSSOOS, SSSSOSOSSOOS, SSSSSOSOSSOOSS, OOSSSSSSOSOSSOOSSSSSS, RS, SR, SRS, SRSS, SSRS, RR, RRR, RRRR, RRRRR, SRR, RRS, SRRS, SSRRS, SRRSS, SRRR, RRRS, SRRRS, SSRRRS, SSRRRS, RSRRR, SRRRSR, SSSRSSS, SSSSRSSSS, SSSSSRSSSSS, SSSSSSRSSSSSS, SSSSSSSRSSSSSSS, SSSSSSSSRSSSSSSSS, SSSSSSSSSRSSSSSSSSS, SRSRSRSRS, SSRSRSRSRSS, SSSRSRSRSRSSS, SSSSRSRSRSRSSSS, SSSSSRSRSRSRSSSSS, SSSSSSRSRSRSRSSSSSS, SRSRSSRRS, SSRSRSSRRSS, SSSRSRSSRRSSS, SSSSRSRSSRRSSSS, SSSSSRSRSSRRSSSSS, SSSSSSRSRSSRRSSSSSS, SRSRRSRRS, SSRSRRSRRSS, SSSRSRRSRRSSS, SSSSRSRRSRRSSSS, SSSSSRSRRSRRSSSSS, SSSSSSRSRRSRRSSSSSS, SRSRSSRRS, SSRSRSSRRSR, SSSRSRSSRRSRS, SSSSRSRSSRRSRSS, SSSSSRSRSSRRSRSSS, SSSSSSRSRSSRRSRSSSS, SRSRRSRRSR, SSRSRRSRRSRS, SSSRSRRSRRSRS, SSSSRSRRSRRSRSS, SSSSSRSRRSRRSRSSS, SSSSSSRSRRSRRSRSSSS, SSRSRSSRR, SSSRSRSSRRS, SSSSRSRSSRRS, SSSSSRSRSSRRSS, SSSSSSRSRSSRRSSS, RSSSSSSRSRSSRRSSS, RRSSSSSSRSRSSRRS, RRSSSSSSRSRSSRRSS, RRSSSSSSRSRSSRRSSS, RRSSSSSSRSRSSRRSSSS, RRSSSSSSRSRSSRRSSSSS, (R).sub.n(S).sub.m, (S).sub.t(R).sub.n, (O).sub.t(R).sub.n(S).sub.m, (S).sub.t(O).sub.m, (O).sub.m(S).sub.t, (S).sub.t(R).sub.n(S).sub.m, (S).sub.t(O).sub.m(S).sub.n, (S).sub.t(O).sub.m, wherein t, m and n are independently 1 to 20, O is a non-chiral internucleotidic linkage, R is a Rp chiral internucleotidic linkage, and S is an Sp chiral internucleotidic linkage. In some embodiments, the non-chiral center is a phosphodiester linkage. In some embodiments, the chiral center in a Sp configuration is a phosphorothioate linkage.

(114) In some embodiments, the 5-end region of provided oligonucleotides, e.g., a 5-wing, comprises a stereochemistry pattern of S, SS, SSS, SSSS, SSSSS, SSSSSS, or SSSSSS. In some embodiments, each S is or represents an Sp phosphorothioate internucleotidic linkage. In some embodiments, the 5-end region of provided oligonucleotides, e.g., a 5-wing, comprises a stereochemistry pattern of S, SS, SSS, SSSS, SSSSS, SSSSSS, or SSSSSS, wherein the first S represents the first (the 5-end) internucleotidic linkage of a provided oligonucleotide. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the 5-end region independently comprise F. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 5-end region independently comprises F. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the 5-end region independently comprise a sugar modification. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 5-end region independently comprises a sugar modification. In some embodiments, each 2-modification is the same. In some embodiments, a sugar modification is a 2-modification. In some embodiments, a 2-modification is 2-OR.sup.1. In some embodiments, a 2-modification is 2-F. In some embodiments, the 3-end region of provided oligonucleotides, e.g., a 3-wing, comprises a stereochemistry pattern of S, SS, SSS, SSSS, SSSSS, SSSSSS, or SSSSSS. In some embodiments, each S is or represents an Sp phosphorothioate internucleotidic linkage. In some embodiments, the 3-end region of provided oligonucleotides, e.g., a 3-wing, comprises a stereochemistry pattern of S, SS, SSS, SSSS, SSSSS, SSSSSS, or SSSSSS, wherein the last S represents the last (the 3-end) internucleotidic linkage of a provided oligonucleotide. In some embodiments, each S represents an Sp phosphorothioate internucleotidic linkage. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the 3-end region independently comprise F. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 3-end region independently comprises F. In some embodiments, one or more nucleotidic units comprising an Sp internucleotidic linkage in the 3-end region independently comprise a sugar modification. In some embodiments, each nucleotidic unit comprising an Sp internucleotidic linkage in the 3-end region independently comprises a sugar modification. In some embodiments, each 2-modification is the same. In some embodiments, a sugar modification is a 2-modification. In some embodiments, a 2-modification is 2-OR.sup.1. In some embodiments, a 2-modification is 2-F. In some embodiments, provided oligonucleotides comprise both a 5-end region, e.g., a 5-wing, and a 3-end region, e.g., a 3-end wing, as described herein. In some embodiments, the 5-end region comprises a stereochemistry pattern of SS, wherein the first S represents the first internucleotidic linkage of a provided oligonucleotide, the 3-end region comprises a stereochemistry pattern of SS, wherein one or more nucleotidic unit comprising an Sp internucleotidic linkage in the 5- or 3-end region comprise F. In some embodiments, the 5-end region comprises a stereochemistry pattern of SS, wherein the first S represents the first internucleotidic linkage of a provided oligonucleotide, the 3-end region comprises a stereochemistry pattern of SS, wherein one or more nucleotidic unit comprising an Sp internucleotidic linkage in the 5- or 3-end region comprise a 2-F sugar modification. In some embodiments, provided oligonucleotides further comprise a middle region between the 5-end and 3-end regions, e.g., a core region, which comprises one or more natural phosphate linkages. In some embodiments, provided oligonucleotides further comprise a middle region between the 5-end and 3-end regions, e.g., a core region, which comprises one or more natural phosphate linkages and one or more internucleotidic linkages. In some embodiments, a middle region comprises one or more sugar moieties, wherein each sugar moiety independently comprises a 2-OR.sup.1 modification. In some embodiments, a middle region comprises one or more sugar moieties comprising no 2-F modification. In some embodiments, a middle region comprises one or more Sp internucleotidic linkages. In some embodiments, a middle region comprises one or more Sp internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, a middle region comprises one or more Rp internucleotidic linkages. In some embodiments, a middle region comprises one or more Rp internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, a middle region comprises one or more Rp internucleotidic linkages and one or more Sp internucleotidic linkages.

(115) In some embodiments, provided oligonucleotides comprise one or more modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more chiral modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more chirally controlled chiral modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more natural phosphate linkages. In some embodiments, provided oligonucleotides comprise one or more modified internucleotidic linkages and one or more natural phosphate linkages. In some embodiments, a modified internucleotidic linkage is a phosphorothioate linkage. In some embodiments, each modified internucleotidic linkage is a phosphorothioate linkage. In some embodiments, a modified internucleotidic linkage comprises a triazole, substituted triazole, alkyne or Tmg.

(116) In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a triazole or alkyne moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising an optionally substituted triazolyl or alkynyl. In some embodiments, such a nucleic acid is a siRNA, double-straned siRNA, single-stranded siRNA, oligonucleotide, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNA, antimir, supermir, ribozyme, Ul adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a modified internucleotidic linkage comprising a triazole or alkyne moiety. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which comprises a modified internucleotidic linkage comprising a triazole or alkyne moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a triazole moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising optionally substituted triazolyl. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a substituted triazole moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising an alkyne moiety. In some embodiments, the present disclosure pertains to a nucleic acid or oligonucleotide which comprises, at a 5 end, a structure of the formula:

(117) ##STR00013##
wherein W is O or S. In some embodiments, an oligonucleotide is a single-stranded siRNA which comprises, at a 5 end, a structure of the formula:

(118) ##STR00014##
wherein W is O or S. In some embodiments, a modified internucleotidic linkage is any modified internucleotidic linkage described in Krishna et al. 2012 J. Am. Chem. Soc. 134:11618-11631.

(119) In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which comprises a guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which comprises a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which comprises a cyclic guanidine moiety and has the structure of:

(120) ##STR00015##
wherein W is O or S. In some embodiments, a neutral internucleotidic linkage or internucleotidic linkage comprising a cyclic guanidine is chirally controlled. In some embodiments, a nucleic acid comprising a non-negatively charged internucleotidic linkage or a modified internucleotidic linkage comprising a cyclic guanidine moiety is a siRNA, double-straned siRNA, single-stranded siRNA, oligonucleotide, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNA, antimir, supermir, ribozyme, Ul adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a modified internucleotidic linkage which comprises a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a modified internucleotidic linkage which has the structure of:

(121) ##STR00016##
wherein W is O or S. In some embodiments, a neutral internucleotidic linkage or internucleotidic linkage comprising a cyclic guanidine moiety is chirally controlled. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which comprises a modified internucleotidic linkage comprising a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which comprises a modified internucleotidic linkage which has the structure of:

(122) ##STR00017##
wherein W is O or S. In some embodiments, a neutral internucleotidic linkage or internucleotidic linkage comprising a cyclic guanidine moiety is chirally controlled. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage comprising a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid which comprises a modified internucleotidic linkage which has the structure of:

(123) ##STR00018##
wherein W is O or S. In some embodiments, the present disclosure pertains to a nucleic acid or oligonucleotide which comprises, at a 5 end, a structure comprising a cyclic guanidine moiety. In some embodiments, the present disclosure pertains to a nucleic acid or oligonucleotide which comprises, at a 5 end, a structure of the formula:

(124) ##STR00019##
wherein W is O or S. In some embodiments, the oligonucleotide is a single-stranded siRNA which comprises, at a 5 end, a structure comprising a cyclic guanidine moiety. In some embodiments, the oligonucleotide is a single-stranded siRNA which comprises, at a 5 end, a structure of the formula:

(125) ##STR00020##
wherein W is O or S. In some embodiments, the internucleotidic linkage comprises

(126) ##STR00021##
(wherein W is O or S) and is chirally controlled.

(127) In some embodiments, provided oligonucleotides can bind to a transcript, and change the splicing pattern of the transcript. In some embodiments, provided oligonucleotides provides exon-skipping of an exon, with efficiency greater than a comparable oligonucleotide under one or more suitable conditions, e.g., as described herein. In some embodiments, a provided skipping efficiency is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% more than, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more fold of, that of a comparable oligonucleotide under one or more suitable conditions, e.g., as described herein. In some embodiments, a comparable oligonucleotide is an oligonucleotide which has fewer or no chirally controlled internucleotidic linkages and/or fewer or no non-negatively charged internucleotidic linkages but is otherwise identical.

(128) In some embodiments, the present disclosure demonstrates that 2-F modifications, among other things, can improve exon-skipping efficiency. In some embodiments, the present disclosure demonstrates that Sp internucleotidic linkages, among other things, at the 5- and 3-ends can improve oligonucleotide stability. In some embodiments, the present disclosure demonstrates that, among other things, natural phosphate linkages and/or Rp internucleotidic linkages can improve removal of oligonucleotides from a system. As appreciated by a person having ordinary skill in the art, various assays known in the art can be utilized to assess such properties in accordance with the present disclosure.

(129) In some embodiments, provided oligonucleotides comprise one or more modified sugar moieties. In some embodiments, a modified sugar moiety comprises a 2-modification. In some embodiments, a modified sugar moiety comprises a 2-modification. In some embodiments, a 2-modification is 2-OR.sup.1. In some embodiments, a 2-modification is a 2-OMe. In some embodiments, a 2-modification is a 2-MOE. In some embodiments, a 2-modification is an LNA sugar modification. In some embodiments, a 2-modification is 2-F. In some embodiments, each sugar modification is independently a 2-modification. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein at least one is 2-F. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl, and wherein at least one is 2-OR.sup.1. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein at least one is 2-F, and at least one is 2-OR.sup.1. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl, and wherein at least one is 2-F, and at least one is 2-OR.sup.1.

(130) In some embodiments, 5% or more of the sugar moieties of provided oligonucleotides are modified. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the sugar moieties of provided oligonucleotides are modified. In some embodiments, each sugar moiety of provided oligonucleotides is modified. In some embodiments, a modified sugar moiety comprises a 2-modification. In some embodiments, a modified sugar moiety comprises a 2-modification. In some embodiments, a 2-modification is 2-OR.sup.1. In some embodiments, a 2-modification is a 2-OMe. In some embodiments, a 2-modification is a 2-MOE. In some embodiments, a 2-modification is an LNA sugar modification. In some embodiments, a 2-modification is 2-F. In some embodiments, each sugar modification is independently a 2-modification. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein at least one is 2-F. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl, and wherein at least one is 2-OR.sup.1. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein at least one is 2-F, and at least one is 2-OR.sup.1. In some embodiments, each sugar modification is independently 2-OR.sup.1 or 2-F, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl, and wherein at least one is 2-F, and at least one is 2-OR.sup.1.

(131) In some embodiments, provided oligonucleotides comprise one or more 2-F. In some embodiments, provided oligonucleotides comprise two or more 2-F.

(132) In some embodiments, provided oligonucleotides comprise alternating 2-F modified sugar moieties and 2-OR.sup.1 modified sugar moieties. In some embodiments, provided oligonucleotides comprise alternating 2-F modified sugar moieties and 2-OMe modified sugar moieties, e.g., [(2-F)(2-OMe)]x, [(2-OMe)(2-F)]x, etc., wherein x is 1-50. In some embodiments, provided oligonucleotides comprise at least two pairs of alternating 2-F and 2-OMe modifications. In some embodiments, provided oligonucleotides comprises alternating phosphodiester and phosphorothioate internucleotidic linkages, e.g., [(PO)(PS)]x, [(PS)(PO)]x, etc., wherein x is 1-50. In some embodiments, provided oligonucleotides comprise at least two pairs of alternating phosphodiester and phosphorothioate internucleotidic linkages.

(133) In some embodiments, provided oligonucleotides comprise one or more natural phosphate linkages and one or more modified internucleotidic linkages. In some embodiments, provided oligonucleotides comprise one or more natural phosphate linkages and one or more modified internucleotidic linkages and one or more non-negatively charged internucleotidic linkages.

(134) In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides, wherein: oligonucleotides of the plurality have the same base sequence; and oligonucleotides of the plurality comprise one or more modified sugar moieties, or comprise one or more natural phosphate linkages and one or more modified internucleotidic linkages.

(135) In some embodiments, oligonucleotides of a plurality comprise one or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise one or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 2 or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise 3 or more modified sugar moieties.

(136) In some embodiments, provided compositions alter transcript splicing so that an undesired target and/or biological function are suppressed.

(137) In some embodiments, provided compositions alter transcript splicing so a desired target and/or biological function is enhanced.

(138) In some embodiments, each oligonucleotide of a plurality comprises one or more modified sugar moieties and modified internucleotidic linkages.

(139) In some embodiments, each oligonucleotide of a plurality comprises no more than about 25 consecutive unmodified sugar moieties

(140) In some embodiments, each oligonucleotide of a plurality comprises no more than about 95% unmodified sugar moieties. In some embodiments, each oligonucleotide of a plurality comprises no more than about 90% unmodified sugar moieties. In some embodiments, each oligonucleotide of a plurality comprises no more than about 85% unmodified sugar moieties. In some embodiments, each oligonucleotide of a plurality comprises no more than about 15 consecutive unmodified sugar moieties.

(141) In some embodiments, each oligonucleotide of a plurality comprises no more than about 95% unmodified sugar moieties.

(142) In some embodiments, each oligonucleotide of a plurality comprises two or more modified internucleotidic linkages.

(143) In some embodiments, about 5% of the internucleotidic linkages in each oligonucleotide of a plurality are modified internucleotidic linkages.

(144) In some embodiments, each oligonucleotide of a plurality comprises no more than about 25 consecutive natural phosphate linkages. In some embodiments, each oligonucleotide of a plurality comprises no more than about 20 natural phosphate linkages.

(145) In some embodiments, oligonucleotides of a plurality comprise no natural DNA nucleotide units. In some embodiments, oligonucleotides of a plurality comprise no more than 30 natural DNA nucleotides. In some embodiments, oligonucleotides of a plurality comprise no more than 30 consecutive DNA nucleotides.

(146) In some embodiments, compared to a reference condition, provided chirally controlled oligonucleotide compositions are surprisingly effective. In some embodiments, desired biological effects (e.g., as measured by increased levels of desired mRNA, proteins, etc., decreased levels of undesired mRNA, proteins, etc.) can be enhanced by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100 fold. In some embodiments, a change is measured by increase of a desired mRNA level compared to a reference condition. In some embodiments, a change is measured by decrease of an undesired mRNA level compared to a reference condition. In some embodiments, a reference condition is absence of oligonucleotide treatment. In some embodiments, a reference condition is a stereorandom composition of oligonucleotides having the same base sequence and chemical modifications.

(147) In some embodiments, a desired biological effect is: improved skipping of one or more exons, increased stability, increased activity, increased stability and activity, low toxicity, low immune response, improved protein binding profile, increased binding to certain proteins, and/or enhanced delivery. In some embodiments, a desired biological effect is enhanced by more than 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, or 500 fold.

(148) In some embodiments, the structure of a DMD oligonucleotide is or comprises a wing-core-wing, wing-core, or core-wing structure. In some embodiments, a 5-wing is a 5-end region. In some embodiments, a 3-wing is a 3-end region. In some embodiments, a core is a middle region. In some embodiments, a 5-end region is a 5-wing region. In some embodiments, a 3-end region is a 3-wing region. In some embodiments, a middle region is a core region.

(149) In some embodiments, an oligonucleotide having a wing-core-wing structure is designated a gapmer. In some embodiments, a gapmer is asymmetric, in that the chemistry of one wing is different from the chemistry of the other wing. In some embodiments, a gapmer is asymmetric, in that the chemistry of one wing is different from the chemistry of the other wing, wherein the wings differ in sugar modifications and/or internucleotidic linkages, or patterns thereof. In some embodiments, a gapmer is asymmetric, in that the chemistry of one wing is different from the chemistry of the other wing, wherein the wings differ in sugar modifications, wherein one wing comprises a sugar modification not present in the other wing; or both wings each comprise a sugar modification not found in the other wing; or both wings comprise different patterns of the same types of sugar modifications; or one wing comprises only one type of sugar modification, while the other wing comprises two types of sugar modifications; etc.

(150) In some embodiments, an internucleotidic linkage between a wing region and a core region is considered part of the wing region. In some embodiments, an internucleotidic linkage between a 5-wing region and a core region is considered part of the wing region. In some embodiments, an internucleotidic linkage between a 3-wing region and a core region is considered part of the wing region. In some embodiments, an internucleotidic linkage between a wing region and a core region is considered part of the core region. In some embodiments, an internucleotidic linkage between a 5-wing region and a core region is considered part of the core region. In some embodiments, an internucleotidic linkage between a 3-wing region and a core region is considered part of the core region.

(151) In some embodiments, a region (e.g., a wing region, a core region, a 5-end region, a middle region, a 3-end region, etc.) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleoside units.

(152) In some embodiments, provided oligonucleotides comprise two wing and one core regions. In some embodiments, provided oligonucleotides comprises a 5-wing-core-wing-3 structure. In some embodiments, provided oligonucleotides are of a 5-wing-core-wing-3 gapmer structure. In some embodiments, the two wing regions are identical. In some embodiments, the two wing regions are different. In some embodiments, the two wing regions are identical in chemical modifications. In some embodiments, the two wing regions are identical in 2-modifications. In some embodiments, the two wing regions are identical in internucleotidic linkage modifications. In some embodiments, the two wing regions are identical in patterns of backbone chiral centers. In some embodiments, the two wing regions are identical in pattern of backbone linkages. In some embodiments, the two wing regions are identical in pattern of backbone linkage types. In some embodiments, the two wing regions are identical in pattern of backbone phosphorus modifications.

(153) A wing region can be differentiated from a core region in that a wing region contains a different structure feature than a core region. For example, in some embodiments, a wing region differs from a core region in that they have different sugar modifications, base modifications, internucleotidic linkages, internucleotidic linkage stereochemistry, etc. In some embodiments, a wing region differs from a core region in that they have different 2-modifications of the sugars.

(154) In some embodiments, a region (e.g., a wing region, a core region, a 5-end region, a middle region, a 3-end region, etc.) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more modified internucleotidic linkages. In some embodiments, a region comprises 2 or more modified internucleotidic linkages. In some embodiments, a region comprises 3 or more modified internucleotidic linkages. In some embodiments, a region comprises 4 or more modified internucleotidic linkages. In some embodiments, a region comprises 5 or more modified internucleotidic linkages. In some embodiments, a region comprises 6 or more modified internucleotidic linkages. In some embodiments, a region comprises 7 or more modified internucleotidic linkages. In some embodiments, a region comprises 8 or more modified internucleotidic linkages. In some embodiments, a region comprises 9 or more modified internucleotidic linkages. In some embodiments, a region comprises 10 or more modified internucleotidic linkages.

(155) In some embodiments, provided oligonucleotides comprise consecutive nucleoside units each of which comprises no 2-OR.sup.1 modifications (wherein R.sup.1 is not hydrogen). In some embodiments, provided oligonucleotides comprise consecutive nucleoside units whose 2-positions are independently unsubstituted or substituted with 2-F. In some embodiments, such an oligonucleotide is a DMD oligonucleotide. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a modified internucleotidic linkage. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a phosphorothioate linkage. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a chirally controlled modified internucleotidic linkage. In some embodiments, each of the consecutive nucleoside units is independently preceded and/or followed by a chirally controlled phosphorothioate linkage.

(156) In some embodiments, a modified internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof. In some embodiments, a modified internucleotidic linkage has a structure of formula I or a salt form thereof. In some embodiments, a modified internucleotidic linkage has a structure of formula I-a or a salt form thereof.

(157) In some embodiments, a modified internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a positively-charged internucleotidic linkage. In some embodiments, a modified internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, such a heterocyclyl or heteroaryl group is of a 5-membered ring. In some embodiments, such a heterocyclyl or heteroaryl group is of a 6-membered ring.

(158) In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a heteroaryl group is directly bonded to a linkage phosphorus. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an unsubstituted triazolyl group, e.g.,

(159) ##STR00022##
In some embodiments, a non-negatively charged internucleotidic linkage comprises a substituted triazolyl group, e.g.,

(160) ##STR00023##

(161) In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-20 membered heterocyclyl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-6 membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted 5-membered heterocyclyl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, a heterocyclyl group is directly bonded to a linkage phosphorus. In some embodiments, a heterocyclyl group is bonded to a linkage phosphorus through a linker, e.g., N when the heterocyclyl group is part of a guanidine moiety who directed bonded to a linkage phosphorus through its N. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted

(162) ##STR00024##
group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted

(163) ##STR00025##
group. In some embodiments, a non-negatively charged internucleotidic linkage comprises an substituted

(164) ##STR00026##
group. In some embodiments, a non-negatively charged internucleotidic linkage comprises a

(165) ##STR00027##
group. In some embodiments, each R.sup.1 is independently optionally substituted C.sub.1-20 alkyl. In some embodiments, each R.sup.1 is independently optionally substituted C.sub.1-6 alkyl. In some embodiments, each R.sup.1 is independently methyl. In some embodiments, the two R.sup.1 groups are different; for example, in some embodiments, one R.sup.1 is methyl, and the other is CH.sub.2(CH.sub.2).sub.10CH.sub.3.

(166) In some embodiments, a modified internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, comprises a triazole or alkyne moiety, each of which is optionally substituted. In some embodiments, a modified internucleotidic linkage comprises a triazole moiety. In some embodiments, a modified internucleotidic linkage comprises a unsubstituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises a substituted triazole moiety. In some embodiments, a modified internucleotidic linkage comprises an alkyl moiety. In some embodiments, a modified internucleotidic linkage comprises an optionally substituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises an unsubstituted alkynyl group. In some embodiments, a modified internucleotidic linkage comprises a substituted alkynyl group. In some embodiments, an alkynyl group is directly bonded to a linkage phosphorus.

(167) In some embodiments, an oligonucleotide comprising a non-negatively charged internucleotidic linkage can comprise any structure, format, or portion thereof described herein. In some embodiments, an oligonucleotide comprising a non-negatively charged internucleotidic linkage can comprise any structure, format, or portion thereof described herein as being a component of a DMD oligonucleotide. In some embodiments, any structure, format, or portion thereof described as being a component of any DMD oligonucleotide can be used in any oligonucleotide comprising a non-negatively charged internucleotidic linkage, whether or not that oligonucleotide targets DMD or not, or whether the oligonucleotide is capable of mediating skipping of a DMD exon or not. In some embodiments, an oligonucleotide comprising a non-negatively charged internucleotidic is double-stranded or single-stranded.

(168) In some embodiments, a provided oligonucleotide composition is characterized in that, when it is contacted with the transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under reference conditions selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof. In some embodiments, a desired splicing product is increased 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 fold or more. In some embodiments, a desired splicing reference is absent (e.g., cannot be reliably detected by quantitative PCR) under reference conditions. In some embodiments, as exemplified in the present disclosure, levels of the plurality of oligonucleotides, e.g., a plurality of oligonucleotides, in provided compositions are pre-determined.

(169) In some embodiments, provided oligonucleotides, e.g., oligonucleotides of a plurality in a provided composition, comprise two or more regions. In some embodiments, provided comprise a 5-end region, a 3-end region, and a middle region in between. In some embodiments, provided oligonucleotides have two wing and one core regions. In some embodiments, provided oligonucleotides are of a wing-core-wing structure. In some embodiments, the two wing regions are identical. In some embodiments, the two wing regions are different. In some embodiments, a 5-end region is a 5-wing region. In some embodiments, a 5-wing region is a 5-end region. In some embodiments, a 3-end region is a 3-wing region. In some embodiments, a 3-wing region is a 3-end region. In some embodiments, a core region is a middle region.

(170) In some embodiments, a region (e.g., a 5-wing region, a 3-wing, a core region, a 5-end region, a middle region, etc.) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleoside units. In some embodiments, a region comprises 2 or more nucleoside units. In some embodiments, a region comprises 3 or more nucleoside units. In some embodiments, a region comprises 4 or more nucleoside units. In some embodiments, a region comprises 5 or more nucleoside units. In some embodiments, a region comprises 6 or more nucleoside units. In some embodiments, a region comprises 7 or more nucleoside units. In some embodiments, a region comprises 8 or more nucleoside units. In some embodiments, a region comprises 9 or more nucleoside units. In some embodiments, a region comprises 10 or more nucleoside units.

(171) In some embodiments, a region (e.g., a 5-wing region, a 3-wing, a core region, a 5-end region, a middle region, etc.) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more modified internucleotidic linkages. In some embodiments, a region comprises 2 or more modified internucleotidic linkages. In some embodiments, the one or more modified internucleotidic linkages are consecutive. In some embodiments, a region comprises 2 or more consecutive modified internucleotidic linkages. In some embodiments, each internucleotidic linkage in a region is independently a modified internucleotidic linkage, wherein each chiral internucleotidic linkage is optionally and independently chirally controlled. In some embodiments, a chiral internucleotidic linkage or a modified internucleotidic linkage has the structure of formula I or a salt form thereof. In some embodiments, a chiral internucleotidic linkage or a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, each chiral internucleotidic linkage or a modified internucleotidic linkage independently has the structure of formula I or a salt form thereof. In some embodiments, each chiral internucleotidic linkage or a modified internucleotidic linkage is a phosphorothioate internucleotidic linkage. In some embodiments, a region comprises 3 or consecutive modified internucleotidic linkages.

(172) In some embodiments, a wing region comprises one or more natural phosphate linkages. In some embodiments, a core region comprises one or more natural phosphate linkages. In some embodiments, a 5-end region comprises one or more natural phosphate linkages. In some embodiments, a 3-end region comprises one or more natural phosphate linkages. In some embodiments, a middle region comprises one or more natural phosphate linkages. In some embodiments, the one or more natural phosphate linkages are consecutive.

(173) In some embodiments, a natural phosphate linkage follows (e.g., connected to a 3-position of a sugar moiety) or precedes (e.g., connected to a 5-position of a sugar moiety) a nucleoside unit whose sugar moiety comprises a 2-OR.sup.1 modification, wherein R.sup.1 is not hydrogen. In some embodiments, R.sup.1 is optionally substituted C.sub.1-6 aliphatic. In some embodiments, a modified internucleotidic linkage follows (e.g., connected to a 3-position of a sugar moiety) or precedes (e.g., connected to a 5-position of a sugar moiety) all or most (e.g., more than 55%, 60%, 70%, 80%, 90%, 95%, etc.) nucleoside units whose sugar moiety comprises no 2-OR.sup.1 modification, wherein R.sup.1 is not hydrogen (e.g., those having two 2-H at the 2-position, those having a 2-H and a 2-F at the 2-position (2-F modified), etc.).

(174) In some embodiments, a region comprises one or more nucleoside units comprising sugar modifications, e.g., 2-F, 2-OR.sup.1, LNA sugar modifications, etc. In some embodiments, each sugar in a region is independently modified. In some embodiments, each sugar moiety in a wing, a 5-end region, and/or a 3-end region is modified. In some embodiments, a modification is a 2-modification. In some embodiments, a modification can increase stability, e.g., 2-OR.sup.1 where in R.sup.1 is not H (e.g., is optionally substituted C.sub.1-6 aliphatic), LNA sugar modifications, etc. In some embodiments, a region, e.g., a core region or a middle region, comprise no sugar modifications (or no 2-OR.sup.1 sugar modifications/LNA modifications etc.). In some embodiments, such a core/middle region can form a duplex with a RNA for recognition/binding of a protein, e.g., RNase H, for the protein to perform one or more of its functions (e.g., in the case of RNase H, its binding and cleavage of DNA/RNA duplex).

(175) A region and/or a provided oligonucleotide may have various patterns of backbone chiral centers. In some embodiments, each internucleotidic linkage in a region is a chirally controlled internucleotidic linkage and is Sp. In some embodiments, the 5-end and/or the 3-end internucleotidic linkage is a chirally controlled internucleotidic linkage and is Sp. In some embodiments, the pattern of backbone chiral centers of a wing region, a 5-end region, and/or a 3-end region is or comprises a 5-end and/or a 3-end internucleotidic linkage which is a chirally controlled internucleotidic linkage and is Sp, with the other internucleotidic linkages in the region independently being an natural phosphate linkage, a modified internucleotidic linkage, or a chirally controlled internucleotidic linkage (Sp or Rp). In some embodiments, such patterns provide stability. Many example patterns of backbone chiral centers are described in the present disclosure.

(176) In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides defined by having: 1) a common base sequence; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that a controlled level of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

(177) In some embodiments, oligonucleotides having a common base sequence may have the same pattern of nucleoside modifications, e.g., sugar modifications, base modifications, etc. In some embodiments, a pattern of nucleoside modifications may be represented by a combination of locations and modifications. In some embodiments, all non-chiral linkages (e.g., PO) may be omitted. In some embodiments, oligonucleotides having the same base sequence have the same constitution.

(178) As understood by a person having ordinary skill in the art, a stereorandom or racemic preparation of oligonucleotides is prepared by non-stereoselective and/or low-stereoselective coupling of nucleotide monomers, typically without using any chiral auxiliaries, chiral modification reagents, and/or chiral catalysts. In some embodiments, in a substantially racemic (or chirally uncontrolled) preparation of oligonucleotides, all or most coupling steps are not chirally controlled in that the coupling steps are not specifically conducted to provide enhanced stereoselectivity. An example substantially racemic preparation of oligonucleotides is the preparation of phosphorothioate oligonucleotides through sulfurizing phosphite triesters from commonly used phosphoramidite oligonucleotide synthesis with either tetraethylthiuram disulfide or (TETD) or 3H-1,2-bensodithiol-3-one 1,1-dioxide (BDTD), a well-known process in the art. In some embodiments, substantially racemic preparation of oligonucleotides provides substantially racemic oligonucleotide compositions (or chirally uncontrolled oligonucleotide compositions). In some embodiments, at least one coupling of a nucleotide monomer has a diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, each internucleotidic linkage independently has a diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, a diastereoselectivity is lower than about 60:40. In some embodiments, a diastereoselectivity is lower than about 70:30. In some embodiments, a diastereoselectivity is lower than about 80:20. In some embodiments, a diastereoselectivity is lower than about 90:10. In some embodiments, a diastereoselectivity is lower than about 91:9. In some embodiments, at least one internucleotidic linkage has a diastereoselectivity lower than about 90:10. In some embodiments, at least two internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, at least three internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, at least four internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, at least five internucleotidic linkages have a diastereoselectivity lower than about 90:10. In some embodiments, each internucleotidic linkage independently has a diastereoselectivity lower than about 90:10. In some embodiments, a non-chirally controlled internucleotidic linkage has a diastereomeric purity no more than 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 55%. In some embodiments, the purity is no more than 90%. In some embodiments, the purity is no more than 85%. In some embodiments, the purity is no more than 80%.

(179) In contrast, in chirally controlled oligonucleotide composition, at least one and typically each chirally controlled internucleotidic linkage, such as those of oligonucleotides of chirally controlled oligonucleotide compositions, independently has a diastereomeric purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more with respect to the chiral linkage phosphorus. In some embodiments, a diastereomeric purity is 95% or more. In some embodiments, a diastereomeric purity is 96% or more. In some embodiments, a diastereomeric purity is 97% or more. In some embodiments, a diastereomeric purity is 98% or more. In some embodiments, a diastereomeric purity is 99% or more. Among other things, technologies of the present disclosure routinely provide chirally controlled internucleotidic linkages with high diastereomeric purity.

(180) As appreciated by a person having ordinary skill in the art, diastereoselectivity of a coupling or diastereomeric purity (diastereopurity) of an internucleotidic linkage can be assessed through the diastereoselectivity of a dimer formation/diastereomeric purity of the internucleotidic linkage of a dimer formed under the same or comparable conditions, wherein the dimer has the same 5- and 3-nucleosides and internucleotidic linkage.

(181) In some embodiments, the present disclosure provides chirally controlled (and/or stereochemically pure) oligonucleotide compositions comprising a plurality of oligonucleotides defined by having: 1) a common base sequence; 2) a common pattern of backbone linkages; and 3) a common pattern of backbone chiral centers, which composition is a substantially pure preparation of a single oligonucleotide in that at least about 10% of the oligonucleotides in the composition have the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

(182) In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of a plurality of oligonucleotides, wherein the composition is enriched, relative to a substantially racemic preparation of the same oligonucleotides, for oligonucleotides of a single oligonucleotide type. In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of a plurality of oligonucleotides wherein the composition is enriched, relative to a substantially racemic preparation of the same oligonucleotides, for oligonucleotides of a single oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications.

(183) In some embodiments, the present disclosure provides a chirally controlled oligonucleotide composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications.
wherein the composition is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type.

(184) In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have identical structures.

(185) In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides of an oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides of a particular type have the same constitution. In some embodiments, oligonucleotides of an oligonucleotide type are identical.

(186) In some embodiments, a chirally controlled oligonucleotide composition is a substantially pure preparation of an oligonucleotide type in that oligonucleotides in the composition that are not of the oligonucleotide type are impurities form the preparation process of said oligonucleotide type, in some case, after certain purification procedures.

(187) In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the oligonucleotides in the composition have a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

(188) In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are identical.

(189) In some embodiments, purity of a chirally controlled oligonucleotide composition of an oligonucleotide type is expressed as the percentage of oligonucleotides in the composition that are of the oligonucleotide type. In some embodiments, at least about 10% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 20% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 30% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 40% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 50% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 60% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 70% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 80% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 90% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 92% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 94% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 95% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 96% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the same oligonucleotide type. In some embodiments, at least about 97% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 98% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type. In some embodiments, at least about 99% of the oligonucleotides in a chirally controlled oligonucleotide composition are of the oligonucleotide type.

(190) In some embodiments, purity of a chirally controlled oligonucleotide composition can be controlled by stereoselectivity of each coupling step in its preparation process. In some embodiments, a coupling step has a stereoselectivity (e.g., diastereoselectivity) of 60% (60% of the new internucleotidic linkage formed from the coupling step has the intended stereochemistry). After such a coupling step, the new internucleotidic linkage formed may be referred to have a 60% purity. In some embodiments, each coupling step has a stereoselectivity of at least 60%. In some embodiments, each coupling step has a stereoselectivity of at least 70%. In some embodiments, each coupling step has a stereoselectivity of at least 80%. In some embodiments, each coupling step has a stereoselectivity of at least 85%. In some embodiments, each coupling step has a stereoselectivity of at least 90%. In some embodiments, each coupling step has a stereoselectivity of at least 91%. In some embodiments, each coupling step has a stereoselectivity of at least 92%. In some embodiments, each coupling step has a stereoselectivity of at least 93%. In some embodiments, each coupling step has a stereoselectivity of at least 94%. In some embodiments, each coupling step has a stereoselectivity of at least 95%. In some embodiments, each coupling step has a stereoselectivity of at least 96%. In some embodiments, each coupling step has a stereoselectivity of at least 97%. In some embodiments, each coupling step has a stereoselectivity of at least 98%. In some embodiments, each coupling step has a stereoselectivity of at least 99%. In some embodiments, each coupling step has a stereoselectivity of at least 99.5%. In some embodiments, each coupling step has a stereoselectivity of virtually 100%. In some embodiments, a coupling step has a stereoselectivity of virtually 100% in that all detectable product from the coupling step by an analytical method (e.g., NMR, HPLC, use of a nuclease which stereoselectively cleaves phosphorothioates, etc) has the intended stereoselectivity. In some embodiments, stereoselectivity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5-nucleoside of the dimer is the same as the nucleoside to the 5-end of the chiral internucleotidic linkage, and the 3-nucleoside of the dimer is the same as the nucleoside to the 3-end of the chiral internucleotidic linkage (e.g., for fU*SfU*SfC*SfU, through the dimer of fU*SfC). As appreciated by a person having ordinary skill in the art, percentage of oligonucleotides of a particular type having n chirally controlled internucleotidic linkages in a preparation may be calculated as DP.sup.1*DP .sup.2*DP.sup.3* . . . DP.sup.n, wherein each of DP.sup.1, DP.sup.2, DP.sup.3, . . . , and DP.sup.n is independently the diastereomeric purity of the 1.sup.st, 2.sup.nd, 3.sup.rd, . . . , and n.sup.th chirally controlled internucleotidic linkage. In some embodiments, each of DP.sup.1, DP.sup.2, DP.sup.3, . . . , and DP.sup.n is independently 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% or more. In some embodiments, each of DP.sup.1, DP.sup.2, DP.sup.3, . . . , and DP.sup.n is independently 95% or more.

(191) In some embodiments, in provided compositions, at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% of oligonucleotides that have the base sequence of a particular oligonucleotide type (defined by 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications) are oligonucleotides of the particular oligonucleotide type. In some embodiments, at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of a particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.

(192) In some embodiments, oligonucleotides of a particular type in a chirally controlled oligonucleotide composition is enriched at least 5 fold (oligonucleotides of the particular type have a fraction of 5* (.sup.n) of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type, wherein n is the number of chiral internucleotidic linkages; or oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type but are not of the particular oligonucleotide type are no more than [1-(.sup.n)]/5 of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type) compared to a stereorandom preparation of the oligonucleotides (oligonucleotides of the particular type are typically considered to have a fraction of .sup.n of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type, wherein n is the number of chiral internucleotidic linkages, and oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type but are not of the particular oligonucleotide type are typically considered to have a fraction of [1-(.sup.n)] of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type). In some embodiments, the enrichment is at least 20 fold. In some embodiments, the enrichment is at least 30 fold. In some embodiments, the enrichment is at least 40 fold. In some embodiments, the enrichment is at least 50 fold. In some embodiments, the enrichment is at least 60 fold. In some embodiments, the enrichment is at least 70 fold. In some embodiments, the enrichment is at least 80 fold. In some embodiments, the enrichment is at least 90 fold. In some embodiments, the enrichment is at least 100 fold. In some embodiments, the enrichment is at least 20,000 fold. In some embodiments, the enrichment is at least (1.5).sup.n. In some embodiments, the enrichment is at least (1.6).sup.n. In some embodiments, the enrichment is at least (1.7).sup.n. In some embodiments, the enrichment is at least (1.1).sup.n. In some embodiments, the enrichment is at least (1.8).sup.n. In some embodiments, the enrichment is at least (1.9).sup.n. In some embodiments, the enrichment is at least 2.sup.n. In some embodiments, the enrichment is at least 3.sup.n. In some embodiments, the enrichment is at least 4.sup.n. In some embodiments, the enrichment is at least 5.sup.n. In some embodiments, the enrichment is at least 6.sup.n. In some embodiments, the enrichment is at least 7.sup.n. In some embodiments, the enrichment is at least 8.sup.n. In some embodiments, the enrichment is at least 9.sup.n. In some embodiments, the enrichment is at least 10.sup.n. In some embodiments, the enrichment is at least 15.sup.n. In some embodiments, the enrichment is at least 20.sup.n. In some embodiments, the enrichment is at least 25.sup.n. In some embodiments, the enrichment is at least 30.sup.n. In some embodiments, the enrichment is at least 40.sup.n. In some embodiments, the enrichment is at least 50.sup.n. In some embodiments, the enrichment is at least 100.sup.n. In some embodiments, enrichment is measured by increase of the fraction of oligonucleotides of the particular oligonucleotide type in oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type. In some embodiments, an enrichment is measured by decrease of the fraction of oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type but are not of the particular oligonucleotide type in oligonucleotides that have the base sequence, the pattern of backbone linkages, and the pattern of backbone phosphorus modifications of the particular oligonucleotide type.

(193) In some embodiments, provided oligonucleotides are antisense oligonucleotides. In some embodiments, provided oligonucleotides are siRNA oligonucleotides. In some embodiments, a provided chirally controlled oligonucleotide composition is of oligonucleotides that can be antisense oligonucleotide, antagomir, microRNA, pre-microRNA, antimir, supermir, ribozyme, Ul adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, a chirally controlled oligonucleotide composition is of antisense oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of siRNA oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of antagomir oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of microRNA oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of pre-microRNA oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of antimir oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of supermir oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of ribozyme oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of Ul adaptor oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of RNA activator oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of RNAi agent oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of decoy oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of triplex forming oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of aptamer oligonucleotides. In some embodiments, a chirally controlled oligonucleotide composition is of adjuvant oligonucleotides.

(194) In some embodiments, a provided oligonucleotide comprises one or more chiral, modified phosphate linkages. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of oligonucleotides that include one or more modified backbone linkages, bases, and/or sugars.

(195) In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 80%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 85%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 90%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 91%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 92%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 93%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 94%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 95%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 96%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 97%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 98%. In some embodiments, provided chirally controlled (and/or stereochemically pure) preparations are of a stereochemical purity of greater than about 99%.

(196) In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the internucleotidic linkages of an oligonucleotide are independently chiral internucleotidic linkages. In some embodiments, all chiral, modified internucleotidic linkages are chiral phosphorothioate internucleotidic linkages. In some embodiments, all chiral, modified internucleotidic linkages except non-negatively charged internucleotidic linkages are chiral phosphorothioate internucleotidic linkages. In some embodiments, each chiral internucleotidic linkage is chirally controlled. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Sp conformation. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% phosphorothioate internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Sp conformation. In some embodiments, the percentage is at least about 10%. In some embodiments, the percentage is at least about 20%. In some embodiments, the percentage is at least about 30%. In some embodiments, the percentage is at least about 40%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 90%.

(197) In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Rp conformation. In some embodiments, at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral phosphorothioate internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Rp conformation. In some embodiments, the percentage is at least about 10%. In some embodiments, the percentage is at least about 20%. In some embodiments, the percentage is at least about 30%. In some embodiments, no more than 10, 20, 30, 40, 50, 60, 70, 80, or 90% chiral internucleotidic linkages of an oligonucleotide are chirally controlled and are of the Rp conformation. In some embodiments, no more than 10, 20, 30, 40, 50, 60, 70, 80, or 90% phosphorothioate internucleotidic linkages of an oligonucleotide are of the Rp conformation. In some embodiments, the percentage is no more than 10%. In some embodiments, the percentage is no more than 20%. In some embodiments, the percentage is no more than 30%.

(198) In some embodiments, provided chirally controlled (and/or stereochemically pure) compositions are of oligonucleotides that contain one or more modified bases. In some embodiments, provided chirally controlled (and/or stereochemically pure) compositions are of oligonucleotides that contain no modified bases. As appreciated by those skilled in the art, many types of modified bases can be utilized in accordance with the present disclosure. Example modified bases are described herein.

(199) In some embodiments, oligonucleotides of provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least one natural phosphate linkage. In some embodiments, oligonucleotides of provided compositions comprise at least two natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least three natural phosphate linkages.

(200) In some embodiments, oligonucleotides of provided compositions comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise one natural phosphate linkage. In some embodiments, oligonucleotides of provided compositions comprise two natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise three natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise four natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise five natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise six natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise seven natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise eight natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise nine natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise ten natural phosphate linkages.

(201) In some embodiments, oligonucleotides of provided compositions comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least two consecutive natural phosphate linkages. In some embodiments, oligonucleotides of provided compositions comprise at least three consecutive natural phosphate linkages.

(202) In some embodiments, oligonucleotides of the present disclosure have at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nucleobases in length. In some embodiments, oligonucleotides of the present disclosure comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nucleobases in length, wherein each nucleobase is independently optionally substituted A, T, C, G, U, or a tautomer thereof.

(203) In some embodiments, provided compositions comprise oligonucleotides containing one or more residues which are modified at the sugar moiety. In some embodiments, provided compositions comprise oligonucleotides containing one or more residues which are modified at the 2 position of the sugar moiety (referred to herein as a 2-modification). Examples of such modifications are described herein and include, but are not limited to, 2-OMe, 2-MOE, 2-LNA, 2-F, FRNA, FANA, S-cEt, etc. In some embodiments, provided compositions comprise oligonucleotides containing one or more residues which are 2-modified. For example, in some embodiments, provided oligonucleotides contain one or more residues which are 2-O-methoxyethyl (2-MOE)-modified residues. In some embodiments, provided compositions comprise oligonucleotides which do not contain any 2-modifications. In some embodiments, provided compositions are oligonucleotides which do not contain any 2-MOE residues. That is, in some embodiments, provided oligonucleotides are not MOE-modified. Additional example sugar modifications are described in the present disclosure.

(204) In some embodiments, one or more is one. In some embodiments, one or more is two. In some embodiments, one or more is three. In some embodiments, one or more is four. In some embodiments, one or more is five. In some embodiments, one or more is six. In some embodiments, one or more is seven. In some embodiments, one or more is eight. In some embodiments, one or more is nine. In some embodiments, one or more is ten. In some embodiments, one or more is at least one. In some embodiments, one or more is at least two. In some embodiments, one or more is at least three. In some embodiments, one or more is at least four. In some embodiments, one or more is at least five. In some embodiments, one or more is at least six. In some embodiments, one or more is at least seven. In some embodiments, one or more is at least eight. In some embodiments, one or more is at least nine. In some embodiments, one or more is at least ten.

(205) In some embodiments, a base sequence, e.g., a common base sequence of a plurality of oligonucleotide, a base sequence of a particular oligonucleotide type, etc., comprises or is a sequence complementary to a gene or transcript (e.g., of Dystrophin or DMD). In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a gene. In some embodiments, a common base sequence comprises or is a sequence complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from a similar sequence sharing homology with the gene. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from another allele of the gene. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from a similar sequence sharing homology with the gene. In some embodiments, a common base sequence comprises or is a sequence complementary to characteristic sequence element of a target gene, which characteristic sequences comprises a mutation that is not found in other copies of the gene, e.g., the wild-type copy of the gene, another mutant copy the gene, etc. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to characteristic sequence element of a target gene, which characteristic sequences comprises a mutation that is not found in other copies of the gene, e.g., the wild-type copy of the gene, another mutant copy the gene, etc. In some embodiments, a common base sequence comprises or is a sequence 100% complementary to a characteristic sequence element of a gene, which characteristic sequences differentiate the gene from another allele of the gene. In some embodiments, a characteristic sequence element is a mutation. In some embodiments, a characteristic sequence element is a SNP.

(206) In some embodiments, a chiral internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof. In some embodiments, linkage phosphorus of chiral internucleotidic linkages are chirally controlled. In some embodiments, a chiral internucleotidic linkage is phosphorothioate internucleotidic linkage. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition independently has the structure of formula I. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition independently has the structure of formula II. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition independently has the structure of formula III. In some embodiments, each chiral internucleotidic linkage in an oligonucleotide of a provided composition is a phosphorothioate internucleotidic linkage.

(207) As appreciated by those skilled in the art, internucleotidic linkages, e.g., those of formula I, natural phosphate linkages, phosphorothioate internucleotidic linkages, etc. may exist in their salt forms depending on pH of their environment. Unless otherwise indicated, such salt forms are included in the present application when such internucleotidic linkages are referred to.

(208) In some embodiments, oligonucleotides of the present disclosure comprise one or more modified sugar moieties. In some embodiments, oligonucleotides of the present disclosure comprise one or more modified base moieties. As known by a person of ordinary skill in the art and described in the disclosure, various modifications can be introduced to sugar and base moieties. For example, in some embodiments, a modification is a modification described in U.S. Pat. No. 9,006,198, WO2014/012081, WO/2015/107425, and WO/2017/062862, the sugar and base modifications of each of which are incorporated herein by reference.

(209) In some embodiments, a sugar modification is a 2-modification. Commonly used 2-modifications include but are not limited to 2-OR.sup.1, wherein R.sup.1 is not hydrogen. In some embodiments, a modification is 2-OR, wherein R is optionally substituted aliphatic. In some embodiments, a modification is 2-OMe. In some embodiments, a modification is 2-O-MOE. In some embodiments, the present disclosure demonstrates that inclusion and/or location of particular chirally pure internucleotidic linkages can provide stability improvements comparable to or better than those achieved through use of modified backbone linkages, bases, and/or sugars. In some embodiments, a provided single oligonucleotide of a provided composition has no modifications on the sugars. In some embodiments, a provided single oligonucleotide of a provided composition has no modifications on 2-positions of the sugars (i.e., the two groups at the 2-position are either H/H or H/OH). In some embodiments, a provided single oligonucleotide of a provided composition does not have any 2-MOE modifications.

(210) In some embodiments, a 2-modification is O-L- or -L- which connects the 2-carbon of a sugar moiety to another carbon of a sugar moiety. In some embodiments, a 2-modification is O-L- or -L- which connects the 2-carbon of a sugar moiety to the 4-carbon of a sugar moiety. In some embodiments, a 2-modification is S-cEt. In some embodiments, a modified sugar moiety is an LNA sugar moiety.

(211) In some embodiments, a 2-modification is F. In some embodiments, a 2-modification is FANA. In some embodiments, a 2-modification is FRNA.

(212) In some embodiments, a sugar modification is a 5-modification. In some embodiments, a modification is 5-R.sup.1, wherein R.sup.1 is not hydrogen. In some embodiments, a sugar modification is 5-R, wherein R is not hydrogen and is otherwise as described in the present disclosure. In some embodiments, a sugar modification is 5-R, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, a sugar modification is 5-R, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, a sugar modification is 5-R, wherein R is optionally substituted methyl. In some embodiments, a sugar modification is 5-R, wherein R is optionally substituted methyl, wherein no substituents of the methyl group comprises a carbon atom. In some embodiments, a 5-modification is methyl. In some embodiments, each substituent is independently halogen. In some embodiments, a substituted 5-carbon is diastereomerically pure. In some embodiments, a substituted 5-carbon has the R configuration. In some embodiments, a substituted 5-carbon has the S configuration. In some embodiments, a 5-modification is 5-(R)-Me. In some embodiments, a 5-modification is 5-(S)-Me.

(213) In some embodiments, a sugar moiety has one and no more than one modification at a position, e.g., a 2-position, 5-position, etc. In some embodiments, a 2-modification takes the position corresponding to the position of the 2-OH in a natural RNA sugar moiety. In some embodiments, a 2-modification takes the position corresponding to the position of the 2-H in a natural RNA sugar moiety.

(214) In some embodiments, a sugar modification changes the size of the sugar ring. In some embodiments, a sugar modification changes the conformation of the sugar ring. In some embodiments, a sugar modification is the sugar moiety in FHNA.

(215) In some embodiments, a sugar modification replaces a sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art, including but not limited to those used in Morpholino, glycol nucleic acids, etc.

(216) Certain Embodiments of Internucleotidic Linkages, Chirally Controlled Oligonucleotides and Chirally Controlled Oligonucleotide Compositions

(217) Among other things, the present disclosure provides chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions. In some embodiments, the present disclosure provides chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions which are of high crude purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotides, and chirally controlled oligonucleotide compositions which are of high diastereomeric purity. Chirally controlled oligonucleotides are oligonucleotides comprise one or more chirally controlled internucleotidic linkages, such as oligonucleotides of a plurality in chirally controlled oligonucleotide compositions. In some embodiments, chirally controlled oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more chirally controlled internucleotidic linkages. In some embodiments, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more chiral internucleotidic linkages of a chirally controlled oligonucleotide are independently chirally controlled internucleotidic linkages. In some embodiments, each chiral internucleotidic linkage in a chirally controlled oligonucleotide is a chirally controlled internucleotidic linkage, and a chirally controlled oligonucleotide is diastereomerically pure.

(218) In some embodiments, a chirally controlled oligonucleotide composition is a substantially pure composition of an oligonucleotide type in that oligonucleotides in the composition that are not of the oligonucleotide type are impurities. In some embodiments, such impurities are formed during the preparation process of oligonucleotides of said oligonucleotide type, in some case, after certain purification procedures.

(219) In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages with respect to the chiral linkage phosphorus (e.g., linkage phosphorus of chirally controlled internucleotidic linkages). In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages with respect to the chiral linkage phosphorus, and one or more natural phosphate linkages (unless otherwise indicated, reference in the present application to internucleotidic linkages, such as natural phosphate linkages and other types of internucleotidic linkages when applicable, includes salt forms of such linkages). Thus, diastereomerically pure internucleotidic linkages here include salt forms of diastereomerically pure internucleotidic linkages; natural phosphate linkages here include salt forms of natural phosphate linkages. A person having ordinary skill in the art appreciates that many internucleotidic linkages, such as natural phosphate linkages, exist as salt forms when at physiological pH, in many buffers (e.g., PBS buffers having a pH around 7, e.g., PH 7.4), etc.). In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof, and one or more natural phosphate linkages. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotidic linkages having the structure of formula I-c, and one or more phosphate diester linkages. In some embodiments, such oligonucleotides are prepared by using stereoselective oligonucleotide synthesis, as described in this application, to form designed diastereomerically pure internucleotidic linkages with respect to the chiral linkage phosphorus.

(220) In some embodiments, an oligonucleotide of the present disclosure comprises at least one internucleotidic linkage, e.g., a modified (non-natural) internucleotidic linkage (e.g., non-negatively charged internucleotidic linkage) within or at the terminus (e.g. 5 or 3) of the oligonucleotide. In some embodiments, an oligonucleotide comprises a P-modification moiety within or at the terminus (e.g. 5 or 3) of the oligonucleotide.

(221) In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, and at least one natural phosphate linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, at least one natural phosphate linkage, and at least one phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, and at least one phosphorothioate triester internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least one chirally controlled internucleotidic linkage within the oligonucleotide, at least one natural phosphate linkage, and at least one phosphorothioate triester internucleotidic linkage.

(222) In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different stereochemistry and/or different P-modifications relative to one another. In some embodiments, such at least two internucleotidic linkages have different stereochemistry. In some embodiments, such at least two internucleotidic linkages have different P-modifications. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, and at least one natural phosphate linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, at least one natural phosphate linkage, and at least one phosphorothioate internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, and at least one phosphorothioate triester internucleotidic linkage. In some embodiments, an oligonucleotide of the present disclosure comprises at least two chirally controlled internucleotidic linkages within the oligonucleotide that have different P-modifications relative to one another, at least one natural phosphate linkage, and at least one phosphorothioate triester internucleotidic linkage.

(223) In certain embodiments, an internucleotidic linkage (e.g., a modified (non-natural) internucleotidic linkage when formula I is not a natural phosphate linkage) has the structure of formula I:

(224) ##STR00028##
or a salt form thereof, wherein: P.sup.L is P(W), P, or P.fwdarw.B(R).sub.3; W is O, N(-L-R.sup.5), S or Se; each of R.sup.1 and R.sup.5 is independently H, -L-R, halogen, CN, NO.sub.2, -L-Si(R).sub.3, OR, SR, or N(R).sub.2; each of X, Y and Z is independently O, S, N(-L-R.sup.5), or L; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

(225) In some embodiments, a linkage of formula I is chiral at the linkage phosphorus (P in P.sup.L). In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different P-modifications relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different X-L-R.sup.1 relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different X relative to one another. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more modified internucleotidic linkages of formula I, and wherein individual internucleotidic linkages of formula I within the oligonucleotide have different -L-R.sup.1 relative to one another. In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a provided composition that is of the particular oligonucleotide type. In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a provided composition that has the common base sequence and length, the common pattern of backbone linkages, and the common pattern of backbone chiral centers.

(226) As extensively described herein, in some embodiments, X-L-R.sup.1 is a moiety useful for oligonucleotide preparation. For example, in some embodiments, X-L-R.sup.1 is OCH.sub.2CH.sub.2CN (e.g., in non-chirally controlled internucleotidic linkages); in some embodiments, X-L-R.sup.1 is of such a structure that HX-L-R.sup.1 is a chiral auxiliary, optionally capped, as described herein (e.g., DPSE, PSM, etc.; particularly in chirally controlled internucleotidic linkages, although may also in non-chirally controlled internucleotidic linkages (e.g., precursors of natural phosphate linkages)).

(227) In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a chirally controlled composition that is of a particular oligonucleotide type, and the chirally controlled oligonucleotide is of the type. In some embodiments, a chirally controlled oligonucleotide is an oligonucleotide in a provided composition that comprises a controlled level of a plurality of oligonucleotides that share a common base sequence, a common pattern of backbone linkages, a common pattern of backbone chiral centers, and a common pattern of backbone phosphorus modifications, and the chirally controlled oligonucleotide shares the common base sequence, the common pattern of backbone linkages, the common pattern of backbone chiral centers, and the common pattern of backbone phosphorus modifications.

(228) In some embodiments, the present disclosure provides a chirally controlled oligonucleotide, wherein at least two chirally controlled internucleotidic linkages within the oligonucleotide have different P-modifications relative to one another, in that they have different X atoms in their XLR.sup.1 moieties, and/or in that they have different L groups in their XLR.sup.1 moieties, and/or that they have different R.sup.1 atoms in their XLR.sup.1 moieties, and/or in that they have different XLR.sup.1 moieties.

(229) In some embodiments, the present disclosure provides a chirally controlled oligonucleotide, wherein at least two of the individual internucleotidic linkages within the oligonucleotide have different stereochemistry and/or different P-modifications relative to one another and the oligonucleotide has a structure represented by the following formula:
[S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . S.sup.BnxR.sup.Bny]
wherein: each R.sup.B independently represents a block of nucleotide units having the R configuration at the linkage phosphorus; each S.sup.B independently represents a block of nucleotide units having the S configuration at the linkage phosphorus; each of n1-ny is zero or an integer, with the requirement that at least one odd n and at least one even n must be non-zero so that the oligonucleotide includes at least two individual internucleotidic linkages with different stereochemistry relative to one another; and wherein the sum of n1-ny is between 2 and 200, and in some embodiments is between a lower limit selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more and an upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upper limit being larger than the lower limit.

(230) In some such embodiments, each n has the same value; in some embodiments, each even n has the same value as each other even n; in some embodiments, each odd n has the same value each other odd n; in some embodiments, at least two even ns have different values from one another; in some embodiments, at least two odd ns have different values from one another.

(231) In some embodiments, at least two adjacent ns are equal to one another, so that a provided oligonucleotide includes adjacent blocks of S stereochemistry linkages and R stereochemistry linkages of equal lengths. In some embodiments, provided oligonucleotides include repeating blocks of S and R stereochemistry linkages of equal lengths. In some embodiments, provided oligonucleotides include repeating blocks of S and R stereochemistry linkages, where at least two such blocks are of different lengths from one another; in some such embodiments each S stereochemistry block is of the same length, and is of a different length from each R stereochemistry length, which may optionally be of the same length as one another.

(232) In some embodiments, at least two skip-adjacent ns are equal to one another, so that a provided oligonucleotide includes at least two blocks of linkages of a first stereochemistry that are equal in length to one another and are separated by a block of linkages of the other stereochemistry, which separating block may be of the same length or a different length from the blocks of first stereochemistry.

(233) In some embodiments, ns associated with linkage blocks at the ends of a provided oligonucleotide are of the same length. In some embodiments, provided oligonucleotides have terminal blocks of the same linkage stereochemistry. In some such embodiments, the terminal blocks are separated from one another by a middle block of the other linkage stereochemistry.

(234) In some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] is a stereoblockmer. In some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] is a stereoskipmer. In some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] is a stereoaltmer. In some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] is a gapmer.

(235) In some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] is of any of the above described patterns and further comprises patterns of P-modifications. For instance, in some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] and is a stereoskipmer and P-modification skipmer. In some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] and is a stereoblockmer and P-modification altmer. In some embodiments, a provided oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . . S.sup.BnxR.sup.Bny] and is a stereoaltmer and P-modification blockmer.

(236) In some embodiments, an internucleotidic linkage of formula I has the structure of:

(237) ##STR00029##
wherein: P* is an asymmetric phosphorus atom and is either Rp or Sp; W is O, S or Se; each of X, Y and Z is independently O, S, N(-L-R.sup.1), or L; L is a covalent bond or an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, a C.sub.1-C.sub.6 heteroaliphatic moiety, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2 SC(O), C(O)S, OC(O), and C(O)O; R.sup.1 is halogen, R, or an optionally substituted C.sub.1-C.sub.50 aliphatic wherein one or more methylene units are optionally and independently replaced by C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, a C.sub.1-C.sub.6 heteroaliphatic moiety, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2 SC(O), C(O)S, OC(O), and C(O)O; each R is independently R, C(O)R, CO.sub.2R, or SO.sub.2R, or: two R are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring; Cy is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, and heterocyclylene; each R is independently hydrogen, or an optionally substituted group selected from C.sub.1-C.sub.6 aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl; and each

(238) ##STR00030##
independently represents a connection to a nucleoside.

(239) In some embodiments, L is a covalent bond or an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O; R.sup.1 is halogen, R, or an optionally substituted C.sub.1-C.sub.50 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O; each R is independently R, C(O)R, CO.sub.2R, or SO.sub.2R, or: two R on the same nitrogen are taken together with their intervening atoms to form an optionally substituted heterocyclic or heteroaryl ring, or two R on the same carbon are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring; Cy is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene; each R is independently hydrogen, or an optionally substituted group selected from C.sub.1-C.sub.6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each

(240) ##STR00031##
independently represents a connection to a nucleoside.

(241) In some embodiments, a chirally controlled oligonucleotide comprises one or more modified internucleotidic linkages. In some embodiments, a chirally controlled oligonucleotide comprises, e.g., a phosphorothioate or a phosphorothioate triester internucleotidic linkage. In some embodiments, a chirally controlled oligonucleotide comprises a chirally controlled phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled phosphorothioate triester internucleotidic linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled phosphorothioate internucleotidic linkages (OP(O)(SH)O or salt forms thereof).

(242) In some embodiments, an oligonucleotide comprises different types of internucleotidic phosphorus linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one natural phosphate linkage and at least one modified (non-natural) internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least one phosphorothioate. In some embodiments, an oligonucleotide comprises at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one phosphorothioate internucleotidic linkage and at least one non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide comprises at least one phosphorothioate internucleotidic linkage, at least one natural phosphate linkage, and at least one non-negatively charged internucleotidic linkage.

(243) In some embodiments, an internucleotidic linkage comprises a chiral auxiliary. In some embodiments, an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., comprises a chiral auxiliary, wherein P.sup.L is PS. In some embodiments, an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., comprises a chiral auxiliary, wherein P.sup.L is PO. In some embodiments, a phosphorothioate triester linkage comprises a chiral auxiliary, which, for example, is used to control the stereoselectivity of a reaction. In some embodiments, a phosphorothioate triester linkage does not comprise a chiral auxiliary. Example chiral auxiliaries that can be utilized in accordance with the present disclosure include those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, US20130178612, US20150211006, U.S. Pat. No. 9,598,458, US20170037399, WO 2017/015555, WO 2017/062862, WO 2018/237194, WO 2019/055951, the chiral auxiliaries of each of which is incorporated herein by reference. In some embodiments, one or more X-L-R.sup.1 independently comprise or are an optionally substituted chiral auxiliary. In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is a chiral reagent/chiral auxiliary described herein (e.g., one having the structure of formula 3-I, formula 3-AA, etc.). In some embodiments, HX-L-R.sup.1 is a capped chiral reagent/chiral auxiliary described herein (e.g., one having the structure of formula 3-I, formula 3-AA, etc.), which is capped in that an amino group of the chiral reagent/chiral auxiliary (e.g., HW.sup.1 and HW.sup.2 is or comprises HNG.sup.5-) is capped (e.g., forming R.sup.1NG.sup.5-(e.g., RC(O)NG.sup.5-, RS(O).sub.2NG.sup.5-, etc.)). In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is methyl. In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is

(244) ##STR00032##
In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is

(245) ##STR00033##
In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is

(246) ##STR00034##

(247) In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof. In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is

(248) ##STR00035##
In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is

(249) ##STR00036##
In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is

(250) ##STR00037##
In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof, wherein the NH of the 5-membered pyrrolidinyl is replaced with N(R.sup.1). In some embodiments, one or more X-L-R.sup.1 are independently

(251) ##STR00038##
In some embodiments, one or more X-L-R.sup.1 are independently

(252) ##STR00039##
In some embodiments, one or more X-L-R.sup.1 are independently

(253) ##STR00040##
In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof, wherein the connection to the linkage phosphorus is through the alcohol hydroxyl group. In some embodiments, one or more X-L-R.sup.1 are independently

(254) ##STR00041##
In some embodiments, one or more X-L-R.sup.1 are independently

(255) ##STR00042##
In some embodiments, one or more X-L-R.sup.1 are independently

(256) ##STR00043##
In some embodiments, one or more X-L-R.sup.1 are each independently of such a structure that HX-L-R.sup.1 is a compound selected from Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12, or CA-13, or a related (having the same constitution) diastereomer or enantiomer thereof, wherein the NH of the 5-membered pyrrolidinyl is replaced with N(R.sup.1), and wherein the connection to the linkage phosphorus is through the alcohol hydroxyl group. In some embodiments, one or more X-L-R.sup.1 are independently

(257) ##STR00044##
and one or more X-L-R.sup.1 are independently

(258) ##STR00045##
In some embodiments, one or more X-L-R.sup.1 are independently

(259) ##STR00046##
and one or more X-L-R.sup.1 are independently

(260) ##STR00047##
In some embodiments, one or more X-L-R.sup.1 are independently

(261) ##STR00048##
and one or more X-L-R.sup.1 are independently

(262) ##STR00049##
In some embodiments, R.sup.1 is a capping group utilized in oligonucleotide synthesis. In some embodiments, R.sup.1 is C(O)R. In some embodiments, R.sup.1 is C(O)R, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1 is C(O)CH.sub.3.

(263) In some embodiments, an oligonucleotide, e.g., a chirally controlled oligonucleotide, an oligonucleotide of a plurality, etc. is linked to a solid support. In some embodiments, an oligonucleotide is not linked to a solid support.

(264) In some embodiments, an oligonucleotide comprises at least one natural phosphate linkage and at least two consecutive chirally controlled modified internucleotidic linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one natural phosphate linkage and at least two consecutive chirally controlled phosphorothioate internucleotidic linkages.

(265) In some embodiments, a chirally controlled oligonucleotide is a blockmer. In some embodiments, a chirally controlled oligonucleotide is a stereoblockmer. In some embodiments, a chirally controlled oligonucleotide is a P-modification blockmer. In some embodiments, a chirally controlled oligonucleotide is a linkage blockmer.

(266) In some embodiments, a chirally controlled oligonucleotide is an altmer. In some embodiments, a chirally controlled oligonucleotide is a stereoaltmer. In some embodiments, a chirally controlled oligonucleotide is a P-modification altmer. In some embodiments, a chirally controlled oligonucleotide is a linkage altmer.

(267) In some embodiments, a chirally controlled oligonucleotide is a unimer.

(268) In some embodiments, in a unimer, all nucleotide units within a strand share at least one common structural feature at the internucleotidic phosphorus linkage. In some embodiments, a common structural feature is a common stereochemistry at the linkage phosphorus or a common modification at the linkage phosphorus. In some embodiments, a chirally controlled oligonucleotide is a stereounimer. In some embodiments, a chirally controlled oligonucleotide is a P-modification unimer. In some embodiments, a chirally controlled oligonucleotide is a linkage unimer.

(269) In some embodiments, a chirally controlled oligonucleotide is a gapmer.

(270) In some embodiments, a chirally controlled oligonucleotide is a skipmer.

(271) In some embodiments, the present disclosure provides oligonucleotides comprising one or more modified internucleotidic linkages independently having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof.

(272) In some embodiments, L is a covalent bond or an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O; R.sup.1 is halogen, R, or an optionally substituted C.sub.1-C.sub.50 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O; each R is independently R, C(O)R, CO.sub.2R, or SO.sub.2R, or: two R on the same nitrogen are taken together with their intervening atoms to form an optionally substituted heterocyclic or heteroaryl ring, or two R on the same carbon are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring; Cy is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene; each R is independently hydrogen, or an optionally substituted group selected from C.sub.1-C.sub.6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each

(273) ##STR00050##
independently represents a connection to a nucleoside.

(274) In some embodiments, a chirally controlled oligonucleotide comprises one or more modified internucleotidic phosphorus linkages. In some embodiments, a chirally controlled oligonucleotide comprises, e.g., a phosphorothioate or a phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises a phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least two phosphorothioate triester linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least three phosphorothioate triester linkages. Example modified internucleotidic phosphorus linkages are described further herein. In some embodiments, a chirally controlled oligonucleotide comprises different internucleotidic phosphorus linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least one modified internucleotidic linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least one phosphorothioate triester linkage. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least two phosphorothioate triester linkages. In some embodiments, a chirally controlled oligonucleotide comprises at least one phosphate diester internucleotidic linkage and at least three phosphorothioate triester linkages.

(275) In some embodiments, P* is an asymmetric phosphorus atom and is either Rp or Sp. In some embodiments, P* is Rp. In other embodiments, P* is Sp. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages of formula I wherein each P* is independently Rp or Sp. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages of formula I wherein each P* is Rp. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages of formula I wherein each P* is Sp. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein P* is Rp. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein P* is Sp. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein P* is Rp, and at least one internucleotidic linkage of formula I wherein P* is Sp.

(276) In some embodiments, W is O, S, or Se. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is O. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is S. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is Se.

(277) In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is O. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein W is S.

(278) In some embodiments, X is O. In some embodiments, X is S. In some embodiments, X is O or S. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is O. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is S. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is O, and at least one internucleotidic linkage of formula I wherein X is S. In some embodiments, an oligonucleotide comprises at least one internucleotidic linkage of formula I wherein X is O, and at least one internucleotidic linkage of formula I wherein X is S, and at least one internucleotidic linkage of formula I wherein L is an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O.

(279) In some embodiments, X is N(-L-R.sup.1). In some embodiments, X is N(R.sup.1). In some embodiments, X is N(R). In some embodiments, X is N(R). In some embodiments, X is NH.

(280) In some embodiments, X is L. In some embodiments, X is a covalent bond. In some embodiments, X is or an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O. In some embodiments, X is an optionally substituted C.sub.1-C.sub.10 alkylene or C.sub.1-C.sub.10 alkenylene. In some embodiments, X is methylene.

(281) In some embodiments, Y is O. In some embodiments, Y is S.

(282) In some embodiments, Y is N(-L-R.sup.1). In some embodiments, Y is N(R.sup.1). In some embodiments, Y is N(R). In some embodiments, Y is N(R). In some embodiments, Y is NH.

(283) In some embodiments, Y is L. In some embodiments, Y is a covalent bond. In some embodiments, Y is or an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O. In some embodiments, Y is an optionally substituted C.sub.1-C.sub.10 alkylene or C.sub.1-C.sub.10 alkenylene. In some embodiments, Y is methylene.

(284) In some embodiments, Z is O. In some embodiments, Z is S.

(285) In some embodiments, Z is N(-L-R.sup.1). In some embodiments, Z is N(R.sup.1). In some embodiments, Z is N(R). In some embodiments, Z is N(R). In some embodiments, Z is NH.

(286) In some embodiments, Z is L. In some embodiments, Z is a covalent bond. In some embodiments, Z is or an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O. In some embodiments, Z is an optionally substituted C.sub.1-C.sub.10 alkylene or C.sub.1-C.sub.10 alkenylene. In some embodiments, Z is methylene.

(287) In some embodiments, L is a covalent bond or an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O.

(288) In some embodiments, L is a covalent bond. In some embodiments, L is an optionally substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O.

(289) In some embodiments, L has the structure of -L.sup.1-V, wherein: L is an optionally substituted group selected from

(290) ##STR00051##
C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, carbocyclylene, arylene, C.sub.1-C.sub.6 heteroalkylene, heterocyclylene, and heteroarylene; V is selected from O, S, NR, C(R).sub.2, SS, BSSC,

(291) ##STR00052##
or an optionally substituted group selected from C.sub.1-C.sub.6 alkylene, arylene, C.sub.1-C.sub.6 heteroalkylene, heterocyclylene, and heteroarylene; A is O, S, NR, or C(R).sub.2; each of B and C is independently O, S, NR, C(R).sub.2, or an optionally substituted group selected from C.sub.1-C.sub.6 alkylene, carbocyclylene, arylene, heterocyclylene, or heteroarylene; and each R is independently as defined above and described herein.

(292) In some embodiments, L.sup.1 is

(293) ##STR00053##

(294) In some embodiments, L.sup.1 is

(295) ##STR00054##
wherein Ring Cy is an optionally substituted arylene, carbocyclylene, heteroarylene, or heterocyclylene. In some embodiments, L.sup.1 is optionally substituted

(296) ##STR00055##
In some embodiments, L.sup.1 is

(297) ##STR00056##

(298) In some embodiments, L.sup.1 is connected to X. In some embodiments, L.sup.1 is an optionally substituted group selected from

(299) ##STR00057##
and the sulfur atom is connect to V. In some embodiments, L.sup.1 is an optionally substituted group selected from

(300) ##STR00058##
and the carbon atom is connect to X.

(301) In some embodiments, L has the structure of:

(302) ##STR00059##
wherein: E is O, S, NR or C(R).sub.2; is a single or double bond; the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, carbocyclic, heteroaryl or heterocyclic ring; and each R is independently as defined above and described herein.

(303) In some embodiments, L has the structure of:

(304) ##STR00060##
wherein: G is O, S, or NR; is a single or double bond; and the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C.sub.3-C.sub.10 carbocyclic, heteroaryl or heterocyclic ring.

(305) In some embodiments, L has the structure of:

(306) ##STR00061##
wherein: E is O, S, NR or C(R).sub.2; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(307) In some embodiments, L has the structure of:

(308) ##STR00062##
wherein: G is O, S, or NR; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3).

(309) In some embodiments, L has the structure of:

(310) ##STR00063##
wherein: E is O, S, NR or C(R).sub.2; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(311) In some embodiments, L has the structure of:

(312) ##STR00064##
wherein: G is O, S, or NR; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3).

(313) In some embodiments, L has the structure of:

(314) ##STR00065##
wherein: E is O, S, NR or C(R).sub.2; is a single or double bond; the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C.sub.3-C.sub.10 carbocyclic, heteroaryl or heterocyclic ring; and each R is independently as defined above and described herein.

(315) In some embodiments, L has the structure of:

(316) ##STR00066##
wherein: G is O, S, or NR; is a single or double bond; the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C.sub.3-C.sub.10 carbocyclic, heteroaryl or heterocyclic ring; and each R is independently as defined above and described herein.

(317) In some embodiments, L has the structure of:

(318) ##STR00067##
wherein: E is O, S, NR or C(R).sub.2; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(319) In some embodiments, L has the structure of:

(320) ##STR00068##
wherein: G is O, S, or NR; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(321) In some embodiments, L has the structure of:

(322) ##STR00069##
wherein: E is O, S, NR or C(R).sub.2; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(323) In some embodiments, L has the structure of:

(324) ##STR00070##
wherein: G is O, S, or NR; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(325) In some embodiments, L has the structure of:

(326) ##STR00071##
wherein: E is O, S, NR or C(R).sub.2; is a single or double bond; the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C.sub.3-C.sub.10 carbocyclic, heteroaryl or heterocyclic ring; and each R is independently as defined above and described herein.

(327) In some embodiments, L has the structure of:

(328) ##STR00072##
wherein: G is O, S, or NR; is a single or double bond; the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C.sub.3-C.sub.10 carbocyclic, heteroaryl or heterocyclic ring; and each R is independently as defined above and described herein.

(329) In some embodiments, L has the structure of:

(330) ##STR00073##
wherein: E is O, S, NR or C(R).sub.2; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(331) In some embodiments, L has the structure of:

(332) ##STR00074##
wherein: G is O, S, or NR; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and R is as defined above and described herein.

(333) In some embodiments, L has the structure of:

(334) ##STR00075##
wherein: E is O, S, NR or C(R).sub.2; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and each R is independently as defined above and described herein.

(335) In some embodiments, L has the structure of:

(336) ##STR00076##
wherein: G is O, S, or NR; D is N, C(F), C(Cl), C(Br), C(I), C(CN), C(NO.sub.2), C(CO.sub.2(C.sub.1-C.sub.6 aliphatic)), or C(CF.sub.3); and R is as defined above and described herein.

(337) In some embodiments, L has the structure of:

(338) ##STR00077##
wherein the phenyl ring is optionally substituted. In some embodiments, the phenyl ring is not substituted. In some embodiments, the phenyl ring is substituted.

(339) In some embodiments, L has the structure of:

(340) ##STR00078##
wherein the phenyl ring is optionally substituted. In some embodiments, the phenyl ring is not substituted. In some embodiments, the phenyl ring is substituted.

(341) In some embodiments, L has the structure of:

(342) ##STR00079##
wherein: is a single or double bond; and the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C.sub.3-C.sub.10 carbocyclic, heteroaryl or heterocyclic ring.

(343) In some embodiments, L has the structure of:

(344) ##STR00080##
wherein: G is O, S, or NR; is a single or double bond; and the two R.sup.L1 are taken together with the two carbon atoms to which they are bound to form an optionally substituted aryl, C.sub.3-C.sub.10 carbocyclic, heteroaryl or heterocyclic ring.

(345) In some embodiments, E is O, S, NR or C(R).sub.2, wherein each R independently as defined above and described herein. In some embodiments, E is O, S, or NR. In some embodiments, E is O, S, or NH. In some embodiments, E is O. In some embodiments, E is S. In some embodiments, E is NH.

(346) In some embodiments, G is O, S, or NR, wherein each R independently as defined above and described herein. In some embodiments, G is O, S, or NH. In some embodiments, G is O. In some embodiments, G is S. In some embodiments, G is NH.

(347) In some embodiments, L is -L.sup.3-G, wherein: L.sup.3 is an optionally substituted C.sub.1-C.sub.5 alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by O, S, N(R), C(O), C(S), C(NR), S(O), S(O).sub.2, or

(348) ##STR00081##
and wherein each of G, R and Ring Cy is independently as defined above and described herein.

(349) In some embodiments, L is -L.sup.3-S, wherein L.sup.3 is as defined above and described herein. In some embodiments, L is -L.sup.3-O, wherein L.sup.3 is as defined above and described herein. In some embodiments, L is -L.sup.3-N(R), wherein each of L.sup.3 and R is independently as defined above and described herein. In some embodiments, L is -L.sup.3-NH, wherein each of L.sup.3 and R is independently as defined above and described herein.

(350) In some embodiments, L.sup.3 is an optionally substituted C.sub.5 alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by O, S, N(R), C(O), C(S), C(NR), S(O), S(O).sub.2, or

(351) ##STR00082##
and each of R and Ring Cy is independently as defined above and described herein. In some embodiments, L.sup.3 is an optionally substituted C.sub.5 alkylene. In some embodiments, -L.sup.3-G is

(352) ##STR00083##

(353) In some embodiments, L.sup.3 is an optionally substituted C.sub.4 alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by O, S, N(R), C(O), C(S), C(NR), S(O), S(O).sub.2, or

(354) ##STR00084##
and each of R and Cy is independently as defined above and described herein.

(355) In some embodiments, -L.sup.3-G is

(356) ##STR00085##

(357) In some embodiments, L.sup.3 is an optionally substituted C.sub.3 alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by O, S, N(R), C(O), C(S), C(NR), S(O), S(O).sub.2, or

(358) ##STR00086##
and each of R and Cy is independently as defined above and described herein.

(359) In some embodiments, -L.sup.3-G is

(360) ##STR00087##

(361) In some embodiments, L is

(362) ##STR00088##
In some embodiments, L is

(363) ##STR00089##
In some embodiments, L is

(364) ##STR00090##

(365) In some embodiments, L.sup.3 is an optionally substituted C.sub.2 alkylene or alkenylene, wherein one or more methylene units are optionally and independently replaced by O, S, N(R), C(O), C(S), C(NR), S(O), S(O).sub.2, or

(366) ##STR00091##
and each of R and Cy is independently as defined above and described herein.

(367) In some embodiments, -L.sup.3-G is

(368) ##STR00092##
wherein each of G and Cy is independently as defined above and described herein. In some embodiments, L is

(369) ##STR00093##

(370) In some embodiments, L is -L.sup.4-G, wherein L.sup.4 is an optionally substituted C.sub.1-C.sub.2 alkylene; and G is as defined above and described herein. In some embodiments, L is -L.sup.4-G, wherein L.sup.4 is an optionally substituted C.sub.1-C.sub.2 alkylene; G is as defined above and described herein; and G is connected to R.sup.1. In some embodiments, L is -L.sup.4-G, wherein L.sup.4 is an optionally substituted methylene; G is as defined above and described herein; and G is connected to R.sup.1. In some embodiments, L is -L.sup.4-G, wherein L.sup.4 is methylene; G is as defined above and described herein; and G is connected to R.sup.1. In some embodiments, L is -L.sup.4-G, wherein L.sup.4 is an optionally substituted (CH.sub.2).sub.2; G is as defined above and described herein; and G is connected to R.sup.1. In some embodiments, L is -L.sup.4-G, wherein L.sup.4 is (CH.sub.2).sub.2; G is as defined above and described herein; and G is connected to R.sup.1.

(371) In some embodiments, L is

(372) ##STR00094##
wherein G is as defined above and described herein, and G is connected to R.sup.1. In some embodiments, L is

(373) ##STR00095##
wherein G is as defined above and described herein, and G is connected to R.sup.1. In some embodiments, L is

(374) ##STR00096##
wherein G is as defined above and described herein, and G is connected to R.sup.1. In some embodiments, L is

(375) ##STR00097##
wherein the sulfur atom is connected to R.sup.1. In some embodiments, L is

(376) ##STR00098##
wherein the oxygen atom is connected to R.sup.1.

(377) In some embodiments, L is

(378) ##STR00099##
wherein G is as defined above and described herein.

(379) In some embodiments, L is SR.sup.L3 or SC(O)R.sup.L3, wherein R.sup.L3 is an optionally substituted, linear or branched, C.sub.1-C.sub.9 alkylene, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, wherein each of R and Cy is independently as defined above and described herein. In some embodiments, L is SR.sup.L3 or SC(O)R.sup.L3, wherein R.sup.L3 is an optionally substituted C.sub.1-C.sub.6 alkylene. In some embodiments, L is SR.sup.L3 or SC(O)R.sup.L3, wherein R.sup.L3 is an optionally substituted C.sub.1-C.sub.6 alkenylene. In some embodiments, L is SR.sup.L3 or SC(O)R.sup.L3, wherein R.sup.L3 is an optionally substituted C.sub.1-C.sub.6 alkylene wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkenylene, arylene, or heteroarylene. In some embodiments, In some embodiments, R.sup.L3 is an optionally substituted S(C.sub.1-C.sub.6 alkenylene)-, S(C.sub.1-C.sub.6 alkylene)-, S(C.sub.1-C.sub.6 alkylene)-arylene-(C.sub.1-C.sub.6 alkylene)-, SCO-arylene-(C.sub.1-C.sub.6 alkylene)-, or SCO(C.sub.1-C.sub.6 alkylene)-arylene-(C.sub.1-C.sub.6 alkylene)-.

(380) In some embodiments, L is

(381) ##STR00100##

(382) In some embodiments, L is

(383) ##STR00101##
In some embodiments, L is

(384) ##STR00102##
In some embodiments,

(385) ##STR00103##

(386) In some embodiments, the sulfur atom in the L embodiments described above and herein is connected to X. In some embodiments, the sulfur atom in the L embodiments described above and herein is connected to R.sup.1.

(387) In some embodiments, R.sup.1 is halogen, R, or an optionally substituted C.sub.1-C.sub.50 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, wherein each variable is independently as defined above and described herein. In some embodiments, R.sup.1 is halogen, R, or an optionally substituted C.sub.1-C.sub.10 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, wherein each variable is independently as defined above and described herein.

(388) In some embodiments, R.sup.1 is hydrogen. In some embodiments, R.sup.1 is halogen. In some embodiments, R.sup.1 is F. In some embodiments, R.sup.1 is Cl. In some embodiments, R.sup.1 is Br. In some embodiments, R.sup.1 is I.

(389) In some embodiments, R.sup.1 is R wherein R is as defined above and described herein.

(390) In some embodiments, R.sup.1 is hydrogen. In some embodiments, R.sup.1 is an optionally substituted group selected from C.sub.1-C.sub.50 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl.

(391) In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.50 aliphatic. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.10 aliphatic. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.6 aliphatic. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.6 alkyl. In some embodiments, R.sup.1 is optionally substituted, linear or branched hexyl. In some embodiments, R.sup.1 is optionally substituted, linear or branched pentyl. In some embodiments, R.sup.1 is optionally substituted, linear or branched butyl. In some embodiments, R.sup.1 is optionally substituted, linear or branched propyl. In some embodiments, R.sup.1 is optionally substituted ethyl. In some embodiments, R.sup.1 is optionally substituted methyl.

(392) In some embodiments, R.sup.1 is optionally substituted phenyl. In some embodiments, R.sup.1 is substituted phenyl. In some embodiments, R.sup.1 is phenyl.

(393) In some embodiments, R.sup.1 is optionally substituted carbocyclyl. In some embodiments, R.sup.1 is optionally substituted C.sub.3-C.sub.10 carbocyclyl. In some embodiments, R.sup.1 is optionally substituted monocyclic carbocyclyl. In some embodiments, R.sup.1 is optionally substituted cycloheptyl. In some embodiments, R.sup.1 is optionally substituted cyclohexyl. In some embodiments, R.sup.1 is optionally substituted cyclopentyl. In some embodiments, R.sup.1 is optionally substituted cyclobutyl. In some embodiments, R.sup.1 is an optionally substituted cyclopropyl. In some embodiments, R.sup.1 is optionally substituted bicyclic carbocyclyl.

(394) In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.50 polycyclic hydrocarbon. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.50 polycyclic hydrocarbon wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, wherein each variable is independently as defined above and described herein. In some embodiments, R.sup.1 is optionally substituted

(395) ##STR00104##
In some embodiments, R.sup.1 is

(396) ##STR00105##
In some embodiments, R.sup.1 is optionally substituted

(397) ##STR00106##

(398) In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.50 aliphatic comprising one or more optionally substituted polycyclic hydrocarbon moieties. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.50 aliphatic comprising one or more optionally substituted polycyclic hydrocarbon moieties, wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, wherein each variable is independently as defined above and described herein. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.50 aliphatic comprising one or more optionally substituted

(399) ##STR00107##
In some embodiments, R.sup.1 is

(400) ##STR00108##
In some embodiments, R.sup.1 is

(401) ##STR00109##
In some embodiments, R.sup.1 is

(402) ##STR00110##
In some embodiments, R.sup.1 is

(403) ##STR00111##
In some embodiments, R.sup.1 is

(404) ##STR00112##

(405) In some embodiments, R.sup.1 is an optionally substituted aryl. In some embodiments, R.sup.1 is an optionally substituted bicyclic aryl ring.

(406) In some embodiments, R.sup.1 is an optionally substituted heteroaryl. In some embodiments, R.sup.1 is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, or oxygen. In some embodiments, R.sup.1 is a substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, or oxygen.

(407) In some embodiments, R.sup.1 is an optionally substituted 5 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R.sup.1 is an optionally substituted 6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

(408) In some embodiments, R.sup.1 is an optionally substituted 5-membered monocyclic heteroaryl ring having 1 heteroatom selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is selected from pyrrolyl, furanyl, or thienyl.

(409) In some embodiments, R.sup.1 is an optionally substituted 5-membered heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R.sup.1 is an optionally substituted 5-membered heteroaryl ring having 1 nitrogen atom, and an additional heteroatom selected from sulfur or oxygen. Example R.sup.1 groups include optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.

(410) In some embodiments, R.sup.1 is a 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments, R.sup.1 is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R.sup.1 is an optionally substituted 6-membered heteroaryl ring having 2 nitrogen atoms. In certain embodiments, R.sup.1 is an optionally substituted 6-membered heteroaryl ring having 1 nitrogen. Example R.sup.1 groups include optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

(411) In certain embodiments, R.sup.1 is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, R.sup.1 is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R.sup.1 is an optionally substituted 5,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted indolyl. In some embodiments, R.sup.1 is an optionally substituted azabicyclo[3.2.1]octanyl. In certain embodiments, R.sup.1 is an optionally substituted 5,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted azaindolyl. In some embodiments, R.sup.1 is an optionally substituted benzimidazolyl. In some embodiments, R.sup.1 is an optionally substituted benzothiazolyl. In some embodiments, R.sup.1 is an optionally substituted benzoxazolyl. In some embodiments, R.sup.1 is an optionally substituted indazolyl. In certain embodiments, R.sup.1 is an optionally substituted 5,6-fused heteroaryl ring having 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

(412) In certain embodiments, R.sup.1 is an optionally substituted 6,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In other embodiments, R.sup.1 is an optionally substituted 6,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted quinolinyl. In some embodiments, R.sup.1 is an optionally substituted isoquinolinyl. According to one aspect, R.sup.1 is an optionally substituted 6,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is a quinazoline or a quinoxaline.

(413) In some embodiments, R.sup.1 is an optionally substituted heterocyclyl. In some embodiments, R.sup.1 is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

(414) In some embodiments, R.sup.1 is an optionally substituted heterocyclyl. In some embodiments, R.sup.1 is an optionally substituted 6 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted 6 membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted 6 membered partially unsaturated heterocyclic ring having 2 oxygen atoms.

(415) In certain embodiments, R.sup.1 is a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R is oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl, azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl. In some embodiments, R.sup.1 is an optionally substituted 5 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

(416) In certain embodiments, R.sup.1 is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, R.sup.1 is an optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

(417) In some embodiments, R.sup.1 is an optionally substituted 8-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 is an optionally substituted indolinyl. In some embodiments, R.sup.1 is an optionally substituted isoindolinyl. In some embodiments, R.sup.1 is an optionally substituted 1, 2, 3, 4-tetrahydroquinoline. In some embodiments, R.sup.1 is an optionally substituted 1, 2, 3, 4-tetrahydroisoquinoline.

(418) In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.10 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, wherein each variable is independently as defined above and described herein. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.10 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, OC(O), or C(O)O, wherein each R is independently as defined above and described herein. In some embodiments, R.sup.1 is an optionally substituted C.sub.1-C.sub.10 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally Cy, O, S, SS, N(R), C(O), OC(O), or C(O)O, wherein each R is independently as defined above and described herein.

(419) In some embodiments, R.sup.1 is

(420) ##STR00113## ##STR00114## ##STR00115## ##STR00116##

(421) In some embodiments, R.sup.1 is CH.sub.3,

(422) ##STR00117##

(423) In some embodiments, R.sup.1 comprises a terminal optionally substituted (CH.sub.2).sub.2 moiety which is connected to L. Examples of such R.sup.1 groups are depicted below:

(424) ##STR00118##

(425) In some embodiments, R.sup.1 comprises a terminal optionally substituted (CH.sub.2) moiety which is connected to L. Example such R.sup.1 groups are depicted below:

(426) ##STR00119##

(427) In some embodiments, R.sup.1 is SR.sup.L2, wherein R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, and each of R and Cy is independently as defined above and described herein. In some embodiments, R.sup.1 is SR.sup.L2, wherein the sulfur atom is connected with the sulfur atom in L group.

(428) In some embodiments, R.sup.1 is C(O)R.sup.L2, wherein R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, and each of R and Cy is independently as defined above and described herein. In some embodiments, R.sup.1 is C(O)R.sup.L2, wherein the carbonyl group is connected with G in L group. In some embodiments, R.sup.1 is C(O)R.sup.L2, wherein the carbonyl group is connected with the sulfur atom in L group.

(429) In some embodiments, R.sup.L2 is optionally substituted C.sub.1-C.sub.9 aliphatic. In some embodiments, R.sup.L2 is optionally substituted C.sub.1-C.sub.9 alkyl. In some embodiments, R.sup.L2 is optionally substituted C.sub.1-C.sub.9 alkenyl. In some embodiments, R.sup.L2 is optionally substituted C.sub.1-C.sub.9 alkynyl. In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by Cy or C(O). In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by Cy. In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted heterocycylene. In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted arylene. In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted heteroarylene. In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.3-C.sub.10 carbocyclylene. In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein two methylene units are optionally and independently replaced by Cy or C(O). In some embodiments, R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein two methylene units are optionally and independently replaced by Cy or C(O). Example R.sup.L2 groups are depicted below:

(430) ##STR00120##

(431) In some embodiments, R.sup.1 is hydrogen, or an optionally substituted group selected from

(432) ##STR00121##
S(C.sub.1-C.sub.10 aliphatic), C.sub.1-C.sub.10 aliphatic, aryl, C.sub.1-C.sub.6 heteroalkyl, heteroaryl and heterocyclyl. In some embodiments, R.sup.1 is

(433) ##STR00122##
or S(C.sub.1-C.sub.10 aliphatic). In some embodiments, R.sup.1 is

(434) ##STR00123##

(435) In some embodiments, R.sup.1 is an optionally substituted group selected from S(C.sub.1-C.sub.6 aliphatic), C.sub.1-C.sub.10 aliphatic, C.sub.1-C.sub.6 heteroaliphatic, aryl, heterocyclyl and heteroaryl. In some embodiments, R.sup.1 is

(436) ##STR00124##

(437) In some embodiments, the sulfur atom in the R.sup.1 embodiments described above and herein is connected with the sulfur atom, G, E, or C(O) moiety in the L embodiments described above and herein. In some embodiments, the C(O) moiety in the R.sup.1 embodiments described above and herein is connected with the sulfur atom, G, E, or C(O) moiety in the L embodiments described above and herein.

(438) In some embodiments, -L-R.sup.1 is any combination of the L embodiments and R.sup.1 embodiments described above and herein.

(439) In some embodiments, -L-R.sup.1 is -L.sup.3-GR.sup.1 wherein each variable is independently as defined above and described herein.

(440) In some embodiments, -L-R.sup.1 is -L.sup.4-GR.sup.1 wherein each variable is independently as defined above and described herein.

(441) In some embodiments, -L-R.sup.1 is -L.sup.3-GSR.sup.L2, wherein each variable is independently as defined above and described herein.

(442) In some embodiments, -L-R.sup.1 is -L.sup.3-GC(O)R.sup.L2, wherein each variable is independently as defined above and described herein.

(443) In some embodiments, -L-R.sup.1 is

(444) ##STR00125##
wherein R.sup.L2 is an optionally substituted C.sub.1-C.sub.9 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), or C(O)O, and each G is independently as defined above and described herein.

(445) In some embodiments, -L-R.sup.1 is R.sup.L3SSR.sup.L2, wherein each variable is independently as defined above and described herein. In some embodiments, -L-R.sup.1 is R.sup.L3C(O)SSR.sup.L2, wherein each variable is independently as defined above and described herein.

(446) In some embodiments, -L-R.sup.1 has the structure of:

(447) ##STR00126##
wherein each variable is independently as defined above and described herein.

(448) In some embodiments, -L-R.sup.1 has the structure of:

(449) ##STR00127##
wherein each variable is independently as defined above and described herein.

(450) In some embodiments, -L-R.sup.1 has the structure of:

(451) ##STR00128##
wherein each variable is independently as defined above and described herein.

(452) In some embodiments, -L-R.sup.1 has the structure of:

(453) ##STR00129##
wherein each variable is independently as defined above and described herein.

(454) In some embodiments, -L-R.sup.1 has the structure of:

(455) ##STR00130##
wherein each variable is independently as defined above and described herein.

(456) In some embodiments, -L-R.sup.1 has the structure of:

(457) ##STR00131##
wherein each variable is independently as defined above and described herein.

(458) In some embodiments, -L-R.sup.1 has the structure of:

(459) ##STR00132##
wherein each variable is independently as defined above and described herein.

(460) In some embodiments, -L-R.sup.1 has the structure of:

(461) ##STR00133##
wherein each variable is independently as defined above and described herein.

(462) In some embodiments, -L-R.sup.1 has the structure of:

(463) ##STR00134##
wherein each variable is independently as defined above and described herein.

(464) In some embodiments, -L-R.sup.1 has the structure of:

(465) ##STR00135##
wherein each variable is independently as defined above and described herein.

(466) In some embodiments, -L-R.sup.1 has the structure of:

(467) ##STR00136##
wherein each variable is independently as defined above and described herein.

(468) In some embodiments, -L-R.sup.1 has the structure of:

(469) ##STR00137##
wherein each variable is independently as defined above and described herein.

(470) In some embodiments, -L-R.sup.1 has the structure of:

(471) ##STR00138##
wherein each variable is independently as defined above and described herein.

(472) In some embodiments, -L-R.sup.1 has the structure of:

(473) ##STR00139##
wherein each variable is independently as defined above and described herein.

(474) In some embodiments, -L-R.sup.1 has the structure of:

(475) ##STR00140##
wherein each variable is independently as defined above and described herein.

(476) In some embodiments, -L-R.sup.1 has the structure of:

(477) ##STR00141##
wherein each variable is independently as defined above and described herein.

(478) In some embodiments, -L-R.sup.1 has the structure of:

(479) ##STR00142##
wherein each variable is independently as defined above and described herein.

(480) In some embodiments, -L-R.sup.1 has the structure of:

(481) ##STR00143##
wherein each variable is independently as defined above and described herein.

(482) In some embodiments, -L-R.sup.1 has the structure of:

(483) ##STR00144##
wherein each variable is independently as defined above and described herein.

(484) In some embodiments, -L-R.sup.1 has the structure of:

(485) ##STR00145##
wherein each variable is independently as defined above and described herein.

(486) In some embodiments, -L-R.sup.1 has the structure of:

(487) ##STR00146##
wherein each variable is independently as defined above and described herein.

(488) In some embodiments, L has the structure of:

(489) ##STR00147##
wherein each variable is independently as defined above and described herein.

(490) In some embodiments, X-L-R.sup.1 has the structure of:

(491) ##STR00148##
wherein: the phenyl ring is optionally substituted, and each of R.sup.1 and X is independently as defined above and described herein.

(492) In some embodiments, -L-R.sup.1 is

(493) ##STR00149## ##STR00150## ##STR00151## ##STR00152##

(494) In some embodiments, -L-R.sup.1 is:

(495) ##STR00153##

(496) In some embodiments, -L-R.sup.1 is CH.sub.3,

(497) ##STR00154##
In some embodiments, -L-R.sup.1 is

(498) ##STR00155##

(499) In some embodiments, -L-R.sup.1 comprises a terminal optionally substituted (CH.sub.2).sub.2 moiety which is connected to X. In some embodiments, -L-R.sup.1 comprises a terminal (CH.sub.2).sub.2 moiety which is connected to X. Examples of such-L-R.sup.1 moieties are depicted below:

(500) ##STR00156##

(501) In some embodiments, -L-R.sup.1 comprises a terminal optionally substituted (CH.sub.2) moiety which is connected to X. In some embodiments, -L-R.sup.1 comprises a terminal (CH.sub.2) moiety which is connected to X. Examples of such -L-R.sup.1 moieties are depicted below:

(502) ##STR00157##

(503) In some embodiments, -L-R.sup.1 is

(504) ##STR00158##

(505) In some embodiments, -L-R.sup.1 is CH.sub.3,

(506) ##STR00159##
and X is S.

(507) In some embodiments, -L-R.sup.1 is CH.sub.3,

(508) ##STR00160##
X is S, Wis O, Y is O, and Z is O.

(509) In some embodiments, R.sup.1 is

(510) ##STR00161##
or S(C.sub.1-C.sub.10 aliphatic).

(511) In some embodiments, R.sup.1 is

(512) ##STR00162##

(513) In some embodiments, X is O or S, and R.sup.1 is

(514) ##STR00163##
or S(C.sub.1-C.sub.10 aliphatic).

(515) In some embodiments, X is O or S, and R.sup.1 is

(516) ##STR00164##
S(C.sub.1-C.sub.10 aliphatic) or S(C.sub.1-C.sub.50 aliphatic).

(517) In some embodiments, L is a covalent bond and -L-R.sup.1 is R.sup.1.

(518) In some embodiments, -L-R.sup.1 is not hydrogen.

(519) In some embodiments, X-L-R.sup.1 is R.sup.1 is

(520) ##STR00165##
S(C.sub.1-C.sub.10 aliphatic) or S(C.sub.1-C.sub.50 aliphatic).

(521) In some embodiments, X-L-R.sup.1 has the structure of

(522) ##STR00166##
wherein the

(523) ##STR00167##
moiety is optionally substituted. In some embodiments, X-L-R.sup.1 is

(524) ##STR00168##

(525) In some embodiments, X-L-R.sup.1 is

(526) ##STR00169##

(527) In some embodiments, X-L-R.sup.1 is

(528) ##STR00170##

(529) In some embodiments, X-L-R.sup.1 has the structure of

(530) ##STR00171##
wherein X is O or S, Y is O, S or NR, and the

(531) ##STR00172##
moiety is optionally substituted. In some embodiments, Y is O, S or NH. In some embodiments,

(532) ##STR00173##
is

(533) ##STR00174##
In some embodiments,

(534) ##STR00175##
is

(535) ##STR00176##
In some embodiments,

(536) ##STR00177##
is

(537) ##STR00178##
In some embodiments, X-L-R.sup.1 has the structure of

(538) ##STR00179##
wherein X is O or S, and the

(539) ##STR00180##
moiety is optionally substituted. In some embodiments,

(540) ##STR00181##
is

(541) ##STR00182##
In some embodiments, X-L-R.sup.1 is

(542) ##STR00183##
wherein the

(543) ##STR00184##
is optionally substituted. In some embodiments, X-L-R.sup.1 is

(544) ##STR00185##
wherein the

(545) ##STR00186##
is substituted. In some embodiments, X-L-R.sup.1 is

(546) ##STR00187##
wherein the

(547) ##STR00188##
is unsubstituted.

(548) In some embodiments, X-L-R.sup.1 is R.sup.1C(O)S-L.sup.x-S, wherein L.sup.x is an optionally substituted group selected from

(549) ##STR00189##
In some embodiments, L.sup.x is

(550) ##STR00190##
In some embodiments, X-L-R.sup.1 is (CH.sub.3).sub.3CSS-L.sup.x-S. In some embodiments, X-L-R.sup.1 is R.sup.1C(X)YC(R).sub.2S-L.sup.x-S. In some embodiments, X-L-R.sup.1 is RC(X)YCH.sub.2S-L.sup.x-S. In some embodiments, X-L-R.sup.1 is

(551) ##STR00191##

(552) As will be appreciated by a person skilled in the art, many of the X-L-R.sup.1 groups described herein are cleavable and can be converted to X.sup. after administration to a subject. In some embodiments, X-L-R.sup.1 is cleavable. In some embodiments, X-L-R.sup.1 is S-L-R.sup.1, and is converted to S.sup. after administration to a subject. In some embodiments, the conversion is promoted by an enzyme of a subject. As appreciated by a person skilled in the art, methods of determining whether the S-L-R.sup.1 group is converted to S.sup. after administration is widely known and practiced in the art, including those used for studying drug metabolism and pharmacokinetics.

(553) In some embodiments, the internucleotidic linkage having the structure of formula I is

(554) ##STR00192##

(555) In some embodiments, the internucleotidic linkage of formula I has the structure of formula I-a:

(556) ##STR00193##
wherein each variable is independently as defined above and described herein.

(557) In some embodiments, the internucleotidic linkage of formula I has the structure of formula I-b:

(558) ##STR00194##
wherein each variable is independently as defined above and described herein.

(559) In some embodiments, the internucleotidic linkage of formula I is an phosphorothioate triester linkage having the structure of formula I-c:

(560) ##STR00195##
wherein R.sup.1 is not H when L is a covalent bond.

(561) In some embodiments, the internucleotidic linkage having the structure of formula I is

(562) ##STR00196## ##STR00197##

(563) In some embodiments, the internucleotidic linkage having the structure of formula I-c is

(564) ##STR00198## ##STR00199##

(565) In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising one or more natural phosphate linkages, and one or more modified internucleotidic linkages having the formula of I-a, I-b, or I-c.

(566) In some embodiments, a modified internucleotidic linkage has the structure of I. In some embodiments, a modified internucleotidic linkage has the structure of I-a. In some embodiments, a modified internucleotidic linkage has the structure of I-b. In some embodiments, a modified internucleotidic linkage has the structure of I-c.

(567) In some embodiments, a modified internucleotidic linkage is phosphorothioate internucleotidic linkage. Examples of internucleotidic linkages having the structure of formula I that can be utilized in accordance with the present disclosure include those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, US20130178612, US20150211006, U.S. Pat. No. 9,598,458, US20170037399, WO 2017/015555, WO 2017/062862, the internucleotidic linkages of each of which is incorporated herein by reference.

(568) Non-limiting examples of internucleotidic linkages that can be utilized in accordance with the present disclosure also include those described in the art, including, but not limited to, those described in any of: Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143, Jones et al. J. Org. Chem. 1993, 58, 2983, Koshkin et al. 1998 Tetrahedron 54: 3607-3630, Lauritsen et al. 2002 Chem. Comm. 5: 530-531, Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256, Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226, Petersen et al. 2003 TRENDS Biotech. 21: 74-81, Schultz et al. 1996 Nucleic Acids Res. 24: 2966, Tso et al. Ann. N. Y. Acad. Sci. 1988, 507, 220, and Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006.

(569) In some embodiments, oligonucleotides comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is not negatively charged in that at a given pH in an aqueous solution less than 50%, 40%, 40%, 30%, 20%, 10%, 5%, or 1% of the internucleotidic linkage exists in a negatively charged salt form. In some embodiments, a pH is about pH 7.4. In some embodiments, a pH is about 4-9. In some embodiments, the percentage is less than 10%. In some embodiments, the percentage is less than 5%. In some embodiments, the percentage is less than 1%. In some embodiments, an internucleotidic linkage is a non-negatively charged internucleotidic linkage in that the neutral form of the internucleotidic linkage has no pKa that is no more than about 1, 2, 3, 4, 5, 6, or 7 in water. In some embodiments, no pKa is 7 or less. In some embodiments, no pKa is 6 or less. In some embodiments, no pKa is 5 or less. In some embodiments, no pKa is 4 or less. In some embodiments, no pKa is 3 or less. In some embodiments, no pKa is 2 or less. In some embodiments, no pKa is 1 or less. In some embodiments, pKa of the neutral form of an internucleotidic linkage can be represented by pKa of the neutral form of a compound having the structure of CH.sub.3the internucleotidic linkageCH.sub.3. For example, pKa of the neutral form of an internucleotidic linkage having the structure of formula I may be represented by the pKa of the neutral form of a compound having the structure of

(570) ##STR00200##
pKa of

(571) ##STR00201##
can be represented by pKa

(572) ##STR00202##
In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage is a positively-charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage comprises a guanidine moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a heteroaryl base moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises a triazole moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises an alkynyl moiety.

(573) In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises P.sup.L (N), wherein P.sup.L is as described in the present disclosure. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises P(N). In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises P()(N). In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises P(O)(N). In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises P(S)(N).

(574) In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises

(575) ##STR00203##
wherein P.sup.L is as described in the present disclosure. For example, in some embodiments, P.sup.L is P; in some embodiments, P.sup.L is P(O); in some embodiments, P.sup.L is P(S); etc. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, comprises

(576) ##STR00204##

(577) In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof (not negatively charged). In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-1 or a salt form thereof:

(578) ##STR00205##

(579) In some embodiments, X is a covalent bond and XCyR.sup.1 is CyR.sup.1. In some embodiments, Cy is an optionally substituted bivalent group selected from a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms. In some embodiments, Cy is an optionally substituted bivalent 5-20 membered heteroaryl ring having 1-10 heteroatoms. In some embodiments, CyR.sup.1 is optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, CyR.sup.1 is optionally substituted 5-membered heteroaryl ring having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, CyR.sup.1 is optionally substituted 6-membered heteroaryl ring having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, CyR.sup.1 is optionally substituted triazolyl.

(580) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-2 or a salt form thereof:

(581) ##STR00206##

(582) In some embodiments, R.sup.1 is R. In some embodiments, L is a covalent bond. In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-3 or a salt form thereof:

(583) ##STR00207##

(584) In some embodiments, two R on different nitrogen atoms are taken together to form a ring as described. In some embodiments, a formed ring is 5-membered. In some embodiments, a formed ring is 6-membered. In some embodiments, a formed ring is substituted. In some embodiments, the two R group that are not taken together to form a ring are each independently R. In some embodiments, the two R group that are not taken together to form a ring are each independently hydrogen or an optionally substituted C.sub.1-6 aliphatic. In some embodiments, the two R group that are not taken together to form a ring are each independently hydrogen or an optionally substituted C.sub.1-6 alkyl. In some embodiments, the two R group that are not taken together to form a ring are the same. In some embodiments, the two R group that are not taken together to form a ring are different. In some embodiments, both of them are CH.sub.3.

(585) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula I-n-4 or a salt form thereof:

(586) ##STR00208##
wherein each of L.sup.a and L.sup.b is independently L or N(R.sup.1), and each other variable is independently as described in the present disclosure. In some embodiments, L is a covalent bond, and an internucleotidic linkage of formula I-n-4 has the structure of:

(587) ##STR00209##
or a salt form thereof, wherein each variable is independently as described in the present disclosure.

(588) In some embodiments, L.sup.a is N(R.sup.1). In some embodiments, L.sup.a is L as described in the present disclosure. In some embodiments, L.sup.a is a covalent bond. In some embodiments, L.sup.a is N(R). In some embodiments, L.sup.a is N(R). In some embodiments, L.sup.a is O. In some embodiments, L.sup.a is S. In some embodiments, L.sup.a is S(O). In some embodiments, L.sup.a is S(O).sub.2. In some embodiments, L.sup.a is S(O).sub.2N(R). In some embodiments, L.sup.b is N(R.sup.1). In some embodiments, L.sup.b is L as described in the present disclosure. In some embodiments, L.sup.b is a covalent bond. In some embodiments, L.sup.b is N(R). In some embodiments, L.sup.b is N(R). In some embodiments, L.sup.b is O. In some embodiments, L.sup.b is S. In some embodiments, L.sup.b is S(O). In some embodiments, L.sup.b is S(O).sub.2. In some embodiments, L.sup.b is S(O).sub.2N(R). In some embodiments, L.sup.a and L.sup.b are the same. In some embodiments, L.sup.a and L.sup.b are different. In some embodiments, at least one of L.sup.a and L.sup.b is N(R.sup.1). In some embodiments, at least one of L.sup.a and L.sup.b is O. In some embodiments, at least one of L.sup.a and L.sup.b is S. In some embodiments, at least one of L.sup.a and L.sup.b is a covalent bond. In some embodiments, as described herein, R.sup.1 is R. In some embodiments, R.sup.1 is H. In some embodiments, R.sup.1 is optionally substituted C.sub.1-10 aliphatic. In some embodiments, R.sup.1 is optionally substituted C.sub.1-10 alkyl. In some embodiments, a structure of formula I-n-4 is a structure of formula I-n-2. In some embodiments, a structure of formula I-n-4 is a structure of formula I-n-3. In some embodiments, a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, has the structure of formula I. In some embodiments, X, e.g., in formula I, II, etc., is N(-L-R.sup.5), wherein R.sup.5 is R as described herein. In some embodiments, X is NH. In some embodiments, L, e.g., in X-L- of formula I, II, etc., comprises SO.sub.2. In some embodiments, L is SO.sub.2. In some embodiments, L is a covalent bond. In some embodiments, L is C(O)O(C.sub.1-4 alkylene)- wherein the alkylene is optionally substituted. In some embodiments, L is C(O) OCH.sub.2. In some embodiments, R.sup.1, e.g., in formula I, III, etc., comprise an optionally substituted ring. In some embodiments, R.sup.1 is R as described herein. In some embodiments, R.sup.1 is optionally substituted phenyl. In some embodiments, R.sup.1 is 4-methylphenyl. In some embodiments, R.sup.1 is 4-methoxyphenyl. In some embodiments, R.sup.1 is 4-aminophenyl. In some embodiments, R.sup.1 is an optionally substituted heteroaliphatic ring. In some embodiments, R.sup.1 is an optionally substituted 3-10 (e.g., 3, 4, 5, 6, 7, or 8) membered heteroaliphatic ring. In some embodiments, R.sup.1 is an optionally substituted 5- or 6-membered saturated monocyclic heteroaliphatic ring having 1-3 heteroatoms. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the number of ring heteroatom(s) is 1. In some embodiments, the number of ring heteroatoms is 2. In some embodiments, a heteroatom is oxygen. In some embodiments, R.sup.1 is optionally substituted

(589) ##STR00210##
In some embodiments, R.sup.1 is optionally substituted

(590) ##STR00211##
In some embodiments, R.sup.1 is

(591) ##STR00212##
In some embodiments, R.sup.1 is optionally substituted C.sub.1-30 aliphatic. In some embodiments, R.sup.1 is optionally substituted C.sub.1-10 alkyl.

(592) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, has the structure of formula II or a salt form thereof:

(593) ##STR00213##
or a salt form thereof, wherein: P.sup.L is P(W), P, or P.fwdarw.B(R).sub.3; W is O, N(-L-R.sup.5), S or Se;
each of X, Y and Z is independently O, S, N(-L-R.sup.5), or L; R.sup.5 is H, -L-R, halogen, CN, NO.sub.2, -L-Si(R).sub.3, OR, SR, or N(R).sub.2; Ring A.sup.L is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R.sup.s is independently H, halogen, CN, N.sub.3, NO, NO.sub.2, -L-R, -L-Si(R).sub.3, -L-OR, -L-SR, -L-N(R).sub.2, O-L-R, O-L-Si(R).sub.3, O-L-OR, O-L-SR, or O-L-N(R).sub.2; g is 0-20; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

(594) In some embodiments, Ring A.sup.L in various structures of the present disclosure is an optionally substituted aryl ring. In some embodiments, Ring A.sup.L is an optionally substituted phenyl ring. In some embodiments, Ring A.sup.L is an optionally substituted 3-10 (e.g., 3, 4, 5, 6, 7, or 8) membered heteroaliphatic ring. In some embodiments, Ring A.sup.L is an optionally substituted 5- or 6-membered saturated monocyclic heteroaliphatic ring having 1-3 heteroatoms. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the number of ring heteroatom(s) is 1. In some embodiments, the number of ring heteroatoms is 2. In some embodiments, a heteroatom is oxygen. In some embodiments, R.sup.s is optionally substituted C.sub.1-C.sub.6 alkyl group. In some embodiments, R.sup.s is Me. In some embodiments, R.sup.s is OR, wherein R is hydrogen or C.sub.1-C.sub.6 alkyl group. In some embodiments, R.sup.s is OH. In some embodiments, R.sup.s is OMe. In some embodiments, R.sup.s is N(R).sub.2. In some embodiments, R.sup.s is NH.sub.2. In some embodiments,

(595) ##STR00214##
is

(596) ##STR00215##

(597) In some embodiments,

(598) ##STR00216##
is

(599) ##STR00217##
In some embodiments,

(600) ##STR00218##

(601) is

(602) ##STR00219##
In some embodiments,

(603) ##STR00220##
is

(604) ##STR00221##
In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n002

(605) ##STR00222##
which, as one skilled in the art will appreciate, can exist under certain conditions in the form of

(606) ##STR00223##
In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n005 (

(607) ##STR00224##
which, as one skilled in the art will appreciate, can exist under certain conditions in the form of

(608) ##STR00225##
In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n006

(609) ##STR00226##
which, as one skilled in the art will appreciate, can exist under certain conditions in the form of

(610) ##STR00227##
In some embodiments, an internucleotidic linkage, e.g. a neutral internucleotidic linkage of formula I or II, is n007

(611) ##STR00228##
which, as one skilled in the art will appreciate, can exist under certain conditions in a form of

(612) ##STR00229##

(613) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, has the structure of formula II-a-1 or a salt form thereof:

(614) ##STR00230##
or a salt form thereof.

(615) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, has the structure of formula II-a-2 or a salt form thereof:

(616) ##STR00231##
or a salt form thereof.

(617) In some embodiments, A.sup.L is bonded to N or L through a carbon atom. In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II or II-a-1, II-a-2, has the structure of formula II-b-1 or a salt form thereof:

(618) ##STR00232##

(619) In some embodiments, a structure of formula II-a-1 or II-a-2 may be referred to a structure of formula II-a. In some embodiments, a structure of formula II-b-1 or II-b-2 may be referred to a structure of formula II-b. In some embodiments, a structure of formula II-c-1 or II-c-2 may be referred to a structure of formula II-c. In some embodiments, a structure of formula II-d-1 or II-d-2 may be referred to a structure of formula II-d.

(620) In some embodiments, A.sup.L is bonded to N or L through a carbon atom. In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II or II-a-1, II-a-2, has the structure of formula II-b-2 or a salt form thereof:

(621) ##STR00233##

(622) In some embodiments, Ring A.sup.L is an optionally substituted 3-20 membered monocyclic ring having 0-10 heteroatoms (in addition to the two nitrogen atoms for formula II-b). In some embodiments, Ring A.sup.L is an optionally substituted 5-membered monocyclic saturated ring.

(623) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, II-a, or II-b, has the structure of formula II-c-1 or a salt form thereof:

(624) ##STR00234##

(625) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, II-a, or II-b, has the structure of formula II-c-2 or a salt form thereof:

(626) ##STR00235##

(627) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, II-a, II-b, or II-c has the structure of formula II-d-1 or a salt form thereof:

(628) ##STR00236##

(629) In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage of formula II, II-a, II-b, or II-c has the structure of formula II-d-2 or a salt form thereof:

(630) ##STR00237##

(631) In some embodiments, each R is independently optionally substituted C.sub.1-6 aliphatic. In some embodiments, each R is independently optionally substituted C.sub.1-6 alkyl. In some embodiments, each R is independently CH.sub.3. In some embodiments, each R.sup.s is H.

(632) In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(633) ##STR00238##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(634) ##STR00239##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(635) ##STR00240##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(636) ##STR00241##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(637) ##STR00242##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(638) ##STR00243##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(639) ##STR00244##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(640) ##STR00245##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(641) ##STR00246##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(642) ##STR00247##

(643) In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(644) ##STR00248##

(645) In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(646) ##STR00249##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(647) ##STR00250##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(648) ##STR00251##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(649) ##STR00252##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(650) ##STR00253##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(651) ##STR00254##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(652) ##STR00255##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(653) ##STR00256##
embodiments, a non-negatively charged internucleotidic linkage has the structure of

(654) ##STR00257##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(655) ##STR00258##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(656) ##STR00259##

(657) In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(658) ##STR00260##

(659) In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(660) ##STR00261##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(661) ##STR00262##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(662) ##STR00263##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(663) ##STR00264##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(664) ##STR00265##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(665) ##STR00266##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(666) ##STR00267##

(667) In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(668) ##STR00268##
In some embodiments, W is O. In some embodiments, W is S. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled. In some embodiments, the linkage phosphorus is Rp. In some embodiments, the linkage phosphorus is Sp.

(669) In some embodiments, each non-negatively charged internucleotidic linkage or neutral internucleotidic linkage (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2) is independently Rp at its linkage phosphorus. In some embodiments, each negatively charged chiral internucleotidic linkage is Sp at its linkage phosphorus. In some embodiments, each phosphorothioate internucleotidic linkages is Sp at its linkage phosphorus. In some embodiments, each natural phosphate linkage is independently bonded to a sugar comprising a 2-OR modification, wherein R is not H. In some embodiments, each natural phosphate linkage is independently bonded to a sugar comprising a 2-OR modification, wherein R is not H, at a 3-position. In some embodiments, each sugar that contains no 2-OR modification wherein R is not H is independently bonded to at least one non-natural phosphate linkages, in many cases, two non-natural natural phosphate linkages. In some embodiments, each 2-F modified sugar is independently bonded to at least one non-natural phosphate linkages, in many cases, two non-natural natural phosphate linkages. In some embodiments, each non-natural phosphate linkage is a phosphorothioate internucleotidic linkage. In some embodiments, each non-natural phosphate linkage is a Sp phosphorothioate internucleotidic linkage. In some embodiments, each sugar bonded to non-negatively charged internucleotidic linkage or neutral internucleotidic linkage (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2) independently contains no 2-OR. In some embodiments, each sugar bonded to non-negatively charged internucleotidic linkage or neutral internucleotidic linkage (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2) is a 2-F modified sugar.

(670) In some embodiments, the present disclosure provides a compound, e.g., an oligonucleotide, a chirally controlled oligonucleotide, an oligonucleotide of a provided composition (e.g., of a plurality of oligonucleotides), having the structure of formula O-I:

(671) ##STR00269##
or a salt thereof, wherein: R.sup.5s is independently R or OR; each BA is independently an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.5-30 heteroaryl having 1-10 heteroatoms, C.sub.3-30 heterocyclyl having 1-10 heteroatoms, a natural nucleobase moiety, and a modified nucleobase moiety; each R.sup.s is independently H, halogen, CN, N.sub.3, NO, NO.sub.2, -L-R, -L-Si(R).sub.3, -L-OR, -L-SR, -L-N(R).sub.2, O-L-R, O-L-Si(R).sub.3, O-L-OR, O-L-SR, or O-L-N(R).sub.2; each s is independently 0-20; each L.sup.s is independently C(R.sup.5s).sub.2, or L; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; each L.sup.P is independently an internucleotidic linkage; z is 1-1000; L.sup.3E is L or -L-L-; R.sup.3E is R, -L-R, OR, or a solid support; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.

(672) In some embodiments, each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, each internucleotidic linkage independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each internucleotidic linkage independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, an internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, each internucleotidic linkage independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

(673) In some embodiments, each BA is independently an optionally substituted group selected from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon; each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

(674) In some embodiments, each BA is independently an optionally substituted C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein the heteroaryl comprises one or more heteroatoms selected from oxygen and nitrogen; each Ring A is independently an optionally substituted 5-10 membered monocyclic or bicyclic saturated ring having 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein the ring comprises at least one oxygen atom; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

(675) In some embodiments, each BA is independently an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U; each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

(676) In some embodiments, each BA is independently an optionally substituted or protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil and tautomers thereof; each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

(677) In some embodiments, BA is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is optionally substituted C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, BA is optionally substituted natural nucleobases and tautomers thereof. In some embodiments, BA is protected natural nucleobases and tautomers thereof. Various nucleobase protecting groups for oligonucleotide synthesis are known and can be utilized in accordance with the present disclosure. In some embodiments, BA is an optionally substituted nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof. In some embodiments, BA is an optionally protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof.

(678) In some embodiments, BA is optionally substituted C.sub.3-30 cycloaliphatic. In some embodiments, BA is optionally substituted C.sub.6-30 aryl. In some embodiments, BA is optionally substituted C.sub.3-30 heterocyclyl. In some embodiments, BA is optionally substituted C.sub.5-30 heteroaryl. In some embodiments, BA is an optionally substituted natural base moiety. In some embodiments, BA is an optionally substituted modified base moiety. BA is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.3-30 heterocyclyl, and C.sub.5-30 heteroaryl. In some embodiments, BA is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.3-30 heterocyclyl, C.sub.5-30 heteroaryl, and a natural nucleobase moiety.

(679) In some embodiments, BA is connected through an aromatic ring. In some embodiments, BA is connected through a heteroatom. In some embodiments, BA is connected through a ring heteroatom of an aromatic ring. In some embodiments, BA is connected through a ring nitrogen atom of an aromatic ring.

(680) In some embodiments, BA is a natural nucleobase moiety. In some embodiments, BA is an optionally substituted natural nucleobase moiety. In some embodiments, BA is a substituted natural nucleobase moiety. In some embodiments, BA is optionally substituted, or an optionally substituted tautomer of, A, T, C, U, or G. In some embodiments, BA is natural nucleobase A, T, C, U, or G. In some embodiments, BA is an optionally substituted group selected from natural nucleobases A, T, C, U, and G.

(681) In some embodiments, BA is an optionally substituted purine base residue. In some embodiments, BA is a protected purine base residue. In some embodiments, BA is an optionally substituted adenine residue. In some embodiments, BA is a protected adenine residue. In some embodiments, BA is an optionally substituted guanine residue. In some embodiments, BA is a protected guanine residue. In some embodiments, BA is an optionally substituted cytosine residue. In some embodiments, BA is a protected cytosine residue. In some embodiments, BA is an optionally substituted thymine residue. In some embodiments, BA is a protected thymine residue. In some embodiments, BA is an optionally substituted uracil residue. In some embodiments, BA is a protected uracil residue. In some embodiments, BA is an optionally substituted 5-methylcytosine residue. In some embodiments, BA is a protected 5-methylcytosine residue.

(682) In some embodiments, BA is a protected base residue as used in oligonucleotide preparation. In some embodiments, BA is a base residue illustrated in US 2011/0294124, US 2015/0211006, US 2015/0197540, and WO 2015/107425, each of which is incorporated herein by reference.

(683) In some embodiments, R.sup.5s-L.sup.s- is CH.sub.2OH. In some embodiments, R.sup.5s-L.sup.s- is CH(R.sup.5s)OH, wherein R.sup.5s is as described in the present disclosure. In some embodiments, L.sup.s is CH.sub.2. In some embodiments, L is CH(R.sup.5s) wherein R.sup.5s is not H. In some embodiments, L.sup.s is CH(R.sup.5s) wherein R.sup.5s is not H and is otherwise R. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is methyl. In some embodiments, CH(R.sup.5s) wherein R.sup.5s is not H has is R. In some embodiments, CH(R.sup.5s) wherein R.sup.5s is not H has is S.

(684) Example embodiments for variables, e.g., variables of each of the formulae, are additionally described in the present disclosure, and may be independently and optionally combined.

(685) In some embodiments, the present disclosure provides oligonucleotides and oligonucleotide compositions that are chirally controlled. For instance, in some embodiments, a provided composition contains controlled levels of one or more individual oligonucleotide types, wherein an oligonucleotide type is defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone P-modifications. In some embodiments, oligonucleotides of the same oligonucleotide type are identical.

(686) In some embodiments, a provided oligonucleotide is an altmer. In some embodiments, a provided oligonucleotide is a P-modification altmer. In some embodiments, a provided oligonucleotide is a stereoaltmer.

(687) In some embodiments, a provided oligonucleotide is a blockmer. In some embodiments, a provided oligonucleotide is a P-modification blockmer. In some embodiments, a provided oligonucleotide is a stereoblockmer.

(688) In some embodiments, a provided oligonucleotide is a gapmer.

(689) In some embodiments, a provided oligonucleotide is a skipmer.

(690) In some embodiments, a provided oligonucleotide is a hemimer. In some embodiments, a hemimer is an oligonucleotide wherein the 5-end or the 3-end has a sequence that possesses a structure feature that the rest of the oligonucleotide does not have. In some embodiments, the 5-end or the 3-end has or comprises 2 to 20 nucleotides. In some embodiments, a structural feature is a base modification. In some embodiments, a structural feature is a sugar modification. In some embodiments, a structural feature is a P-modification. In some embodiments, a structural feature is stereochemistry of the chiral internucleotidic linkage. In some embodiments, a structural feature is or comprises a base modification, a sugar modification, a P-modification, or stereochemistry of the chiral internucleotidic linkage, or combinations thereof. In some embodiments, a hemimer is an oligonucleotide in which each sugar moiety of the 5-end sequence shares a common modification. In some embodiments, a hemimer is an oligonucleotide in which each sugar moiety of the 3-end sequence shares a common modification. In some embodiments, a common sugar modification of the 5 or 3 end sequence is not shared by any other sugar moieties in the oligonucleotide. In some embodiments, an example hemimer is an oligonucleotide comprising a sequence of substituted or unsubstituted 2-O-alkyl sugar modified nucleosides, bicyclic sugar modified nucleosides, -D-ribonucleosides or -D-deoxyribonucleosides (for example 2-MOE modified nucleosides, and LNA or ENA bicyclic sugar modified nucleosides) at one terminus and a sequence of nucleosides with a different sugar moiety (such as a substituted or unsubstituted 2-O-alkyl sugar modified nucleosides, bicyclic sugar modified nucleosides or natural ones) at the other terminus. In some embodiments, a provided oligonucleotide is a combination of one or more of unimer, altmer, blockmer, gapmer, hemimer and skipmer. In some embodiments, a provided oligonucleotide is a combination of one or more of unimer, altmer, blockmer, gapmer, and skipmer. For instance, in some embodiments, a provided oligonucleotide is both an altmer and a gapmer. In some embodiments, a provided nucleotide is both a gapmer and a skipmer. One of skill in the chemical and synthetic arts will recognize that numerous other combinations of patterns are available and are limited only by the commercial availability and/or synthetic accessibility of constituent parts required to synthesize a provided oligonucleotide in accordance with methods of the present disclosure. In some embodiments, a hemimer structure provides advantageous benefits. In some embodiments, provided oligonucleotides are 5-hemimers that comprises modified sugar moieties in a 5-end sequence. In some embodiments, provided oligonucleotides are 5-hemimers that comprises modified 2-sugar moieties in a 5-end sequence.

(691) In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleotides. In some embodiments, a provided oligonucleotide comprises one or more modified nucleotides. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleosides. In some embodiments, a provided oligonucleotide comprises one or more modified nucleosides. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleosides or sugars of LNAs.

(692) In some embodiments, a provided oligonucleotide comprises one or more optionally substituted nucleobases. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted natural nucleobases. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted modified nucleobases. In some embodiments, a provided oligonucleotide comprises one or more 5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxylcytosine. In some embodiments, a provided oligonucleotide comprises one or more 5-methylcytidine.

(693) In some embodiments, a provided oligonucleotide comprises one or more optionally substituted sugars. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted sugars found in naturally occurring DNA and RNA. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted ribose or deoxyribose. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted ribose or deoxyribose, wherein one or more hydroxyl groups of the ribose or deoxyribose moiety is optionally and independently replaced by halogen, R, N(R).sub.2, OR, or SR, wherein each R is independently as defined above and described herein. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with R.sup.2s, halogen, R, N(R).sub.2, OR, or SR, wherein each R is independently as defined above and described herein. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with halogen. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with one or more F. halogen. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with OR, wherein each R is independently as defined above and described herein. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with OR, wherein each R is independently an optionally substituted C.sub.1-C.sub.6 aliphatic. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with OR, wherein each R is independently an optionally substituted C.sub.1-C.sub.6 alkyl. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with OMe. In some embodiments, a provided oligonucleotide comprises one or more optionally substituted deoxyribose, wherein the 2 position of the deoxyribose is optionally and independently substituted with O-methoxyethyl.

(694) In some embodiments, a provided oligonucleotide is single-stranded oligonucleotide. In some embodiments, a provided oligonucleotide is a hybridized oligonucleotide strand. In certain embodiments, a provided oligonucleotide is a partially hybridized oligonucleotide strand. In certain embodiments, a provided oligonucleotide is a completely hybridized oligonucleotide strand. In certain embodiments, a provided oligonucleotide is a double-stranded oligonucleotide. In certain embodiments, a provided oligonucleotide is a triple-stranded oligonucleotide (e.g., a triplex).

(695) In some embodiments, a provided oligonucleotide is chimeric. For example, in some embodiments, a provided oligonucleotide is DNA-RNA chimera, DNA-LNA chimera, etc.

(696) In some embodiments, an oligonucleotide is a chirally controlled oligonucleotide variant of an oligonucleotide described in WO2012/030683. For example, in some embodiments, a chirally controlled oligonucleotide variant comprises a chirally controlled version of a chiral internucleotidic linkage which is not chirally controlled in WO2012/030683. In some embodiments, a chirally controlled oligonucleotide variant comprises one or more chirally controlled internucleotidic linkages which independently replace one or more natural phosphate linkages or non-chirally controlled modified internucleotidic linkages in WO2012/030683.

(697) In some embodiments, a provided oligonucleotide is or comprises a portion of GNA, LNA, PNA, TNA or Morpholino.

(698) In some embodiments, a provided oligonucleotide is from about 15 to about 25 nucleotide units in length. In some embodiments, a provided oligonucleotide is from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide units in length.

(699) In some embodiments, the present disclosure provides oligonucleotides comprising one or more modified internucleotidic linkage, which can be chiral at linkage phosphorus and chirally controlled. In some embodiments, an oligonucleotide comprises one or more linkages L.sup.PO, L.sup.PA or L.sup.PB, wherein: each L.sup.PO is independently

(700) ##STR00270##
or a salt form thereof; each L.sup.PA is independently an internucleotidic linkage having the structure of

(701) ##STR00271##
or a salt form thereof; each L.sup.PB is independently an internucleotidic linkage having the structure of

(702) ##STR00272##
or a salt form thereof;

(703) ##STR00273##
and W.sup.N is N-L-R.sup.5,

(704) ##STR00274##
wherein each other variable is independently as described herein.

(705) In some embodiments, each L.sup.PO is independently

(706) ##STR00275##
or a salt form thereof.

(707) In some embodiments, O-L-R.sup.1 is OH. In some embodiments, X-L-R.sup.1, e.g., in L.sup.PO is OCH.sub.2CH.sub.2CN. In some embodiments, S-L-R.sup.1 is SH. In some embodiments, L.sup.PA is a phosphorothioate internucleotidic linkage with the specified stereochemistry. In some embodiments, L.sup.PB is a phosphorothioate internucleotidic linkage with the specified stereochemistry. In some embodiments, X is O, and X-L-R.sup.1 is as described in the present disclosure, e.g., X-L-R.sup.1 is

(708) ##STR00276##
wherein each variable is independently in accordance with the present disclosure, or HX-L-R.sup.1 is a chiral auxiliary as described herein. In some embodiments, X-L-R.sup.1 is

(709) ##STR00277##
wherein G.sup.4 and G.sup.5 are taken together to form an optionally substituted ring as described herein. In some embodiments, X-L-R.sup.1 is

(710) ##STR00278##
In some embodiments, G.sup.2 is CH.sub.2Si(R).sub.3 as described herein. In some embodiments, G.sup.2 is CH.sub.2Si(Ph).sub.2Me. In some embodiments, G.sup.2 comprises an electron-withdrawing group as described herein, for example, in some embodiments, G.sup.2 is CH.sub.2SO.sub.2R as described herein. In some embodiments, G.sup.2 is CH.sub.2SO.sub.2Ph.

(711) In some embodiments, N.sup.x is N(-L-R.sup.5)-L-R.sup.1, and an internucleotidic linkage having such a N.sup.x group is an internucleotidic linkage having the structure of formula I wherein P.sup.L is PO, Y and Z are O, and X is N(-L-R.sup.5), wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N.sup.x is

(712) ##STR00279##
and an internucleotidic linkage having such a N.sup.x group is an internucleotidic linkage having the structure of formula II, wherein P.sup.L is PO, Y and Z are O, and X is N(-L-R.sup.5), wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N.sup.x is

(713) ##STR00280##
In some embodiments, N.sup.x is

(714) ##STR00281##
In some embodiments, N.sup.x is

(715) ##STR00282##
In some embodiments, N.sup.x is.

(716) ##STR00283##
In some embodiments, N.sup.x is

(717) ##STR00284##
and an internucleotidic linkage having such a N.sup.x group is an internucleotidic linkage having the structure of formula I-n-3, wherein P.sup.L is PO, and Y and Z are O, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, R.sup.1 is optionally substituted alkyl. In some embodiments, R.sup.1 is methyl. In some embodiments, N.sup.x is

(718) ##STR00285##
In some embodiments, two R.sup.1 on the same nitrogen independently are taken together to form an optionally substituted ring as described herein, e.g., an optionally substituted 5- or 6-membered ring which in addition to the nitrogen atom, has 1-3 heteroatoms. In some embodiments, the ring is saturated. In some embodiments, the ring is monocyclic. In some embodiments, N.sup.x is

(719) ##STR00286##
In some embodiments, N.sup.x is

(720) ##STR00287##
In some embodiments, N.sup.x is

(721) ##STR00288##
Those skilled in the art will appreciate that two N(R.sup.1).sub.2 groups, in any, in a structure or formula can either be the same or different. In some embodiments, N.sup.x is

(722) ##STR00289##
and an internucleotidic linkage having such a N.sup.x group is an internucleotidic linkage having the structure of formula I-n-4, wherein P.sup.L is PO, L is a covalent bond, and Y and Z are O, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N.sup.x is

(723) ##STR00290##
and an internucleotidic linkage having such a N.sup.x group is an internucleotidic linkage having the structure of formula II-a-1, wherein P.sup.L is PO, L is a covalent bond, and Y and Z are O, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N.sup.x is

(724) ##STR00291##
and an internucleotidic linkage having such a N.sup.x group is an internucleotidic linkage having the structure of formula II-b-1, wherein P.sup.L is PO, L is a covalent bond, and Y and Z are O, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N.sup.x is

(725) ##STR00292##
and an internucleotidic linkage having such a N group is an internucleotidic linkage having the structure of formula II-c-1, wherein P.sup.L is PO, L is a covalent bond, and Y and Z are O, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, N.sup.x is

(726) ##STR00293##
and an internucleotidic linkage having such a N.sup.x group is an internucleotidic linkage having the structure of formula II-d-1, wherein P.sup.L is PO, L is a covalent bond, and Y and Z are O, wherein the linkage phosphorus stereochemistry is as specified. In some embodiments, R or R.sup.s is optionally substituted alkyl. In some embodiments, R or R.sup.s is CH.sub.3. In some embodiments, R or R.sup.s is CH.sub.2(CH.sub.2).sub.10CH.sub.3. In some embodiments, R.sup.s is H. In some embodiments, N.sup.x is

(727) ##STR00294##
In some embodiments, N.sup.x is

(728) ##STR00295##

(729) In some embodiments, PW.sup.N is a P.sup.N group as described herein. In some embodiments, W.sup.N is

(730) ##STR00296##
wherein each variable is as described herein (for example, in N.sup.x). In some embodiments, W.sup.N is

(731) ##STR00297##
In some embodiments, as described herein R or R.sup.s is optionally substituted alkyl or H. In some embodiments, R is CH.sub.3. In some embodiments, R is CH.sub.2(CH.sub.2).sub.10CH.sub.3. In some embodiments, R.sup.s is H. In some embodiments, W.sup.N is

(732) ##STR00298##
In some embodiments, W.sup.N is

(733) ##STR00299##
In some embodiments, W.sup.N is N-L-R.sup.5 wherein each variable is as described herein. For example, in some embodiments, L is SO.sub.2. In some embodiments, L is C(O)OCH.sub.2. In some embodiments, as described herein, R.sup.5 is or comprise an optionally substituted ring. In some embodiments, R.sup.5 is R as described herein. In some embodiments, R.sup.5 is optionally substituted phenyl. In some embodiments, R.sup.5 is 4-methylphenyl. In some embodiments, R.sup.5 is 4-methoxyphenyl. In some embodiments, R.sup.5 is 4-aminophenyl. In some embodiments, R.sup.5 is an optionally substituted heteroaliphatic ring. In some embodiments, R.sup.5 is an optionally substituted 3-10 (e.g., 3, 4, 5, 6, 7, or 8) membered heteroaliphatic ring. In some embodiments, R.sup.5 is an optionally substituted 5- or 6-membered saturated monocyclic heteroaliphatic ring having 1-3 heteroatoms. In some embodiments, the ring is 5-membered. In some embodiments, the ring is 6-membered. In some embodiments, the number of ring heteroatom(s) is 1. In some embodiments, the number of ring heteroatoms is 2. In some embodiments, a heteroatom is oxygen. In some embodiments, R.sup.5 is optionally substituted

(734) ##STR00300##
In some embodiments, R.sup.5 is optionally substituted

(735) ##STR00301##
In some embodiments, R.sup.5 is

(736) ##STR00302##
In some embodiments, R.sup.5 is optionally substituted C.sub.1-30 aliphatic. In some embodiments, R.sup.5 is optionally substituted C.sub.1-10 alkyl. In some embodiments, W.sup.N is

(737) ##STR00303##
In some embodiments, W.sup.N is

(738) ##STR00304##
In some embodiments, W.sup.N is

(739) ##STR00305##
In some embodiments, W.sup.N is

(740) ##STR00306##
In some embodiments, W.sup.N is

(741) ##STR00307##
In some embodiments, W.sup.N is

(742) ##STR00308##
In In some embodiments, W.sup.N is

(743) ##STR00309##
In some embodiments, W.sup.N is

(744) ##STR00310##
In some embodiments, W.sup.N is

(745) ##STR00311##
Q.sup.. In some embodiments, W.sup.N is

(746) ##STR00312##
In some embodiments, Q.sup. is PF.sub.6.sup..

(747) In some embodiments, X-L-R.sup.1 in

(748) ##STR00313##
is

(749) ##STR00314##
In some embodiments, X-L-R.sup.1 in

(750) ##STR00315##
is

(751) ##STR00316##
In some embodiments, G.sup.2 is CH.sub.2Si(R).sub.3 as described herein. In some embodiments, G.sup.2 is CH.sub.2Si(Ph).sub.2Me. In some embodiments, X-L-R.sup.1 in

(752) ##STR00317##
is

(753) ##STR00318##
In some embodiments, X-L-R.sup.1 in

(754) ##STR00319##
is

(755) ##STR00320##
In some embodiments, G.sup.2 comprises an electron-withdrawing group as described herein. In some embodiments, G.sup.2 is CH.sub.2SO.sub.2R, wherein R is not H. In some embodiments, R is optionally substituted phenyl. In some embodiments, G.sup.2 is CH.sub.2SO.sub.2Ph. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic, e.g., t-butyl. In some embodiments, as described herein, R.sup.1 is C(O)R. In some embodiments, R.sup.1 is C(O)CH.sub.3. In some embodiments, R.sup.1 is H.

(756) In some embodiments, L.sup.PO is a natural phosphate linkage. In some embodiments, L.sup.PA is a Rp phosphorothioate internucleotidic linkage. In some embodiments, L.sup.PA is a Rp non-negatively charged internucleotidic linkage, e.g., n001. In some embodiments, L.sup.PB is a Sp phosphorothioate internucleotidic linkage. In some embodiments, L.sup.PB is a Sp non-negatively charged internucleotidic linkage, e.g., n001. In some embodiments, an oligonucleotide comprises one or more linkages L.sup.PO. In some embodiments, an oligonucleotide comprises one or more linkages L.sup.PA. In some embodiments, an oligonucleotide comprises one or more linkages L.sup.PB. In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages independently selected from L.sup.PO, L.sup.PA and L.sup.PB. In some embodiments, each internucleotidic linkage is independently selected from L.sup.PO, L.sup.PA and L.sup.PB. In some embodiments, each internucleotidic linkage is independently selected from L.sup.PA and L.sup.PB. In some embodiments, at least one internucleotidic linkage is L.sup.PA or L.sup.PB. In some embodiments, each chirally controlled internucleotidic linkage is independently selected from L.sup.PA and L.sup.PB

(757) In some embodiments, the present disclosure provides oligonucleotides (e.g., chirally controlled oligonucleotides) and compositions thereof (e.g., chirally controlled oligonucleotide compositions), wherein the internucleotidic linkages of the oligonucleotides or regions thereof are or comprise the following consecutive internucleotidic linkages (from 5 to 3): (L.sup.PX/L.sup.PO)t[(L.sup.PA)n(L.sup.PB)m]y, (L.sup.PX/L.sup.PO)t[(L.sup.PO)n(L.sup.PB)m]y, [(L.sup.PA)n(L.sup.PB)m]y, [(L.sup.PO)n(L.sup.PB)m]y, ((L.sup.PB)t[(L.sup.PA)n(L.sup.PB) m]y, (L.sup.PB)t[(L.sup.PO)n(L.sup.PB)m]y, (L.sup.PB)t[(L.sup.PO/_PA)n(L.sup.PB)m]y, [(L.sup.PA)n(L.sup.PB)m]y, [(L.sup.PO)n(L.sup.PB)m]y, [(L.sup.PO/L.sup.PA)n(L.sup.PB)m]y, (L.sup.PA)t(L.sup.PX)n(L.sup.PA)m, (L.sup.PA)t(L.sup.PB)n(L.sup.PA)m, (L.sup.PA)t[(L.sup.PX/L.sup.PO)n]y(L.sup.PA)m, (L.sup.PA)t[(L.sup.PB/L.sup.PX)n]y(L.sup.PA)m, (L.sup.PA)t[(L.sup.PB/L.sup.PO)n]y(L.sup.PA)m, (L.sup.PX/L.sup.PO)t(L.sup.PX)n(L.sup.PX/L.sup.PO)m, (L.sup.PX/L.sup.PO)t(L.sup.PB)n(L.sup.PX/L.sup.PO)m, (L.sup.PX/L.sup.PO)t[(L.sup.PX/L.sup.PO)n]y(L.sup.PX/L.sup.PO)m, (L.sup.PX/L.sup.PO)t[(L.sup.PB/L.sup.PO)n]y(L.sup.PX/L.sup.PO)m, (L.sup.PX/L.sup.PO)t[(L.sup.PB/_PO)n]y(L.sup.PX/L.sup.PO)m, (L.sup.PA/L.sup.PO)t(L.sup.PX)n(L.sup.PA/L.sup.PO)m, (L.sup.PA/L.sup.PO)t(L.sup.PB)n(L.sup.PA/L.sup.PO)m, (L.sup.PA/L.sup.PO)t[(L.sup.PX/L.sup.PO)n]y(L.sup.PA/L.sup.PO)m, (L.sup.PA/L.sup.PO)t[(L.sup.PB/L.sup.PO)n]y(L.sup.PA/L.sup.PO)m, or (L.sup.PA/L.sup.PO)t[(L.sup.PB/L.sup.PO)n]y(L.sup.PA/L.sup.PO)m, or a combination thereof, wherein: each L.sup.PX is independently L.sup.PA or L.sup.PB; and each other variable is independently as described herein.

(758) In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages [(L.sup.PA)n(L.sup.PB)m]y, [(L.sup.PO)n(L.sup.PB)m]y, (L.sup.PB)t[(L.sup.PA)n(L.sup.PB)m]y, or (L.sup.PB)t[(L.sup.PO)n(L.sup.PB)m]y. In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages (L.sup.PA)(L.sup.PB)m. In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages [(L.sup.PA)(L.sup.PB)m]y. In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages (L.sup.PB)t(L.sup.PA)(L.sup.PB)m. In some embodiments, each sugar between two of the consecutive internucleotidic linkages independently contains no 2-modification. In some embodiments, each sugar between two of the consecutive internucleotidic linkages is independently

(759) ##STR00321##
In some embodiments, n is 1. In some embodiments, y is 1. In some embodiments, y is 2-10. In some embodiments, t is 1. In some embodiments, t is 2-10. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, t is 2-10, n is 1 and m is 2-10. In some embodiments, each L.sup.PA is independently

(760) ##STR00322##
or a salt form thereof. In some embodiments, each L.sup.PB is independently

(761) ##STR00323##
or a salt form thereof. In some embodiments, each L.sup.PA is independently

(762) ##STR00324##
or a salt form thereof, and each each L.sup.PB is independently

(763) ##STR00325##
or a salt form thereof.

(764) In some embodiments, internucleotidic linkages of an provided oligonucleotides or regions thereof comprise or are consecutive internucleotidic linkages (from 5 to 3) (L.sup.PO)m(L.sup.PA/L.sup.PB)n, L.sup.PO(L.sup.PA/L.sup.PB)n, (L.sup.PO)m(L.sup.PB)n, L.sup.PO(L.sup.PB)n, [(L.sup.PO)m(L.sup.PA/L.sup.PB)n]y, [L.sup.PO(L.sup.PA/L.sup.PB)n]y, [(L.sup.PO)m(L.sup.PB)n]y, [L.sup.PO(L.sup.PB)n]y, (L.sup.PA/L.sup.PB)t(L.sup.PO)m(L.sup.PA/L.sup.PB)n, (L.sup.PA/L.sup.PB)t(L.sup.PO)m(L.sup.PB)n, (L.sup.PA/L.sup.PB)tL.sup.PO(L.sup.PB)n, (L.sup.PA/L.sup.PB)t[(L.sup.PO)m(L.sup.PA/L.sup.PB)n]y, (L.sup.PA/L.sup.PB)t[(L.sup.PO)m(L.sup.PB)n]y, (L.sup.PA/L.sup.PB)t[L.sup.PO(L.sup.PB)n]y, (L.sup.PO)m(L.sup.PA/L.sup.PB)n(L.sup.PA/L.sup.PB)t, L.sup.PO(L.sup.PA/L.sup.PB)n(L.sup.PA/L.sup.PB)t, (L.sup.PO)m(L.sup.PB)n(L.sup.PA/L.sup.PB)t, L.sup.PO(L.sup.PB)n(L.sup.PA/L.sup.PB)t, [(L.sup.PO)m(L.sup.PA/L.sup.PB)n]y(L.sup.PA/L.sup.PB)t, [L.sup.PO(L.sup.PA/L.sup.PB)n]y(L.sup.PA/L.sup.PB)t, [(L.sup.PO)m(L.sup.PB)n]y(L.sup.PA/L.sup.PB)t, [L.sup.PO(L.sup.PB)n]y(L.sup.PA/L.sup.PB)t, (L.sup.PA/L.sup.PB)t[(L.sup.PO)m(L.sup.PA/L.sup.PB)n]y(L.sup.PA/L.sup.PB)t, L.sup.PB(L.sup.PA/L.sup.PB)t[(L.sup.PO)m(L.sup.PA/L.sup.PB)n]y(L.sup.PA/L.sup.PB)tL.sup.PB, (L.sup.PA/L.sup.PB)t[(L.sup.PO)m(L.sup.PB)n]y(L.sup.PA/L.sup.PB)t, L.sup.PB(L.sup.PA/L.sup.PB)t[(L.sup.PO)m(L.sup.PB)n]y(L.sup.PA/L.sup.PB)tL.sup.PB, (L.sup.PA/L.sup.PB)t[(L.sup.PO)(L.sup.PA/L.sup.PB)]y(L.sup.PA/L.sup.PB)t, L.sup.PB(L.sup.PA/L.sup.PB)t[(L.sup.PO)(L.sup.PA/L.sup.PB)]y(L.sup.PA/L.sup.PB)tL.sup.PB(L.sup.PA/L.sup.PB)t[(L.sup.PO)(L.sup.PB)]y(L.sup.PA/L.sup.PB)t, L.sup.PB(L.sup.PA/L.sup.PB)t[(L.sup.PO)(L.sup.PB)]y(L.sup.PA/L.sup.PB)tL.sup.PB, or a combination thereof, wherein each variable is independently as described herein. In some embodiments, at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)t is L.sup.PA. In some embodiments, at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)t is L.sup.PB. In some embodiments, at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)t is L.sup.PA, and at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)t is L.sup.PB. In some embodiments, at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)m is L.sup.PA. In some embodiments, at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)m is L.sup.PB. In some embodiments, at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)m is L.sup.PA, and at least one L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)m is L.sup.PB. In some embodiments, each L.sup.PA/L.sup.PB of (L.sup.PA/L.sup.PB)m is L.sup.PB. In some embodiments, a sugar bonded to a L.sup.PO linkage at its 3-carbon comprises a 2-modification, wherein the 2-modification is not 2-F. In some embodiments, a sugar bonded to a L.sup.PO linkage at its 3-carbon is independently

(765) ##STR00326##
wherein R.sup.2s is not H or OH. In some embodiments, each sugar bonded to a L.sup.PO linkage at its 3-carbon is independently

(766) ##STR00327##
wherein R.sup.2s is not H or OH. In some embodiments, each sugar bonded to a L.sup.PO linkage at its 3-carbon is independently

(767) ##STR00328##
wherein R.sup.2s is not H or OH. In some embodiments, R.sup.4s is H. In some embodiments, R.sup.2s is not H, For OH. In some embodiments, each sugar bonded to a L.sup.PO linkage at its 3-carbon is independently

(768) ##STR00329##
wherein R.sup.2s is not H, F or OH. In some embodiments, R.sup.2s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.2s is OMe. In some embodiments, a 5-end sugar, a 3-end sugar, and/or a sugar between L.sup.PA/L.sup.PB and L.sup.PA/L.sup.PB comprises a 2-F modification. In some embodiments, a 5-end sugar, a 3-end sugar, and/or a sugar between L.sup.PA/L.sup.PB and L.sup.PA/L.sup.PB is

(769) ##STR00330##
wherein R.sup.2s is F. In some embodiments, each sugar comprises a 2-F is bonded to a modified internucleotidic linkage, e.g., at its 3-carbon. In some embodiments, a modified internucleotidic linkage is L.sup.PA or L.sup.PB. In some embodiments, each L.sup.PA is independently

(770) ##STR00331##
or a salt form thereof. In some embodiments, each L.sup.PB is independently

(771) ##STR00332##
or a salt form thereof. In some embodiments, t is 2-10. In some embodiments, each L.sup.PA is independently

(772) ##STR00333##
or a salt form thereof, and each L.sup.PB is independently

(773) ##STR00334##
or a salt form thereof. In some embodiments, each modified internucleotidic linkage in a provided oligonucleotide is independently L.sup.PO(wherein X-L-R.sup.1 is not H),

(774) ##STR00335##
or a salt form thereof. In some embodiments, each modified internucleotidic linkage is independently

(775) ##STR00336##
or a salt form thereof. In some embodiments, each modified internucleotidic linkage is independently

(776) ##STR00337##
or a salt form thereof. In some embodiments, m is 1. In some embodiments, each m is 1. In some embodiments, n is 2 or more. In some embodiments, each n is 2 or more. In some embodiments, t is 1. In some embodiments, t is 2 or more. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9. In some embodiments, t is 10. In some embodiments, each t is independently 2 or more. In some embodiments, each t is independently 3 or more. In some embodiments, each t is independently 4 or more. In some embodiments, each t is independently 5 or more.

(777) In some embodiments, each of L.sup.PO, L.sup.PA and L.sup.PB independently bonds to a 5-sugar through its 3-carbon, and to a 3-sugar through its 5-carbon, e.g., each L.sup.PA is independently an internucleotidic linkage having the structure of

(778) ##STR00338##
or a salt form thereof; each L.sup.PB is independently an internucleotidic linkage having the structure of

(779) ##STR00339##
or a salt form thereof. Example sugar structures are described herein, e.g., in some embodiments, each sugar moiety independently has the structure of

(780) ##STR00340##
wherein each variable is independently as described in the present disclosure.

(781) In some embodiments, L.sup.PO has a pattern, location, number, percentage, etc. as described herein for a natural phosphate linkage. In some embodiments, L.sup.PA has a pattern, location, number, percentage, etc. as described herein for a Rp internucleotidic linkage. In some embodiments, a Rp internucleotidic linkage is a Rp phosphorothioate internucleotidic linkage. In some embodiments, a Rp internucleotidic linkage is a Rp non-negatively charged internucleotidic linkage (e.g., n001). In some embodiments, L.sup.PB has a pattern, location, number, percentage, etc. as described herein for a Sp internucleotidic linkage. In some embodiments, a Sp internucleotidic linkage is a Sp phosphorothioate internucleotidic linkage. In some embodiments, a Sp internucleotidic linkage is a Sp non-negatively charged internucleotidic linkage (e.g., n001).

(782) In some embodiments, the present disclosure provides an oligonucleotide, wherein the first internucleotidic linkage from the 5-end is an internucleotidic linkage of O.sup.5P, and each other internucleotidic linkage is independently selected from O.sup.P, *.sup.PD, *.sup.PDS, *.sup.PDR, *.sup.N, *.sup.NS and *.sup.NR, wherein: O.sup.5P is

(783) ##STR00341##
L.sup.PO, L.sup.PA, L.sup.PB, or a salt form thereof; each O.sup.P is independently L.sup.PO; each *.sup.PD is independently

(784) ##STR00342##
or a salt form thereof; each *.sup.PDS is independently

(785) ##STR00343##
or a salt form thereof; each *.sup.PDR is independently

(786) ##STR00344##
or a salt form thereof; each *.sup.N is independently

(787) ##STR00345##
or a salt form thereof; each *.sup.NS is independently

(788) ##STR00346##
or a salt form thereof; and each *.sup.NR is independently

(789) ##STR00347##
or a salt form thereof;
wherein each variable in independently as described herein, wherein X-L-R.sup.1 is not OH.

(790) In some embodiments, O.sup.5P is independently

(791) ##STR00348##
L.sup.PO, L.sup.PA, L.sup.PB, or a salt form thereof. In some embodiments, each O.sup.P is independently L.sup.PO. In some embodiments, each *.sup.PD is independently

(792) ##STR00349##
or a salt form thereof. In some embodiments, each *.sup.PDS is independently

(793) ##STR00350##
or a salt form thereof. In some embodiments, each *.sup.PDR is independently

(794) ##STR00351##
or a salt form thereof. In some embodiments, each *.sup.N is independently

(795) ##STR00352##
or a salt form thereof. In some embodiments, each *.sup.NS is independently

(796) ##STR00353##
or a salt form thereof. In some embodiments, each *.sup.NR is independently

(797) ##STR00354##
or a salt form thereof.

(798) In some embodiments, X is O. In some embodiments, -L-R.sup.1 contains an electron-withdrawing group. In some embodiments, -L-R.sup.1 is CH.sub.2G.sup.2, wherein the methylene unit is optionally substituted. In some embodiments, -L-R.sup.1 is CH(R) G.sup.2. In some embodiments, G.sup.2 does not comprise a chiral element, and G.sup.2 comprises an electron-withdrawing group as described herein, e.g., in some embodiments, G.sup.2 is CH.sub.2CN (e.g., in O.sup.5P, O.sup.P, *.sup.PD, or *.sup.N, wherein linkage phosphorus is not chirally controlled). In some embodiments, G.sup.2 comprises a chiral element, e.g., wherein linkage phosphorus is chirally controlled. In some embodiments, X-L-R.sup.1 is of such a structure that HX-L-R.sup.1 is a chiral reagent described herein, or a capped chiral reagent described herein wherein an amino group of the chiral reagent (typically of W.sup.1H or W.sup.2H, which comprises an amino group NHG.sup.5-) is capped, e.g., with C(O)R (replacing a H, e.g., N[C(O)R]G.sup.5-). In some embodiments, X-L-R.sup.1 is

(799) ##STR00355##
wherein each variable is independently in accordance with the present disclosure. In some embodiments, X-L-R.sup.1 is

(800) ##STR00356##
wherein each variable is independently in accordance with the present disclosure. In some embodiments, R.sup.1 is H or C(O)R. In some embodiments, wherein R.sup.1 is H, e.g., in O.sup.5P. In some embodiments, R.sup.1 is C(O)R (e.g., in O.sup.5P, O.sup.P, *.sup.PDS, *.sup.PDR, *.sup.NS, *.sup.NR, etc.). In some embodiments, R.sup.1 is CH.sub.3C(O). In some embodiments, as described herein, G.sup.2 is In some embodiments, G.sup.2 is C(R).sub.2Si(R).sub.3, wherein-C(R).sub.2 is optionally substituted CH.sub.2, and each R of Si(R).sub.3 is independently an optionally substituted group selected from C.sub.1-10 aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, G.sup.2 is CH.sub.2Si(Me)(Ph).sub.2. In some embodiments, e.g., in *.sup.PDS, *.sup.PDR, etc., G.sup.2 is CH.sub.2Si(Me)(Ph).sub.2. In some embodiments, G.sup.2 comprises an electron-withdrawing group as described herein. In some embodiments, G.sup.2 is C(R).sub.2SO.sub.2R, wherein C(R).sub.2 is optionally substituted CH.sub.2, and R is an optionally substituted group selected from C.sub.1-10 aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, R is phenyl. In some embodiments, e.g., in *.sup.NS, *.sup.NR, etc., G.sup.2 is CH.sub.2SO.sub.2Ph.

(801) In some embodiments, the present disclosure provides an oligonucleotide (a first oligonucleotide), which has an identical structure as an oligonucleotide described in a Table herein or an oligonucleotide described in e.g., US20150211006, US20170037399, US20180216107, US 20180216108, US20190008986, WO 2017/015555, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/022473, WO 2018/067973, WO 2018/098264, WO 2018/223056, WO 2018/223073, WO 2018/223081, WO 2018/237194, WO 2019/032607, WO 2019/032612, etc., the oligonucleotide of each of which is incorporated herein by reference (a second oligonucleotide), which second oligonucleotide comprises modified internucleotidic linkages, except that compared to the second oligonucleotide, in the first oligonucleotide: the first internucleotidic linkage from the 5-end is an internucleotidic linkage of O.sup.5P; and for the rest linkages: at each location where there is a phosphate linkage in the second oligonucleotide, there is independently a linkage of O.sup.P in the first oligonucleotide; at each location where there is a stereorandom phosphorothioate linkages in the second oligonucleotide, there is independently a linkage of *.sup.PD in the first oligonucleotide; at each location where there is a Sp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *.sup.PDS in the first oligonucleotide; at each location where there is a Rp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *.sup.PDR in the first oligonucleotide; at each location where there is a stereorandom non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *.sup.N in the first oligonucleotide; at each location where there is a Sp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *.sup.NS in the first oligonucleotide; at each location where there is a Rp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *.sup.NR in the first oligonucleotide, and each nucleobase in the first oligonucleotide is optionally and independently protected (e.g., as in oligonucleotide synthesis), and each additional chemical moiety, if any, in the first oligonucleotide is optionally and independently protected (e.g., OH in a carbohydrate moiety protected as OAc).

(802) In some embodiments, at each location where there is a phosphate linkage in the second oligonucleotide, there is independently a linkage of O.sup.P in the first oligonucleotide; at each location where there is a stereorandom phosphorothioate linkages in the second oligonucleotide, there is independently a linkage of *.sup.PD in the first oligonucleotide; at each location where there is a Sp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *.sup.PDS in the first oligonucleotide; at each location there is a Rp phosphorothioate linkage in the second oligonucleotide, there is independently a linkage of *.sup.PDR in the first oligonucleotide; at each location there is a stereorandom non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *.sup.N in the first oligonucleotide; at each location there is a Sp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *.sup.NS in the first oligonucleotide; at each location there is a Rp non-negatively charged internucleotidic linkage in the second oligonucleotide, there is independently a linkage of *.sup.NR in the first oligonucleotide, and each nucleobase in the first oligonucleotide is optionally and independently protected (e.g., as in oligonucleotide synthesis), and each additional chemical moiety, if any, in the first oligonucleotide is optionally and independently protected (e.g., OH in a carbohydrate moiety protected as OAc); wherein each of O.sup.5P, O.sup.P, *.sup.PD, *.sup.PDS, *.sup.PDR, *.sup.N, *.sup.NS and *.sup.NR is independently as described herein. In some embodiments, such an oligonucleotide is linked to a support optionally through a linker, e.g., a CNA linker to CPG. In some embodiments, as appreciated by those skilled in the art, after a removal process of X-L-R.sup.1, a linkage of O.sup.5P, O.sup.P, *.sup.PD, *.sup.PDS, *.sup.PDR, *.sup.N, *.sup.NS or *.sup.NR becomes a linkage it replaces. In some embodiments, such oligonucleotides (e.g., first oligonucleotides) are useful intermediates for preparing their corresponding oligonucleotides (e.g., second oligonucleotides). In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of a provided first oligonucleotide or a stereoisomer thereof.

(803) In some embodiments, as appreciated by those skilled in the art, W.sup.N is of such a structure that its N-moiety has the same non-hydrogen atoms and connections of non-hydrogen atoms as the N-moiety of the non-negatively charged internucleotidic linkage it replaces (without considering single, double, or triple bond etc.). For example, in some embodiments, P.sup.N in *.sup.N is

(804) ##STR00357##
(such a *.sup.N is n001.sup.P), and its corresponding non-negatively charged internucleotidic linkage is n001.

(805) In some embodiments, a provided oligonucleotide has the same Description as an oligonucleotide listed in a Table herein (e.g., Table A1), except that: the oligonucleotide comprises at least one linkage of O.sup.P, and/or at each location in the oligonucleotide where there is a phosphate linkage, there is independently a linkage of O.sup.P, wherein O.sup.P is

(806) ##STR00358## at each location where there is a stereorandom phosphorothioate linkages, there is independently a linkage of *.sup.PD, wherein *.sup.PD is

(807) ##STR00359## at each location where there is a Sp phosphorothioate linkage, there is independently a linkage of *.sup.PDS, wherein *.sup.PDS is

(808) ##STR00360## at each location where there is a Rp phosphorothioate linkage, there is independently a linkage of *.sup.PDR, wherein *.sup.PDR is

(809) ##STR00361## at each location where there is a stereorandom n001, there is independently a linkage of *.sup.N wherein *.sup.N is

(810) ##STR00362##
(as appreciated by those skilled in the art, it is associated with an anion (e.g., Q.sup. such as PF.sub.6.sup. (which can be an anion in a modification step))); at each location where there is a Sp n001, there is independently a linkage of *.sup.NS, wherein *.sup.NS is

(811) ##STR00363##
(as appreciated by those skilled in the art, it is associated with an anion (e.g., Q.sup. such as PF.sub.6.sup. (which can be an anion in a modification step))); and at each location where there is a Rp n001, there is independently a linkage of *.sup.NR, wherein *.sup.NR is

(812) ##STR00364##
(as appreciated by those skilled in the art, it is associated with an anion (e.g., Q.sup. such as PF.sub.6.sup. (which can be an anion in a modification step))); and the oligonucleotide is optionally connected to a solid support, optionally through a linker.
In some embodiments, the oligonucleotide is connected to a solid support, e.g., CPG, polystyrene support, etc. In some embodiments, the oligonucleotide is connected to a solid support through a linker, e.g., a CNA linker. In some embodiments, such an oligonucleotide is an oligonucleotide of formula O-I or a salt form thereof.
Certain Embodiments of Stereochemistry and Pattern of Backbone Chiral Centers

(813) Among other things, the present disclosure provides oligonucleotides comprising one or more chirally controlled internucleotidic linkages. In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions. In some embodiments, each chiral linkage phosphorus of provided oligonucleotides is independently chirally controlled (stereocontrolled) (e.g., each independently having a stereopurity (diastereopurity) of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (e.g., as typically assessed using an appropriate dimer comprising an internucleotidic linkage containing the linkage phosphorus, and the two nucleoside units being linked by the internucleotidic linkage)). In some embodiments, a stereopurity is at least 90%. In some embodiments, a stereopurity is at least 95%. In some embodiments, a stereopurity is at least 96%. In some embodiments, a stereopurity is at least 97%. In some embodiments, a stereopurity is at least 98%. In some embodiments, a stereopurity is at least 99%. With the capability to fully control stereochemistry and other modifications (e.g., base modifications, sugar modifications, internucleotidic linkage modifications, etc.), the present disclosure provides technologies of improved properties and/or activities compared to corresponding non-chirally controlled technologies.

(814) In some embodiments, pattern of backbone chiral centers of a region, particularly a core region or a middle region, or of an oligonucleotide (e.g., an oligonucleotide of a plurality of oligonucleotides) is or comprises (Np/Op)t[(Rp)n(Sp)m]y, (Np/Op)t[(Op)n(Sp)m]y, (Np/Op)t[(Op/Rp)n(Sp)m]y, (Sp)t[(Rp)n(Sp)m]y, (Sp)t[(Op)n(Sp)m]y, (Sp)t[(Op/Rp)n(Sp)m]y, [(Rp)n(Sp)m]y, [(Op)n(Sp)m]y, [(Op/Rp)n(Sp)m]y, (Rp)t(Np)n(Rp)m, (Rp)t(Sp)n(Rp)m, (Rp)t[(Np/Op)n]y(Rp)m, (Rp)t[(Sp/Np)n]y(Rp)m, (Rp)t[(Sp/Op)n]y(Rp)m, (Np/Op)t(Np)n(Np/Op)m, (Np/Op)t(Sp)n(Np/Op)m, (Np/Op)t[(Np/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Rp/Op)t(Np)n(Rp/Op)m, (Rp/Op)t(Sp)n(Rp/Op)m, (Rp/Op)t[(Np/Op)n]y(Rp/Op)m, (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m, or (Rp/Op)t[(Sp/Op)n]y(Rp/Op) m (unless otherwise specified, description of patterns of modifications and stereochemistry are from 5 to 3 as typically used in the art), wherein Sp indicates S configuration of a chiral linkage phosphorus of a chiral modified internucleotidic linkage, Rp indicates R configuration of a chiral linkage phosphorus of a chiral modified internucleotidic linkage, Op indicates an achiral linkage phosphorus of a natural phosphate linkage, each Np is independently Rp, or Sp, and each of m, n, t and y is independently 1-50 as described in the present disclosure. In some embodiments, a pattern of backbone chiral centers is or comprises [(Rp/Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises [(Rp)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises [(Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Rp/Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Rp)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[(Rp/Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[(Rp)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[(Op)n(Sp)m]y. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t(Np)n(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t(Sp)n(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t[(Np/Op)n]y(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t[(Sp/Np)n]y(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp)t[(Sp/Op)n]y(Rp)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t(Np)n(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t(Sp)n(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Np/Op)n]y(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Sp/Op)n]y(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Np/Op)t[(Sp/Op)n]y(Np/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t(Np)n(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t(Sp)n(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t[(Np/Op)n]y(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m. In some embodiments, a pattern of backbone chiral centers is or comprises (Rp) (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m(Rp). In some embodiments, n is 1. For example, in some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[Op(Sp)m]y; in some embodiments, a pattern of backbone chiral centers is or comprises (Sp)t[Rp(Sp)m]y. In some embodiments, y is 1. In some embodiments, m is 2 or more. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, t is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, n is 1, and m is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 internucleotidic linkages preceding, and there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 internucleotidic linkages after the Rp or Op. In some embodiments, there are at least 2 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 3 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 4 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 5 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 6 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 7 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 8 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 9 internucleotidic linkages preceding and/or following. In some embodiments, there are at least 10 internucleotidic linkages preceding and/or following. In some embodiments, y is 1. In some embodiments, y is 2 or more. In some embodiments, y is 2, 3, 4, or 5. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, a region having such a pattern of backbone chiral centers contains no 2-modifications on its sugar moieties, wherein the 2-modification is 2-OR.sup.1 or 2-O-L-, wherein R.sup.1 is not hydrogen and L comprises a carbon atom and connects to another carbon atom of the sugar moiety. In some embodiments, each sugar moiety of a region having such a pattern of backbone chiral centers is independently a natural DNA sugar moiety

(815) ##STR00365##
As appreciated by a person having ordinary skill in the art, for a natural DNA sugar moiety in natural DNA, C1 is connected to a base, C3 and C5 are each independently connected to internucleotidic linkages or OH (when at the 5- or 3-end)). Certain benefits/advantages provided by such patterns of backbone chiral centers are described in US20170037399, WO 2017/015555, and WO 2017/062862.

(816) In some embodiments, y, t, n and m each are independently 1-20 as described in the present disclosure. In some embodiments, y is 1. In some embodiments, y is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10.

(817) In some embodiments, n is 1. In some embodiments, n is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 1-10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 3, 4, 5, 6, 7 or 8. In some embodiments, n is 4, 5, 6, 7 or 8. In some embodiments, n is 5, 6, 7 or 8. In some embodiments, n is 6, 7 or 8. In some embodiments, n is 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

(818) In some embodiments, m is 0-50. In some embodiments, m is 1-50. In some embodiments, m is 1. In some embodiments, m is 2-50. In some embodiments, m is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, m is 3, 4, 5, 6, 7 or 8. In some embodiments, m is 4, 5, 6, 7 or 8. In some embodiments, m is 5, 6, 7 or 8. In some embodiments, m is 6, 7 or 8. In some embodiments, m is 7 or 8. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18. In some embodiments, m is 19. In some embodiments, m is 20. In some embodiments, m is 21. In some embodiments, m is 22. In some embodiments, m is 23. In some embodiments, m is 24. In some embodiments, m is 25. In some embodiments, m is at least 2. In some embodiments, m is at least 3. In some embodiments, m is at least 4. In some embodiments, m is at least 5. In some embodiments, m is at least 6. In some embodiments, m is at least 7. In some embodiments, m is at least 8. In some embodiments, m is at least 9. In some embodiments, m is at least 10. In some embodiments, m is at least 11. In some embodiments, m is at least 12. In some embodiments, m is at least 13. In some embodiments, m is at least 14. In some embodiments, m is at least 15. In some embodiments, m is at least 16. In some embodiments, m is at least 17. In some embodiments, m is at least 18. In some embodiments, m is at least 19. In some embodiments, m is at least 20. In some embodiments, m is at least 21. In some embodiments, m is at least 22. In some embodiments, m is at least 23. In some embodiments, m is at least 24. In some embodiments, m is at least 25. In some embodiments, m is at least greater than 25.

(819) In some embodiments, t is 1-20. In some embodiments, tis 1. In some embodiments, t is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is 1-5. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9. In some embodiments, t is 10. In some embodiments, t is 11. In some embodiments, t is 12. In some embodiments, t is 13. In some embodiments, t is 14. In some embodiments, t is 15. In some embodiments, t is 16. In some embodiments, t is 17. In some embodiments, t is 18. In some embodiments, t is 19. In some embodiments, t is 20.

(820) In some embodiments, each of t and m is independently at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, each of t and m is independently at least 3. In some embodiments, each of t and m is independently at least 4. In some embodiments, each of t and m is independently at least 5. In some embodiments, each of t and m is independently at least 6. In some embodiments, each of t and m is independently at least 7. In some embodiments, each of t and m is independently at least 8. In some embodiments, each of t and m is independently at least 9. In some embodiments, each of t and m is independently at least 10.

(821) In some embodiments, provided oligonucleotides comprises a block, e.g., a first block, a 5-wing, etc., that has a pattern of backbone chiral centers of or comprising a t-section, e.g., (Sp)t, (Rp)t, (Np/Op)t, (Rp/Op)t, etc., a block, e.g., a second block, a core, etc., that has a pattern of backbone chiral centers of or comprising a y- or n-section, e.g., (Np)n, (Sp)n, [(Np/Op)n]y, [(Rp/Op)n]y, [(Sp/Op)n]y, etc., and a block, e.g., a third block, a 3-wing, etc., that has a pattern of backbone chiral centers of or comprising a m-section, e.g., (Sp)m, (Rp)m, (Np/Op)m, (Rp/Op)m, etc.

(822) In some embodiments, a t-, y-, n-, or m-section that comprises Np or Rp, e.g., (Rp)t, (Np/Op)t, (Rp/Op)t, (Np)n, [(Np/Op)n]y, [(Rp/Op)n]y, (Rp)m, (Np/Op)m, (Rp/Op)m, etc. independently comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% Rp. In some embodiments, a t- or m-section that comprises Np or Rp independently comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% Rp. In some embodiments, provided oligonucleotides comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% Rp. In some embodiments, a percentage is at least 10%. In some embodiments, a percentage is at least 20%. In some embodiments, a percentage is at least 30%. In some embodiments, a percentage is at least 40%. In some embodiments, a percentage is at least 50%. In some embodiments, a percentage is at least 60%. In some embodiments, a percentage is at least 70%. In some embodiments, a percentage is at least 75%. In some embodiments, a percentage is at least 80%. In some embodiments, a percentage is at least 85%. In some embodiments, a percentage is at least 90%. In some embodiments, a percentage is at least 95%. In some embodiments, a percentage is 100%.

(823) In some embodiments, each sugar moiety bonded to a Rp or Op linkage phosphorus at 3 independently comprises a modification. In some embodiments, each sugar moiety bonded to a Rp or Op linkage phosphorus at 5 independently comprises a modification. In some embodiments, each sugar moiety bonded to a Rp linkage phosphorus at 3 independently comprises a modification. In some embodiments, each sugar moiety bonded to a Rp linkage phosphorus at 5 independently comprises a modification. In some embodiments, each sugar moiety bonded to an Op linkage phosphorus at 3 independently comprises a modification. In some embodiments, each sugar moiety bonded to an Op linkage phosphorus at 5 independently comprises a modification. In some embodiments, each sugar moiety bonded to a Sp linkage phosphorus at 3 independently comprises a modification. In some embodiments, each sugar moiety bonded to a Sp linkage phosphorus at 5 independently comprises a modification. In some embodiments, each sugar moiety independently comprises a modification. In some embodiments, a modification is a 2-modification. In some embodiments, a modification is 2-OR, wherein R is not hydrogen. In some embodiments, a modification is 2-OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, a modification is 2-OR, wherein R is substituted C.sub.1-6 alkyl. In some embodiments, a modification is 2-OR, wherein R is optionally substituted C.sub.2-6 alkyl. In some embodiments, a modification is 2-OR, wherein R is substituted C.sub.2-6 alkyl. In some embodiments, R is CH.sub.2CH.sub.2OMe. In some embodiments, a modification is or comprises -L- connecting two sugar carbons, e.g., those found in LNA. In some embodiments, a modification is -L- connecting C.sub.2 and C.sub.4 of a sugar moiety. In some embodiments, L is CH.sub.2CH(R), wherein R is as described in the present disclosure. In some embodiments, L is CH.sub.2CH(R), wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, L is CH.sub.2(R)CH(R), wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, L is CH.sub.2(S)CH(R), wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, a block, a wing, a core, or an oligonucleotide has sugar modifications as described in the present disclosure.

(824) In some embodiments, a provided pattern of backbone chiral centers is or comprises (Rp/Sp)-(All Rp or All Sp)-(Rp/Sp), wherein each Rp/Sp is independently Rp or Sp. In some embodiments, a provided pattern of backbone chiral centers is or comprises (Rp)-(All Sp)-(Rp). In some embodiments, a provided pattern of backbone chiral centers is or comprises (Sp)-(All Sp)-(Sp). In some embodiments, a provided pattern of backbone chiral centers is or comprises (Sp)-(All Rp)-(Sp). In some embodiments, a provided pattern of backbone chiral centers is or comprises (Rp/Sp)-(repeating (Sp)m(Rp)n)-(Rp/Sp). In some embodiments, a provided pattern of backbone chiral centers is or comprises (Rp/Sp)-(repeating SpSpRp)-(Rp/Sp).

(825) Blocks

(826) In some embodiments, provided oligonucleotides comprise one or more blocks, characterized by base modifications, sugar modifications, types of internucleotidic linkages, stereochemistry of linkage phosphorus, etc. In some embodiments, provided oligonucleotides comprises or are of a 5-first block-second block-third block-3 structure. In some embodiments, a first block is a 5-wing. In some embodiments, a first block is 5-end region. In some embodiments, a second block is a core. In some embodiments, a second block is a middle region between a 5-end and a 3-end region. In some embodiments, a third block a 3-wing. In some embodiments, a third block is a 3-end region. Each of a 5-wing, 5-end region, core, middle region, 3-wing, and 3-end region can independently be a block.

(827) In some embodiments, provided oligonucleotides comprises or are of a 5-wing-core-wing-3, 5-wing-core-3 or 5-core-wing-3 structures. In some embodiments, a first block, a second block, a third block, a wing (e.g., a 5-wing, a 3-wing) and/or a core of provided oligonucleotides are each independently a block or comprise one or more blocks as described in the present disclosure.

(828) Various blocks, 5-wings, 3-wings and cores can be utilized in accordance with the present disclosure, including those described in US20150211006, US20150211006, WO 2017015555, WO 2017015575, WO 2017062862, WO 2017160741, blocks, 5-wings, 3-wings and cores of each of which are incorporated herein by reference.

(829) In some embodiments, a block is a linkage phosphorus stereochemistry block. For example, in some embodiments, a block comprises only Rp, Sp, or Op linkage phosphorus. In some embodiments, a block is a Rp block comprising only Rp linkage phosphorus. In some embodiments, a block is a Rp/Op block comprising only Rp/Op linkage phosphorus. In some embodiments, a block is a Sp/Op block comprising only Sp/Op linkage phosphorus. In some embodiments, a block is an Op block. In some embodiments, an oligonucleotide, or a region thereof (a first block, a second block, a third block, a wing, a core, etc.) comprises one or more of a Rp block, a Sp block and/or an Op block. In some embodiments, a block comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, linkage phosphorus.

(830) In some embodiments, a block is a sugar modification block. In some embodiments, a block is a 2-modification block wherein each sugar moiety of the block independently comprises the 2-modification. In some embodiments, a 2-modification is 2-OR wherein R is as described in the present disclosure. In some embodiments, a 2-modification is a 2-OR wherein R is not hydrogen. In some embodiments, a 2-modification is 2-OMe. In some embodiments, a 2-modification is 2-MOE. In some embodiments, a modification is a LNA modification. In some embodiments, an oligonucleotide, or a region thereof (a first block, a second block, a third block, a wing, a core, etc.) comprises one or more sugar modification blocks, each independently of its own sugar modification. In some embodiments, a block comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, sugar moieties.

(831) As illustrated herein, a block can be of various lengths. In some embodiments, a block is of 1-30, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length. In some embodiments, a 5-first block-second-block-third block-3, or a 5-wing-core-wing-3 is of 5-10-5, 3-10-4, 3-10-6, 4-12-4, etc.

(832) In some embodiments, an oligonucleotide or a block or region thereof (e.g., a 5-end region, a 5-wing, a middle region, a core region, a 3-end region, a 3-ring, etc.) comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, non-negatively charged internucleotidic linkages as described in the present disclosure. In some embodiments, a provided oligonucleotide comprises two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, consecutive non-negatively charged internucleotidic linkages. In some embodiments, a block or region comprises two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, consecutive non-negatively charged internucleotidic linkages. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5. In some embodiments, the number is 6. In some embodiments, the number is 7. In some embodiments, the number is 8. In some embodiments, the number is 9. In some embodiments, the number is 10 or more. In some embodiments, each internucleotidic linkage between nucleoside units in a block, e.g., a 5-end region, a 5-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the block from the 5-end of the block. In some embodiments, each internucleotidic linkage between nucleoside units in a block, e.g., a 3-end region, a 3-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the block from the 3-end of the block. In some embodiments, each internucleotidic linkage between nucleoside units in a region, e.g., a 5-end region, a 5-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the region from the 5-end of the region. In some embodiments, each internucleotidic linkage between nucleoside units in a region, e.g., a 3-end region, a 3-wing, is a non-negatively charged internucleotidic linkage except the first internucleotidic linkage between two nucleoside units of the region from the 3-end of the region. In some embodiments, each internucleotidic linkage in a region or block, e.g., a 5-end region, a 5-wing, a middle region, a core region, a 3-end region, a 3-ring, etc., is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage. In some embodiments, each internucleotidic linkage in a region or block is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp phosphorothioate internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block, e.g., a 5-end region, a 5-wing, a middle region, a core region, a 3-end region, a 3-ring, etc., is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp phosphorothioate internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block is independently a non-negatively charged internucleotidic linkage or a natural phosphate internucleotidic linkage. In some embodiments, about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of internucleotidic linkages of an oligonucleotide or a region or block is independently a non-negatively charged internucleotidic linkage. In some embodiments, the percentage is 45% or more. In some embodiments, the percentage is 50% or more. In some embodiments, the percentage is 60% or more. In some embodiments, the percentage is 70% or more. In some embodiments, the percentage is 80% or more. In some embodiments, the percentage is 90% or more. In some embodiments, a region or block is a wing. In some embodiments, a region or block is a 5-wing. In some embodiments, a region or block is a 3-wing. In some embodiments, a region or block is a core. As described herein, a region or block, e.g., a wing, a core, etc., can have various lengths, e.g., comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobases. In some embodiments, each nucleobase is independently optionally substituted A, T, C, G, U or an optionally substituted tautomer of A, T, C, G, or U.

(833) Length

(834) As described in the present disclosure, provided oligonucleotides can be of various lengths, e.g., 2-200, 10-15, 10-25, 15-20, 15-25, 15-40, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 150, nucleobases in length, wherein each nucleobase is independently optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U. In some embodiments, provided oligonucleotides, e.g., oligonucleotide of a plurality in chirally controlled oligonucleotide compositions, are 15 nucleobases in length. In some embodiments, provided oligonucleotides are 16 nucleobases in length. In some embodiments, provided oligonucleotides are 17 nucleobases in length. In some embodiments, provided oligonucleotides are 18 nucleobases in length. In some embodiments, provided oligonucleotides are 19 nucleobases in length. In some embodiments, provided oligonucleotides are 20 nucleobases in length. In some embodiments, provided oligonucleotides are 21 nucleobases in length. In some embodiments, provided oligonucleotides are 22 nucleobases in length. In some embodiments, provided oligonucleotides are 23 nucleobases in length. In some embodiments, provided oligonucleotides are 24 nucleobases in length. In some embodiments, provided oligonucleotides are 25 nucleobases in length.

(835) As described in the present disclosure, provided oligonucleotides, oligonucleotides of a plurality in chirally controlled oligonucleotide compositions, may comprise various modifications, e.g., base modifications, sugar modifications, internucleotidic linkage modifications, etc. In some embodiments, the oligonucleotide composition comprises at least one modified nucleotide, at least one modified sugar moiety, at least one morpholino moiety, at least one 2-deoxy ribonucleotide, at least one locked nucleotide, and/or at least one bicyclic nucleotide.

(836) Nucleobases

(837) In some embodiments, a nucleobase is a natural nucleobase. In some embodiments, a nucleobase is a modified nucleobase (non-natural nucleobase). In some embodiments, a nucleobase, e.g., BA, in provided oligonucleotides is a natural nucleobase (e.g., adenine, cytosine, guanosine, thymine, or uracil) or a modified nucleobase derived from a natural nucleobase, e.g., optionally substituted adenine, cytosine, guanosine, thymine, or uracil, or tautomeric forms thereof. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, and tautomeric forms thereof, having their respective amino groups protected by protecting groups, e.g., one or more of R, C(O)R, etc. Example protecting groups, including those useful for oligonucleotide synthesis, are widely known in the art and can be utilized in accordance with the present disclosure. In some embodiments, a protected nucleobase and/or derivative is selected from nucleobases with one or more acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products). Example modified nucleobases are also disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313. In some embodiments, a modified nucleobase is substituted uracil, thymine, adenine, cytosine, or guanine. In some embodiments, a modified nucleobase is a functional replacement, e.g., in terms of hydrogen bonding and/or base pairing, of uracil, thymine, adenine, cytosine, or guanine. In some embodiments, a nucleobase is optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine. In some embodiments, a nucleobase is uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine.

(838) In some embodiments, a modified base is optionally substituted adenine, cytosine, guanine, thymine, or uracil. In some embodiments, a modified nucleobase is independently adenine, cytosine, guanine, thymine or uracil, modified by one or more modifications by which: (1) a nucleobase is modified by one or more optionally substituted groups independently selected from acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof; (2) one or more atoms of a nucleobase are independently replaced with a different atom selected from carbon, nitrogen or sulfur; (3) one or more double bonds in a nucleobase are independently hydrogenated; or (4) one or more optionally substituted aryl or heteroaryl rings are independently inserted into a nucleobase.

(839) Modified nucleobases also include expanded-size nucleobases in which one or more aryl rings, such as phenyl rings, have been added. Nucleic base replacements described in the Glen Research catalog (available at the Glen Research website); Krueger A T et al, Acc. Chem. Res., 2007, 40, 141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627, are contemplated as useful for oligonucleotides of the present disclosure.

(840) In some embodiments, modified nucleobases include structures such as, but not limited to, corrin- or porphyrin-derived rings. Porphyrin-derived base replacements have been described in Morales-Rojas, H and Kool, E T, Org. Lett., 2002, 4, 4377-4380. Shown below is an example of a porphyrin-derived ring which can be used as a nucleobase replacement:

(841) ##STR00366##

(842) In some embodiments, a modified nucleobase is fluorescent. Examples of such fluorescent modified nucleobases include phenanthrene, pyrene, stillbene, isoxanthine, isozanthopterin, terphenyl, terthiophene, benzoterthiophene, coumarin, lumazine, tethered stillbene, benzo-uracil, and naphtho-uracil.

(843) In some embodiments, a modified nucleobase is a universal base or a degenerate base, e.g., 3-nitropyrrole, 5-nitroindole, P, K, etc.

(844) In some embodiments, other nucleosides can also be used in technologies disclosed in the present disclosure and include nucleosides that incorporate modified nucleobases, or nucleobases covalently bound to modified sugars. Some examples of nucleosides that incorporate modified nucleobases include 4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2-O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2-O-methylpseudouridine; beta,D-galactosylqueosine; 2-O-methylguanosine; N.sup.6-isopentenyladenosine; 1-methyladenosine; 1-methylpseudouridine; 1-methylguanosine; 1-methylinosine; 2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine; N.sup.7-methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5-hydroxymethylcytidine; 5-formylcytosine; 5-carboxylcytosine; N.sup.6-methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine; beta, D-mannosylqueosine; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N.sup.6-isopentenyladenosine; N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine; N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine; uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v); pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2-O-methyl-5-methyluridine; and 2-O-methyluridine.

(845) In some embodiments, a nucleobase is optionally substituted A, T, C, G or U, wherein one or more NH.sub.2 are independently and optionally replaced with C(-L-R.sup.1).sub.3, one or more NH are independently and optionally replaced with C(-L-R.sup.1).sub.2, one or more N are independently and optionally replaced with C(-L-R.sup.1), one or more CH are independently and optionally replaced with N, and one or more O are independently and optionally replaced with S, N(-L-R.sup.1), or C(-L-R.sup.1).sub.2, wherein two or more -L-R.sup.1 are optionally taken together with their intervening atoms to form a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatom ring atoms. In some embodiments, a modified nucleobase is optionally substituted A, T, C, G or U, wherein one or more NH.sub.2 are independently and optionally replaced with C(-L-R.sup.1).sub.3, one or more NH are independently and optionally replaced with C(-L-R.sup.1).sub.2, one or more N are independently and optionally replaced with C(-L-R.sup.1), one or more CH are independently and optionally replaced with N, and one or more O are independently and optionally replaced with S, N(-L-R.sup.1), or C(-L-R.sup.1).sub.2, wherein two or more -L-R.sup.1 are optionally taken together with their intervening atoms to form a 3-30 membered bicyclic or polycyclic ring having 0-10 heteroatom ring atoms, wherein the modified base is different than the natural A, T, C, G and U. In some embodiments, a nucleobase is optionally substituted A, T, C, G or U. In some embodiments, a modified base is substituted A, T, C, G or U, wherein the modified base is different than the natural A, T, C, G and U.

(846) In some embodiments, a modified nucleobase may be optionally substituted. In some embodiments, a modified nucleobase contains one or more, e.g., heteroatoms, alkyl groups, or linking moieties connected to fluorescent moieties, biotin or avidin moieties, or other proteins or peptides. In some embodiments, a nucleobase or modified nucleobase comprises or is conjugated with one or more biomolecule binding moieties such as e.g., antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties. In some embodiments, a modified nucleobase is modified by substitution with a fluorescent or biomolecule binding moiety. In some embodiments, a substituent on a nucleobase or modified nucleobase is a fluorescent moiety. In some embodiments, a substituent on a nucleobase or modified nucleobase is biotin or avidin.

(847) Example nucleobases are also described in US20110294124, US20120316224, US 20140194610, US20150211006, US20150197540, WO 2015107425, WO/2017/015555, WO/2017/015575, and WO/2017/062862, the nucleobases of each of which is incorporated herein by reference.

(848) Sugars

(849) In some embodiments, oligonucleotides comprise one or more modified sugar moieties beside the natural sugar moieties. In some embodiments, a sugar is a natural sugar. In some embodiments, a sugar is a modified sugar (non-natural sugar). The most common naturally occurring nucleotides are comprised of ribose sugars linked to the nucleobases adenosine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U). Also included in the present disclosure are modified nucleotides wherein an internucleotidic linkage is linked to various positions of a sugar or modified sugar. As non-limiting examples, an internucleotidic linkage can be linked to the 2, 3, 4 or 5 position of a sugar.

(850) In some embodiments, a sugar moiety is

(851) ##STR00367##
wherein each variable is independently as described in the present disclosure. In some embodiments, a sugar moiety is

(852) ##STR00368##
wherein L.sup.s is C(R.sup.5s).sub.2, wherein each R.sup.5s is independently as described in the present disclosure. In some embodiments, a sugar moiety has the structure of

(853) ##STR00369##
wherein each variable is independently as described in the present disclosure. In some embodiments, a sugar moiety has the structure of

(854) ##STR00370##
wherein each variable is independently as described in the present disclosure. In some embodiments, a sugar has or is derived from the structure of

(855) ##STR00371##
wherein each variable is independently as described in the present disclosure. In some embodiments, a nucleoside has the structure of

(856) ##STR00372##
wherein each variable is independently as described in the present disclosure. In some embodiments, a nucleoside moiety has or comprises the structure of

(857) ##STR00373##
wherein each variable is independently as described in the present disclosure. In some embodiments, L.sup.s is CH(R), wherein R is as described in the present disclosure. In some embodiments, R is H. In some embodiments, R is not H, and L.sup.s is (R)CH(R). In some embodiments, R is not H, and L.sup.s is (S)CH(R). In some embodiments, R, as described in the present disclosure, is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is methyl.

(858) Various types of sugar modifications are known and can be utilized in accordance with the present disclosure. In some embodiments, a sugar modification is a 2-modification (e.g. R.sup.2s (e.g., in

(859) ##STR00374##
In some embodiments, a 2-modification is 2-F. In some embodiments, a 2-modification is 2-OR, wherein R is not hydrogen. In some embodiments, a 2-modification is 2-OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, a 2-modification is 2-OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, a 2-modification is 2-OMe. In some embodiments, a 2-modification is 2-MOE. In some embodiments, a 2-modification is a LNA sugar modification (C.sub.2OCH.sub.2C4). In some embodiments, a 2-modification is (C.sub.2OC(R).sub.2C4), wherein each R is independently as described in the present disclosure. In some embodiments, a 2-modification is (C.sub.2OCHRC4), wherein R is as described in the present disclosure. In some embodiments, a 2-modification is (C.sub.2O(R)CHRC4), wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, a 2-modification is (C.sub.2O(S)CHRC4), wherein R is as described in the present disclosure and is not hydrogen. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is unsubstituted C.sub.1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, a 2-modification is (C.sub.2OCHRC4), wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, a 2-modification is (C.sub.2OCHRC4), wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, a 2-modification is (C.sub.2OCHRC4), wherein R is methyl. In some embodiments, a 2-modification is (C.sub.2OCHRC4), wherein R is ethyl. In some embodiments, a 2-modification is (C.sub.2O(R)CHRC4), wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, a 2-modification is (C.sub.2O(R)CHRC4), wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, a 2-modification is (C.sub.2O(R)CHRC4), wherein R is methyl. In some embodiments, a 2-modification is (C.sub.2O(R)CHRC4), wherein R is ethyl. In some embodiments, a 2-modification is (C.sub.2O(S)CHRC4), wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, a 2-modification is (C.sub.2O(S)CHRC4), wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, a 2-modification is (C.sub.2O(S)CHRC4), wherein R is methyl. In some embodiments, a 2-modification is (C.sub.2O(S)CHRC4), wherein R is ethyl. In some embodiments, a 2-modification is C.sub.2O(R)CH(CH.sub.2CH.sub.3)C4. In some embodiments, a 2-modification is C.sub.2O(S)CH(CH.sub.2CH.sub.3)C4. In some embodiments, a sugar moiety is a natural DNA sugar moiety. In some embodiments, a sugar moiety is a natural DNA sugar moiety modified at 2 (2-modification). In some embodiments, a sugar moiety is an optionally substituted natural DNA sugar moiety. In some embodiments, a sugar moiety is an 2-substituted natural DNA sugar moiety.

(860) Many modified sugars can be incorporated within oligonucleotides of the present disclosure. In some embodiments, a modified sugar contains one or more substituents at the 2 position including one of the following: F; CF.sub.3, CN, N.sub.3, NO, NO.sub.2, OR, SR, or N(R).sub.2, wherein each R is independently as described in the present disclosure; O(C.sub.1-C.sub.10 alkyl), S(C.sub.1-C.sub.10 alkyl), NH(C.sub.1-C.sub.10 alkyl), or N(C.sub.1-C.sub.10 alkyl).sub.2; O(C.sub.2-C.sub.10 alkenyl), S(C.sub.2-C.sub.10 alkenyl), NH(C.sub.2-C.sub.10 alkenyl), or N(C.sub.2-C.sub.10 alkenyl).sub.2; O(C.sub.2-C.sub.10 alkynyl), S(C.sub.2-C.sub.10 alkynyl), NH(C.sub.2-C.sub.10 alkynyl), or N(C.sub.2-C.sub.10 alkynyl).sub.2; or O(C.sub.1-C.sub.10 alkylene)-O(C.sub.1-C.sub.10 alkyl), O(C.sub.1-C.sub.10 alkylene)-NH(C.sub.1-C.sub.10 alkyl) or O(C.sub.1-C.sub.10 alkylene)-NH(C.sub.1-C.sub.10 alkyl).sub.2, NH(C.sub.1-C.sub.10 alkylene)-O(C.sub.1-C.sub.10 alkyl), or N(C.sub.1-C.sub.10 alkyl)-(C.sub.1-C.sub.10 alkylene)-O(C.sub.1-C.sub.10 alkyl), wherein the alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. Examples of substituents include, and are not limited to, O(CH.sub.2).sub.nOCH.sub.3, and O(CH.sub.2).sub.nNH.sub.2, wherein n is from 1 to about 10, MOE, DMAOE, and DMAEOE. Certain modified sugars are described in WO 2001/088198, WO/2017/062862, and Martin et al., Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments, a modified sugar comprises one or more groups selected from a substituted silyl group, an RNA cleaving group, a reporter group, a fluorescent label, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, a group for improving the pharmacodynamic properties of an oligonucleotide, or other substituents having similar properties. In some embodiments, modifications are made at one or more of the the 2, 3, 4, 5, or 6 positions of a sugar, including the 3 position of a sugar on the 3-terminal nucleoside or in the 5 position of the 5-terminal nucleoside. In some embodiments, a RNA comprises a sugar which has, at the 2 position, a 2-OH, or 2-OR.sup.1, wherein OR.sup.1 is optionally substituted alkyl, including 2-OMe.

(861) In some embodiments, a 2-modification is 2-F.

(862) In some embodiments, the 2-OH of a ribose is replaced with a substituent (e.g., R.sup.2s) including one of the following: H, F; CF.sub.3, CN, N.sub.3, NO, NO.sub.2, OR, SR, or N(R).sub.2, wherein each R is independently as defined above and described herein; O(C.sub.1-C.sub.10 alkyl), S(C.sub.1-C.sub.10 alkyl), NH(C.sub.1-C.sub.10 alkyl), or N(C.sub.1-C.sub.10 alkyl).sub.2; O(C.sub.2-C.sub.10 alkenyl), S(C.sub.2-C.sub.10 alkenyl), NH(C.sub.2-C.sub.10 alkenyl), or N(C.sub.2-C.sub.10 alkenyl).sub.2; O(C.sub.2-C.sub.10 alkynyl), S(C.sub.2-C.sub.10 alkynyl), NH(C.sub.2-C.sub.10 alkynyl), or N(C.sub.2-C.sub.10 alkynyl).sub.2; or O(C.sub.1-C.sub.10 alkylene)-O(C.sub.1-C.sub.10 alkyl), O(C.sub.1-C.sub.10 alkylene)-NH(C.sub.1-C.sub.10 alkyl) or O(C.sub.1-C.sub.10 alkylene)-NH(C.sub.1-C.sub.10 alkyl).sub.2, NH(C.sub.1-C.sub.10 alkylene)-O(C.sub.1-C.sub.10 alkyl), or N(C.sub.1-C.sub.10 alkyl)-(C.sub.1-C.sub.10 alkylene)-O(C.sub.1-C.sub.10 alkyl), wherein the alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. In some embodiments, the 2-OH is replaced with H (deoxyribose). In some embodiments, the 2-OH is replaced with F. In some embodiments, the 2-OH is replaced with OR. In some embodiments, the 2-OH is replaced with OMe. In some embodiments, the 2-OH is replaced with OCH.sub.2CH.sub.2OMe.

(863) In some embodiments, a modified sugars is a sugar in locked nucleic acids (LNAs). In some embodiments, two substituents on sugar carbon atoms are taken together to form a bivalent moiety. In some embodiments, two substituents are on two different sugar carbon atoms. In some embodiments, a formed bivalent moiety has the structure of -L- as defined herein. In some embodiments, -L- is OCH.sub.2, wherein CH.sub.2 is optionally substituted. In some embodiments, -L- is OCH.sub.2. In some embodiments, -L- is OCH(Me)-. In some embodiments, -L- is OCH(Et)-. In some embodiments, -L- is between C.sub.2 and C.sub.4 of a sugar moiety. In some embodiments, a locked nucleic acid sugar has the structure indicated below, wherein R.sup.2s is OCH.sub.2C4-:

(864) ##STR00375##

(865) In some embodiments, a modified sugar is an ENA sugar or modified ENA sugar such as those described in, e.g., Seth et al., J Am Chem Soc. 2010 Oct. 27; 132 (42): 14942-14950. In some embodiments, a modified sugar is any of those found in an XNA (xenonucleic acid), for instance, arabinose, anhydrohexitol, threose, 2fluoroarabinose, or cyclohexene.

(866) In some embodiments, a modified sugar is one described in WO 2017/062862.

(867) In some embodiments, modified sugars are sugar mimetics such as cyclobutyl or cyclopentyl moieties in place of pentofuranosyl. Representative United States patents that teach preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; and 5,359,044. In some embodiments, modified sugars are sugars in which the oxygen atom within the ribose ring is replaced by nitrogen, sulfur, selenium, or carbon. In some embodiments, a modified sugar is a modified ribose wherein the oxygen atom within the ribose ring is replaced with nitrogen, and wherein the nitrogen is optionally substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc).

(868) Non-limiting examples of modified sugars include glycerol, which form glycerol nucleic acid (GNA) analogues. In some embodiments, an GNA analogue is described in Zhang, R et al., J. Am. Chem. Soc., 2008, 130, 5846-5847; Zhang L, et al., J. Am. Chem. Soc., 2005, 127, 4174-4175 and Tsai C H et al., PNAS, 2007, 14598-14603.

(869) In some embodiments, another example of a GNA derived analogue, flexible nucleic acid (FNA) based on the mixed acetal aminal of formyl glycerol, is described in Joyce G F et al., PNAS, 1987, 84, 4398-4402 and Heuberger B D and Switzer C, J. Am. Chem. Soc., 2008, 130, 412-413.

(870) Additional non-limiting examples of modified sugars include hexopyranosyl (6 to 4), pentopyranosyl (4 to 2), pentopyranosyl (4 to 3), or tetrofuranosyl (3 to 2) sugars.

(871) In some embodiments, one or more hydroxyl group in a sugar moiety is optionally and independently replaced with halogen, R N(R).sub.2, OR, or SR, wherein each R is independently as defined above and described herein.

(872) In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more), inclusive, of the sugars in an oligonucleotide, e.g., a chirally controlled oligonucleotide, an oligonucleotide of a plurality of oligonucleotide of an oligonucleotide composition, etc. are modified. In some embodiments, sugars of purine nucleosides and in some embodiments, only purine nucleosides, are modified (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more] of the purine nucleosides are modified). In some embodiments, sugars of pyrimidine nucleosides and in some embodiments, only pyrimidine nucleosides, are modified (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or more [e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more] of the pyrimidine nucleosides are modified). In some embodiments, both purine and pyrimidine nucleosides are modified.

(873) In some embodiments, modified sugars include those described in: A. Eschenmoser, Science (1999), 284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75:1416-1477; M. Egli et al, J. Am. Chem. Soc. (2006), 128 (33): 10847-56; A. Eschenmoser in Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V. Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p.293; K.-U. Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et al, Helv. Chim. Acta (1992), 75:218; J. Hunziker et al, Helv. Chim. Acta (1993), 76:259; G. Otting et al, Helv. Chim. Acta (1993), 76:2701; K. Groebke et al, Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser, Science (1999), 284:2118. Modifications to the 2 modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. In some embodiments, a modified sugar is one described in WO2012/030683. In some embodiments, a modified sugar is any modified sugar described in any of: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256; Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al. 1997 Chem. Soc. Rev. 73; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew; Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Thazha P; Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; et al. From Nucleic Acids Symposium Series (2008), 52 (1), 553-554; Singh et al. 1998 Chem. Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm. 2130-2131; Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; or WO 2016/079181.

(874) In some embodiments, a modified sugar moiety is an optionally substituted pentose or hexose moiety. In some embodiments, a modified sugar moiety is an optionally substituted pentose moiety. In some embodiments, a modified sugar moiety is an optionally substituted hexose moiety. In some embodiments, a modified sugar moiety is an optionally substituted ribose or hexitol moiety. In some embodiments, a modified sugar moiety is an optionally substituted ribose moiety. In some embodiments, a modified sugar moiety is an optionally substituted hexitol moiety.

(875) In some embodiments, a sugar is D-2-deoxyribose. In some embodiments, a sugar is beta-D-deoxyribofuranose. In some embodiments, a sugar moiety is a beta-D-deoxyribofuranose moiety. In some embodiments, a sugar is D-ribose. In some embodiments, a sugar is beta-D-ribofuranose. In some embodiments, a sugar moiety is a beta-D-ribofuranose moiety. In some embodiments, a sugar is optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose. In some embodiments, a sugar moiety is an optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose moiety. In some embodiments, a sugar moiety/unit in an oligonucleotide, nucleic acid, etc. is a sugar which comprises one or more carbon atoms each independently connected to an internucleotidic linkage, e.g., optionally substituted beta-D-deoxyribofuranose or beta-D-ribofuranose whose 5-C and/or 3-C are each independently connected to an internucleotidic linkage (e.g., a natural phosphate linkage, a modified internucleotidic linkage, a chirally controlled internucleotidic linkage, etc.).

(876) In some embodiments, each nucleoside of a provided oligonucleotide comprises a 2-O-methoxyethyl sugar modification.

(877) In some embodiments, the oligonucleotide composition comprises at least one locked nucleic acid (LNA)nucleotide. In some embodiments, the oligonucleotide composition comprises at least one modified nucleotide comprising a modified sugar moiety which is modified at the 2-position.

(878) In some embodiments, the oligonucleotide composition comprises modified sugar moiety which comprises a 2-substituent selected from the group consisting of: H, OR, R, halogen, SH, SR, NH.sub.2, NHR, NR.sub.2, and ON, wherein R is an optionally substituted C.sub.1-C.sub.6 alkyl, alkenyl, or alkynyl and halogen is F, Cl, Br or I.

(879) In some embodiments, a modified nucleobase, sugar, nucleoside, nucleotide, and/or modified internucleotidic linkage is selected from those described in Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Gryaznov, S.; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Jones et al. J. Org. Chem. 1993, 58, 2983; Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Nielsen et al. 1997 Chem. Soc. Rev. 73; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Singh et al. 1998 Chem. Comm. 1247-1248; Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Sorensen 2003 Chem. Comm. 2130-2131; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396; Jepsen et al. 2004 Oligo. 14: 130-146; Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256; WO 20070900071; Seth et al., Nucleic Acids Symposium Series (2008), 52 (1), 553-554; Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; WO 2016/079181; U.S. Pat. Nos. 6,326,199; 6,066,500; and 6,440,739.

(880) In some embodiments, sugars and nucleosides include 6-modified bicyclic sugars and nucleosides, respectively, that have either (R) or (S)-chirality at the 6-position, e.g., those described in U.S. Pat. No. 7,399,845. In other embodiments, sugars and nucleosides include 5-modified bicyclic sugars and nucleosides, respectively, that have either (R) or (S)-chirality at the 5-position, e.g., those described in US Patent Application Publication No. 20070287831.

(881) In some embodiments, modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the sugars, nucleobases, nucleosides, nucleotides, and internucleotidic linkages of each of which are incorporated by reference.

(882) In some embodiments, modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages are those described in any of: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al. 1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12: 3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256; Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Morita et al. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al. 1997 Chem. Soc. Rev. 73; Nielsen et al. 1997 J. Chem. Soc. Perkins Transl. 1: 3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan et al. 2012 Chem. Comm. 48: 8195-8197; Petersen et al. 2003 TRENDS Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et al. 2009 J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol. Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew; Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Thazha P; Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat, Balkrishen; et al. From Nucleic Acids Symposium Series (2008), 52 (1), 553-554; Singh et al. 1998 Chem. Comm. 1247-1248; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Singh et al. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm. 2130-2131; Ts'o et al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Van Aerschot et al. 1995 Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; and WO 2016/079181.

(883) In some embodiments, modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages include, or include those in, HNA, PNA, 2-Fluoro N3-P5-phosphoramidate, LNA, beta-D-oxy-LNA, 2-O,3-C-linked bicyclic, PS-LNA, beta-D-thio-LNA, beta-D-amino-LNA, xylo-LNA [c], alpha-L-LNA, ENA, beta-D-ENA, amide-linked LNA, methylphosphonate-LNA, (R, S)-cEt, (R, S)-cMOE, (R, S)-5-Me-LNA, S-Me cLNA, Methylene-cLNA, 3-Me-alpha-L-LNA, R-6-Me-alpha-L-LNA, S-5-Me-alpha-L-LNA, or R-5-Me-alpha-L-LNA. Certain modified sugars, nucleobases, nucleosides, nucleotides, and/or internucleotidic linkages are described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, US20130178612, US20150211006, U.S. Pat. No. 9,598,458, US20170037399, WO 2017/015555, WO 2017/062862, the modified sugars, nucleobases, nucleosides, nucleotides, and internucleotidic linkages of each of which are incorporated herein by reference.

(884) Dystrophin

(885) In some embodiments, the present disclosure provides technologies, e.g., oligonucleotides, compositions, methods, etc., related to the dystrophin (DMD) gene or a product encoded thereby (a transcript, a protein (e.g., various variants of the dystrophin protein), etc.). In some embodiments, the base sequence of an oligonucleotide is or comprise a sequence which sequence is, or is complementary (e.g., 85%, 90%, 95%, 100%; in many embodiments, 100%) to, a sequence in the DMD gene or a product thereof (e.g., a transcript, mRNA, etc.) (such an oligonucleotide-DMD oligonucleotide). In some embodiments, such a sequence in the DMD gene or a product thereof comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35 or more nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 10 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 15 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 16 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 17 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 18 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 19 nucleobases. In some embodiments, such a sequence in the DMD gene or a product thereof comprises at least 20 nucleobases. In some embodiments, the present disclosure provides technologies, including DMD oligonucleotides and compositions an d methods of use thereof, for treatment of muscular dystrophy, including but not limited to, Duchenne Muscular Dystrophy (also abbreviated as DMD) and Becker Muscular Dystrophy (BMD). In some embodiments, DMD comprises one or more mutations. In some embodiments, such mutations are associated with reduced biological functions of dystrophin protein in a subject suffering from or susceptible to muscular dystrophy.

(886) In some embodiments, the dystrophin (DMD) gene or a product thereof, or a variant or portion thereof, may be referred to as DMD, BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85, or dystrophin; External IDs: OMIM: 300377 MGI: 94909; HomoloGene: 20856; GeneCards: DMD; In Human: Entrez: 1756; Ensembl: ENSG00000198947; UniProt: P11532; RefSeq (mRNA): NM_000109; NM_004006; NM_004007; NM_004009; NM_004010; RefSeq (protein): NP_000100; NP_003997; NP_004000; NP_004001; NP_004002; Location (UCSC): Chr X: 31.1-33.34 Mb; In Mouse: Entrez: 13405; Ensembl: ENSMUSG00000045103; UniProt: P11531; RefSeq (mRNA): NM_007868; NM_001314034; NM_001314035; NM_001314036; NM_001314037; RefSeq (protein): NP_001300963; NP_001300964; NP 001300965; NP_001300966; NP_001300967; Location (UCSC): Chr X: 82.95-85.21 Mb.

(887) The DMD gene reportedly contains 79 exons distributed over 2.3 million bp of genetic real estate on the X chromosome; however, only approximately 14,000 bp (<1%) is reported to be used for translation into protein (coding sequence). It is reported that about 99.5% of the genetic sequence, the intronic sequences, is spliced out of the 2.3 million bp initial heteronuclear RNA transcript to provide a mature 14,000 bp mRNA that includes all key information for dystrophin protein production. In some embodiments, patients with DMD have mutation(s) in the DMD gene that prevent the appropriate construction of the wild-type DMD mRNA and/or the production of the wild-type dystrophin protein, and patients with DMD often show marked dystrophin deficiency in their muscle.

(888) In some embodiments, a dystrophin transcript, e.g., mRNA, or protein encompasses those related to or produced from alternative splicing. For example, sixteen alternative transcripts of the dystrophin gene were reported following an analysis of splicing patterns of the DMD gene in skeletal muscle, brain and heart tissues. Sironi et al. 2002 FEBS Letters 517: 163-166.

(889) It is reported that dystrophin has several isoforms. In some embodiments, dystrophin refers to a specific isoform. At least three full-length dystrophin isoforms have been reported, each controlled by a tissue-specific promoter. Klamut et al. 1990 Mol. Cell. Biol. 10: 193-205; Nudel et al. 1989 Nature 337: 76-78; Gorecki et al. 1992 Hum. Mol. Genet. 1: 505-510. The muscle isoform is reportedly mainly expressed in skeletal muscle but also in smooth and cardiac muscles [Bies, R. D., Phelps, S. F., Cortez, M. D., Roberts, R., Caskey, C. T. and Chamberlain, J. S. 1992 Nucleic Acids Res. 20: 1725-1731], the brain dystrophin is reportedly specific for cortical neurons but can also be detected in heart and cerebellar neurons, while the Purkinje-cell type reportedly accounts for nearly all cerebellar dystrophin [Gorecki et al. 1992 Hum. Mol. Genet. 1: 505-510]. Alternative splicing reportedly provides a means for dystrophin diversification: the 3 region of the gene reportedly undergoes alternative splicing resulting in tissue-specific transcripts in brain neurons, cardiac Purkinje fibers, and smooth muscle cells [Bies et al. 1992 Nucleic Acids Res. 20: 1725-1731; and Feener et al. 1989 Nature 338: 509-511] while 12 patterns of alternative splicing have been reported in the 5 region of the gene in skeletal muscle [Surono et al. 1997 Biochem. Biophys. Res. Commun. 239: 895-899].

(890) In some embodiments, a dystrophin mRNA, gene or protein is a revertant version. Among others, revertant dystrophins were reported in, for example: Hoffman et al. 1990 J. Neurol. Sci. 99: 9-25; Klein et al. 1992 Am. J. Hum. Genet. 50: 950-959; and Chelly et al. 1990 Cell 63: 1239-1348; Arahata et al. 1998 Nature 333: 861-863; Bonilla et al. 1988 Cell 54: 447-452; Fanin et al. 1992 Neur. Disord. 2: 41-45; Nicholson et al. 1989 J. Neurol. Sci. 94: 137-146; Shimizu et al. 1988 Proc. Jpn. Acad. Sci. 64: 205-208; Sicinzki et al. 1989 Science 244: 1578-1580; and Sherratt et al. Am. J. Hum. Genet. 53: 1007-1015.

(891) Various mutations in the DMD gene can and/or were reported to cause muscular dystrophy.

(892) Muscular Dystrophy

(893) Compositions comprising one or more DMD oligonucleotides described herein can be used to treat muscular dystrophy. In some embodiments, muscular dystrophy (MD) is any of a group of muscle conditions, diseases, or disorders that results in (increasing) weakening and breakdown of skeletal muscles over time. The conditions, diseases, or disorders differ in which muscles are primarily affected, the degree of weakness, when symptoms begin, and how quickly symptoms worsen. Many MD patients will eventually become unable to walk. In many cases musuclar dystrophy is fatal. Some types are also associated with problems in other organs, including the central nervous system. In some embodiments, the muscular dystrophy is Duchenne (Duchenne's) Muscular Dystrophy (DMD) or Becker (Becker's) Muscular Dystrophy (BMD).

(894) In some embodiments, a symptom of Duchenne Muscular Dystrophy is muscle weakness associated with muscle wasting, with the voluntary muscles being first affected, especially those of the hips, pelvic area, thighs, shoulders, and calves. Muscle weakness can also occur later, in the arms, neck, and other areas. Calves are often enlarged. Symptoms usually appear before age six and may appear in early infancy. Other physical symptoms are: awkward manner of walking, stepping, or running (in some cases, patients tend to walk on their forefeet, because of an increased calf muscle tone), frequent falls, fatigue, difficulty with motor skills (e.g., running, hopping, jumping), lumbar hyperlordosis, possibly leading to shortening of the hip-flexor muscles, unusual overall posture and/or manner of walking, stepping, or running, muscle contractures of Achilles tendon and hamstrings impair functionality, progressive difficulty walking, muscle fiber deformities, pseudohypertrophy (enlarging) of tongue and calf muscles, higher risk of neurobehavioral disorders (e.g., ADHD), learning disorders (e.g., dyslexia), and non-progressive weaknesses in specific cognitive skills (e.g., short-term verbal memory), which are believed to be the result of absent or dysfunctional dystrophin in the brain, eventual loss of ability to walk (usually by the age of 12), skeletal deformities (including scoliosis in some cases), and trouble getting up from lying or sitting position.

(895) In some embodiments, Becker muscular dystrophy (BMD) is caused by mutations that give rise to shortened but in-frame transcripts resulting in the production of truncated but partially functional protein(s). Such partially functional protein(s) were reported to retain the critical amino terminal, cysteine rich and C-terminal domains but usually lack elements of the central rod domains which were reported to be of less functional significance. England et al. 1990 Nature, 343, 180-182.

(896) In some embodiments, BMD phenotypes range from mild DMD to virtually asymptomatic, depending on the precise mutation and the level of dystrophin produced. Yin et al. 2008 Hum. Mol. Genet. 17: 3909-3918.

(897) In some embodiments, dystrophy patients with out-of-frame mutations are generally diagnosed with the more severe Duchenne Muscular Dystrophy, and dystrophy patients with in-frame mutations are generally diagnosed with the less severe Becker Muscular Dystrophy. However, a minority of patients with in-frame deletions are diagnosed with Duchenne Muscular Dystrophy, including those with deletion mutations starting or ending in exons 50 or 51, which encode part of the hinge region, such as deletions of exons 47 to 51, 48 to 51, and 49 to 53. Without wishing to be bound by any particular theory, the present disclosure notes that the patient-to-patient variability in disease severity despite the presence of the same exon deletion reportedly may be related to the effect of the specific deletion breakpoints on mRNA splicing efficiency and/or patterns; translation or transcription efficiency after genome rearrangement; and stability or function of the truncated protein structure. Yokota et al. 2009 Arch. Neurol. 66: 32.

(898) Exon Skipping as a Treatment for Muscular Dystrophy

(899) In some embodiments, a treatment for muscular dystrophy comprises the use of a DMD oligonucleotide which is capable of mediating skipping of one or more Dystrophin exons. In some embodiments, the present disclosure provides methods for treatment of muscular dystrophy comprising administering to a subject suffering therefrom or susceptible thereto an DMD oligonucleotide, or a composition comprising a DMD oligonucleotide. Particularly, among other things, the present disclosure demonstrates that chirally controlled oligonucleotide/chirally controlled oligonucleotide compositions are unexpectedly effective for modulating exon skipping compared to otherwise identical but non-chirally controlled oligonucleotide/oligonucleotide compositions. In some embodiments, the present disclosure demonstrates incorporation of one or more non-negatively charged internucleotidic linkage can greatly improve delivery and/or overall exon skipping efficiency.

(900) In some embodiments, a treatment for muscular dystrophy employs the use of a DMD oligonucleotide, wherein the oligonucleotide is capable of providing skipping of one or more exons. Skipping of one or more (e.g., multiple) DMD exons can, for example, remove a mutated exon(s), or compensate for a mutation(s) (e.g., restoring the reading frame if the mutation is a frameshift mutation) in an exon which is not skipped. In some embodiments, a DMD oligonucleotide is capable of mediating the skipping of an exon which comprises a mutation (e.g., a frameshift, insertion, deletion, missense, or nonsense mutation, or other mutation), wherein the skipping of the exon maintains (or restores) the proper reading frame of the DMD gene, and translation produces a truncated but functional (or largely functional) DMD protein. In some embodiments, a DMD oligonucleotide compensates for an exon comprising a frameshift mutation by providing skipping of a different exon (not the one comprising the frameshift mutation), and thus restoring the reading frame of the DMD gene. In some embodiments, a patient having muscular dystrophy has a frameshift mutation in one exon of the DMD gene; and this patient is treated with a DMD oligonucleotide which does not cause skipping of the exon having the mutation, but causes skipping of a different exon, which restores the reading frame of the DMD gene, so that a functional DMD protein is produced (and, if the deleted exon is 3 to the exon which has the frameshift mutation, this functional DMD protein will generally have an amino acid of a normal DMD protein, except for a sequence of amino acids not normally found in DMD, spanning from the frameshift mutation to the exon which is 3 to the deleted exon).

(901) In some embodiments, a composition comprising a DMD oligonucleotide is useful for treatment of a Dystrophin-related disorder of the central nervous system. In some embodiments, the present disclosure pertains to a method of treatment of a Dystrophin-related disorder of the central nervous system, wherein the method comprises the step of administering a therapeutically effective amount of a DMD oligonucleotide to a patient suffering from a Dystrophin-related disorder of the central nervous system. In some embodiments, a DMD oligonucleotide is administered outside the central nervous system (as non-limiting examples, intravenously or intramuscularly) to a patient suffering from a Dystrophin-related disorder of the central nervous system, and the DMD oligonucleotide is capable of passing through the blood-brain barrier into the central nervous system. In some embodiments, a DMD oligonucleotide is administered directly into the central nervous system (as non-limiting example, via intrathecal, intraventricular, intracranial, etc., delivery).

(902) In some embodiments, a Dystrophin-related disorder of the central nervous system, or a symptom thereof, can be any one or more of: decreased intelligence, decreased long term memory, decreased short term memory, language impairment, epilepsy, autism spectrum disorder, attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder, learning problem, behavioral problem, a decrease in brain volume, a decrease in grey matter volume, lower white matter fractional anisotropy, higher white matter radial diffusivity, an abnormality of skull shape, or a deleterious change in the volume or structure of the hippocampus, globus pallidus, caudate putamen, hypothalamus, anterior commissure, periaqueductal gray, internal capsule, amygdala, corpus callosum, septal nucleus, nucleus accumbens, fimbria, ventricle, or midbrain thalamus. In some embodiments, a patient exhibiting muscle-related symptoms of muscular dystrophy also exhibits symptoms of a Dystrophin-related disorder of the central nervous system.

(903) In some embodiments, a Dystrophin-related disorder of the central nervous system is related to, associated with and/or caused by an abnormality in the level, activity, expression and/or distribution of a gene product of the Dystrophin gene, such as full-length Dystrophin or a smaller isoform of Dystrophin, including, but not limited to, Dp260, Dp140, Dp116, Dp71 or Dp40. In some embodiments, a DMD oligonucleotide is administered into the central nervous system of a muscular dystrophy patient in order to ameliorate one or more systems of a Dystrophin-related disorder of the central nervous system. In some embodiments, a Dystrophin-related disorder of the central nervous system is related to, associated with and/or caused by an abnormality in the level, activity, expression and/or distribution of a gene product of the Dystrophin gene, such as full-length Dystrophin or a smaller isoform of Dystrophin, including, but not limited to, Dp260, Dp140, Dp116, Dp71 or Dp40. In some embodiments, administration of a DMD oligonucleotide to a patient suffering from a Dystrophin-related disorder of the central nervous system increases the level, activity, and/or expression and/or improves the distribution of a gene product of the Dystrophin gene.

(904) In some embodiments, the present disclosure provides technologies for modulating dystrophin pre-mRNA splicing, whereby selected exons are excised to either remove nonsense mutations or restore the reading frame around frameshifting mutations from the mature mRNA. In some embodiments, a DMD oligonucleotide capable of skipping an exon is capable of restoring the reading frame.

(905) As a non-limiting example, in a patient with Duchenne Muscular Dystrophy who has a deletion of exon 50, an out-of-frame transcript is generated in which exon 49 is spliced to exon 51. As a result, a stop codon is generated in exon 51, which prematurely aborts dystrophin synthesis. In some embodiments, the present disclosure provides oligonucleotides that can mediate skipping of exon 51, restore the open reading frame of the transcript, and allow the production of a truncated dystrophin similar to that in patients with Becker muscular dystrophy (BMD).

(906) In some embodiments, in a DMD patient, a DMD gene comprises an exon comprising a mutation, and the disorder is at least partially treated by skipping of one or more exons (e.g., the exon comprising the mutation, or an exon adjacent to the exon comprising the mutation, or a set of consecutive exons, including the exon comprising the mutation).

(907) In some embodiments, in a DMD patient, a DMD gene or transcript has a mutation in an exon(s), which is a missense or nonsense mutation and/or deletion, insertion, inversion, translocation or duplication. In some embodiments, in a DMD patient, a DMD gene or transcript has a mutation in an exon(s) which results in a frameshift, premature stop codon, or otherwise perturbation of the proper reading frame.

(908) In some embodiments, in a treatment for muscular dystrophy, an exon of DMD is skipped, wherein the exon encodes a string of amino acids not essential for DMD protein function, or whose skipping can provide a fully or partially functional DMD protein. In some embodiments, in a treatment for muscular dystrophy, an exon of DMD is skipped, wherein the exon(s) skipped include an exon which comprises a mutation or is adjacent to (e.g., flanking) an exon comprising a mutation, or wherein multiple exons are skipped, the skipped exons optionally include an exon comprising a mutation. In some embodiments, in a treatment for muscular dystrophy, two or more exons are skipped, wherein the exons skipped include an exon which comprises a mutation or is adjacent to (e.g., flanking) an exon comprising a mutation. In some embodiments, in a treatment for muscular dystrophy, an exon comprises a frameshift mutation, and the skipping of a different exon (while leaving the exon with the frameshift mutation in place) restores the proper reading frame.

(909) In some embodiments, in a treatment for muscular dystrophy, a DMD oligonucleotide is capable of mediating skipping of one or more DMD exons, thereby either restoring or maintaining the proper reading frame, and/or creating an artificially internally truncated DMD which provides at least partially improved or fully restored biological activity.

(910) In some embodiments, an DMD oligonucleotide skips an exon(s) which is not exon 64 and exon 70, portions of which are reportedly important for protein function, and/or which is not first or the last exon. In some embodiments, an DMD oligonucleotide skips an exon(s), but skipping of the exon(s) does not cause deletion of one or more or all actin-binding sites in the N-terminal region.

(911) In some embodiments, an internally truncated DMD protein produced from a dystrophin transcript with a skipped exon(s) is more functional than a terminally truncated DMD protein e.g., produced from a dystrophin transcript with an out-of-frame deletion.

(912) In some embodiments, an internally truncated DMD protein produced from a dystrophin transcript with a skipped exon(s) is more resistant to nonsense-mediated decay, which can degrade a terminally truncated DMD protein, e.g., produced from a dystrophin transcript with an out-of-frame deletion.

(913) In some embodiments, a treatment for muscular dystrophy employs the use of a DMD oligonucleotide, wherein the oligonucleotide is capable of providing skipping of one or more exons. Skipping of one or more (e.g., multiple) DMD exons can, for example, remove a mutated exon, or compensate for a mutation (e.g., restoring from for a frameshift mutation) in an exon which is not skipped.

(914) In some embodiments, the present disclosure encompasses the recognition that the nature and location of a DMD mutation may be utilized to design exon-skipping strategy. In some embodiments, if a DMD patient has a mutation in an exon, skipping of the mutated exon can produce an internally truncated (internally shortened) but at least partially functional DMD protein product.

(915) In some embodiments, a DMD patient has a mutation which alters splicing of a DMD transcript, e.g., by inactivating a site required for splicing, or activating a cryptic site so that it becomes active for splicing, or by creating an alternative (e.g., unnatural) splice site. In some embodiments, such a mutation causes production of proteins with low or no activities. In some embodiments, splicing modulation, e.g., exon skipping, suppression of such a mutation, etc., can be employed to remove or reduce effects of such a mutation, e.g., by restoring proper splicing to produce proteins with restored activities, or producing an internally truncated dystrophin protein with improved or restored activities, etc.

(916) In some embodiments, a DMD patient has a mutation which is a duplication of one or several exons, and the present disclosure provides exon skipping technologies to delete the duplication and/or to restore the reading frame.

(917) In some embodiments, a DMD patient has a mutation which causes the skipping of an exon, which in turn can cause a frameshift. In some embodiments, the present disclosure provides technologies that can provide skipping of an additional exon(s) to restore the reading frame. For example, deletion of exon 51, which causes a frame shift, may be addressed by skipping of exon 50 or 52, which restores the reading frame. In some embodiments, a DMD patient has a mutation in one exon which causes a frame shift, and a deletion of a different exon(s) (e.g., a different exon, or an adjacent or flanking exon(s) immediately 5 or 3 to the mutated exon) restores the reading frame.

(918) In some embodiments, restoring the reading frame can convert an out-of-frame mutation to an in-frame mutation; in some embodiments, in humans, such a change can transform severe Duchenne Muscular Dystrophy into milder Becker Muscular Dystrophy.

(919) In some embodiments, a DMD patient or a patient suspected to have DMD is analyzed for DMD genotype prior to administration of a composition comprising a DMD oligonucleotide.

(920) In some embodiments, a DMD patient or a patient suspected to have DMD is analyzed for DMD phenotype prior to administration of a composition comprising a DMD oligonucleotide.

(921) In some embodiments, a DMD patient is analyzed for genotype and phenotype to determine the relationship of DMD genotype and DMD phenotype prior to administration of a composition comprising a DMD oligonucleotide.

(922) In some embodiments, a patient is genetically verified to have dystrophy prior to administration of a composition comprising a DMD oligonucleotide.

(923) In some embodiments, analysis of DMD genotype or genetic verification of DMD or a patient comprises determining if the patient has one or more deleterious mutations in DMD.

(924) In some embodiments, analysis of DMD genotype or genetic verification of DMD or a patient comprises determining if the patient has one or more deleterious mutations in DMD and/or analyzing DMD splicing and/or detecting splice variants of DMD, wherein a splice variant is produced by an abnormal splicing of DMD.

(925) In some embodiments, analysis of DMD genotype or genetic verification of DMD informs the selection of a composition comprising a DMD oligonucleotide useful for treatment.

(926) In some embodiments, an abnormal or mutant DMD gene or a portion thereof is removed or copied from a patient or a patient's cell(s) or tissue(s) and the abnormal or mutant DMD gene, or a portion thereof comprising the abnormality or mutation, or a copy thereof, is inserted into a cell. In some embodiments, this cell can be used to test various compositions comprising a DMD oligonucleotide to predict if such a composition would be useful as a treatment for the patient. In some embodiments, the cell is a myoblast or myotubule.

(927) In some embodiments, an individual or patient can produce, prior to treatment with a DMD oligonucleotide, one or more splice variants of DMD, often each variant being produced at a very low level. In some embodiments, a method such as that described in Example 20 can be used to detect low levels of splice variants being produced in a patient prior to, during or after administration of a DMD oligonucleotide.

(928) In some embodiments, a patient and/or the tissues thereof are analyzed for production of various splicing variants of a DMD gene prior to administration of a composition comprising a DMD oligonucleotide.

(929) In some embodiments, the present disclosure provides methods for designing a DMD oligonucleotide (e.g., an oligonucleotide capable of mediating skipping of one or more exons of DMD). In some embodiments, the present disclosure utilizes rationale design described herein and optionally sequence walks to design oligonucleotides, e.g., for testing exon skipping in one or more assays and/or conditions. In some embodiments, an efficacious oligonucleotide is developed following rational design, including using various information of a given biological system.

(930) In some embodiments, in a method for developing DMD oligonucleotides, oligonucleotides are designed to anneal to one or more potential splicing-related motifs and then tested for their ability to mediate exon skipping. In some embodiments, splicing-related motifs include, but are not limited to, any one or more of: an acceptor, exon recognition sequence (ERS), exonic splice enhancer (ESE) site, splicing enhancer sequence (SES), branch point sequence, and donor splice site of a target exon. Certain sequences that may be involved in splicing were reported in, for example: Disset et al. 2006 Human Mol. Gen. 15: 999-1013.

(931) In some embodiments, software packages, such as RESCUE-ESE, ESEfinder, and the PESX server, may be utilized to predict putative ESE sites (Fairbrother et al. 2002 Science 297: 1007-1013; Cartegni et al. 2003 Nat. Struct. Biol. 120-125; Zhang and Chasin 2004 Gen. Dev. 18: 1241-1250; Smith et al. 2006 Hum. Mol. Genet. 15: 2490-2508).

(932) In some embodiments, a DMD oligonucleotide which targets or interacts with an acceptor, exon recognition sequence (ERS), exonic splice enhancer (ESE) site, or donor splice site of a DMD exon does not interact or significantly interact with a sequence in another (e.g., off-target) gene.

(933) In some embodiments, in a rational approach to DMD oligonucleotide design, oligonucleotides are designed with consideration of secondary structures of dystrophin transcripts, e.g., mRNA. Designed oligonucleotide can then be assessed for exon skipping. A number of effective DMD oligonucleotides have been designed using rational approaches described in the present disclosure.

(934) In some embodiments, alternatively or additionally, sequence walk, e.g., of an exon sequence can be performed to search for efficacious DMD oligonucleotide sequences.

(935) In some embodiments, provided methods comprise sequence walking. In some embodiments, a set of overlapping oligonucleotides is generated. In some embodiments, oligonucleotides in a set have the same length, and the 5 ends of the oligonucleotides in the set are evenly spaced apart. In some embodiments, a set of overlapping oligonucleotides encompasses an entire exon or a portion(s) thereof. The 5 ends of the oligonucleotides in a walk can be evenly spaced at a suitable distance, e.g., 1 base apart, 2 bases apart, 3 bases apart, etc. Among other things, the present disclosure demonstrates that sequences can be optimized and in combination with chemistry and/or stereochemistry technologies of the present disclosure, highly effective oligonucleotides (and compositions and methods of use thereof) can be prepared.

(936) Example Technologies for Assessing Oligonucleotides and Oligonucleotide Compositions

(937) Various technologies for assessing properties and/or activities of oligonucleotides can be utilized in accordance with the present disclosure, e.g., US20170037399, WO 2017/015555, WO 2017/015575, WO 2017/192664, WO 2017/062862, WO 2017/192679, WO 2017/210647, etc.

(938) For example, DMD oligonucleotides can be evaluated for their ability to mediate exon skipping in various assays, including in vitro and in vivo assays, in accordance with the present disclosure. In vitro assays can be performed in various test cells described herein or known in the art, including but not limited to, A48-50 Patient-Derived Myoblast Cells. In vivo tests can be performed in test animals described herein or known in the art, including but not limited to, a mouse, rat, cat, pig, dog, monkey, or non-human primate.

(939) As non-limiting examples, a number of assays are described below for assessing properties/activities of DMD oligonucleotides. Various other suitable assays are available and may be utilized to assess oligonucleotide properties/activities, including those of oligonucleotides not designed for exon skipping (e.g., for oligonucleotides that may involve RNase H for reducing levels of target transcripts, assays described in US20170037399, WO 2017/015555, WO 2017/015575, WO 2017/192664, WO 2017/192679, WO 2017/210647, etc.).

(940) A DMD oligonucleotide can be evaluated for its ability to mediate skipping of an exon in the Dystrophin RNA, which can be tested, as non-limiting examples, using nested PCR, qRT-PCR, and/or sequencing.

(941) A DMD oligonucleotide can be evaluated for its ability to mediate protein restoration (e.g., production of an internally truncated protein lacking the amino acids corresponding to the codons encoded in the skipped exon, which has improved functions compared to proteins (if any) produced prior to exon skipping), which can be evaluated by a number of methods for protein detection and/or quantification, such as western blot, immunostaining, etc. Antibodies to dystrophin are commercially available or if desired, can be developed for desired purposes.

(942) A DMD oligonucleotide can be evaluated for its ability to mediate production of a stable restored protein. Stability of restored protein can be tested, in non-limiting examples, in assays for serum and tissue stability.

(943) A DMD oligonucleotide can be evaluated for its ability to bind protein, such as albumin. Example related technologies include those described, e.g., in WO 2017/015555, WO 2017/015575, etc.

(944) A DMD oligonucleotide can be evaluated for immuno activity, e.g., through assays for cytokine activation, complement activation, TLR9 activity, etc. Example related technologies include those described, e.g., in WO 2017/015555, WO 2017/015575, WO 2017/192679, WO 2017/210647, etc.

(945) In some embodiments, efficacy of a DMD oligonucleotide can be tested, e.g., in in silico analysis and prediction, a cell-free extract, a cell transfected with artificial constructs, an animal such as a mouse with a human Dystrophin transgene or portion thereof, normal and dystrophic human myogenic cell lines, and/or clinical trials. It may be desirable to utilize more than one assay, as normal and dystrophic human myogenic cell lines may sometimes produce different efficacy results under certain conditions (Mitrpant et al. 2009 Mol. Ther. 17: 1418).

(946) In some embodiments, DMD oligonucleotides can be tested in vitro in cells. In some embodiments, testing in vitro in cells involves gymnotic delivery of the oligonucleotide(s), or delivery using a delivery agent or transfectant, many of which are known in the art and may be utilized in accordance with the present disclosure.

(947) In some embodiments, DMD oligonucleotides can be tested in vitro in normal human skeletal muscle cells (hSkMCs). See, for example, Arechavala et al. 2007 Hum. Gene Ther. 18: 798-810.

(948) In some embodiments, DMD oligonucleotides can be tested in a muscle explant from a DMD patient. Muscle explants from DMD patients are reported in, for example, Fletcher et al. 2006 J. Gene Med. 8: 207-216; McClorey et al. 2006 Neur. Dis. 16: 583-590; and Arechavala et al. 2007 Hum. Gene Ther. 18: 798-810.

(949) In some embodiments, cells are or comprise cultured muscle cells from DMD patients. See, for example: Aartsma-Rus et al. 2003 Hum. Mol. Genet. 8: 907-914.

(950) In some embodiments, an individual DMD oligonucleotide may demonstrate experiment-to-experiment variability in its ability to skip an exon under certain circumstances. In some embodiments, an individual DMD oligonucleotide can demonstrate variability in its ability to skip an exon(s) depending on which cells are used, the growth conditions, and other experimental factors. To control variations, typically oligonucleotides to be tested and control oligonucleotides are assayed under the same or substantially the same conditions.

(951) In vitro experiments also include those conducted with patient-derived myoblasts. Certain results from such experiments were described herein. In certain such experiments, cells were cultured in skeletal growth media to keep them in a dividing/immature myoblast state. The media was then changed to differentiation media (containing insulin and 2% horse serum) concurrent with spiking oligonucleotides in the media for dosing. The cells differentiated into myotubes as they were getting dosed for a suitable period of time, e.g., a total of 4d for RNA experiments and 6d for protein experiments (such conditions referenced as Od pre-differentiation (0d+4d for RNA, 0d+6d for protein)).

(952) Without wishing to be bound by any particular theory, the present disclosure notes that it may be desirable to know if DMD oligonucleotides are able to enter mature myotubes and induce skipping in these cells as well as immature cells. In some embodiments, the present disclosure provided assays to test effects of DMD oligonucleotides in myotubes. In some embodiments, a dosing schedule different from the Od pre-differentiation was used, wherein the myoblasts were pre-differentiated into myotubes in differentiation media for several days (4d or 7d or 10d) and then DMD oligonucleotides were administered. Certain related protocols are described in Example 19.

(953) In some embodiments, the present disclosure demonstrated that, in the pre-differentiation experiments, DMD oligonucleotides (excluding those which are PMOs) usually give about the same level of RNA skipping and dystrophin protein restoration, regardless of the number of days cells were cultured in differentiation media prior to dosing. In some embodiments, the present disclosure provides oligonucleotides that may be able to enter and be active in myoblasts and in myotubes. In some embodiments, a DMD oligonucleotide is tested in vitro in 45-52 DMD patient cells (also designated D45-52 or del45-52) or 52 DMD patient cells (also designated D52 or del52) with 0, 4 or 7 days of pre-differentiation.

(954) In some embodiments, DMD oligonucleotides can be tested in any one or more of various animal models, including non-mammalian and mammalian models; including, as non-limiting examples, Caenorhabditis, Drosophila, zebrafish, mouse, rat, cat, dog and pig. See, for example, a review in McGreevey et al. 2015 Dis. Mod. Mech. 8:195-213.

(955) Example use of mdx mice is reported in, for example: Lu et al. 2003 Nat. Med. 9: 1009; Jearawiriyapaisarn et al. 2008 Mol. Ther., 16, 1624-1629; Yin et al. 2008 Hum. Mol. Genet., 17, 3909-3918; Wu et al. 2009 Mol. Ther., 17, 864-871; Wu et al. 2008 Proc. Natl Acad. Sci. USA, 105, 14814-14819; Mann et al. 2001 Proc. Nat. Acad. Sci. USA 98: 42-47; and Gebski et al. 2003 Hum. Mol. Gen. 12: 1801-1811.

(956) Efficacy of DMD oligonucleotides can be tested in dogs, such as the Golden Retriever Muscular Dystrophy (GRMD) animal model. Lu et al. 2005 Proc. Natl. Acad. Sci. USA 102: 198-203; Alter et al. 2006 Nat. Med. 12: 175-7; McClorey et al. 2006 Gene Ther. 13: 1373-81; and Yokota et al. 2012 Nucl. Acid Ther. 22: 306.

(957) A DMD oligonucleotide can be evaluated in vivo in a test animal for efficient delivery to various tissues (e.g., skeletal, heart and/or diaphragm muscle); this can be tested, in non-limiting examples, by hybridization ELISA and tests for distribution in animal tissue.

(958) A DMD oligonucleotide can be evaluated in vivo in a test animal for plasma PK; this can be tested, as non-limiting examples, by assaying for AUC(area under the curve) and half-life.

(959) In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration a muscle of a test animal.

(960) In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration into the gastrocnemius muscle of a test animal.

(961) In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration into the gastrocnemius muscle of a mouse.

(962) In some embodiments, DMD oligonucleotides can be tested in vivo, via an intramuscular administration into the gastrocnemius muscle of a mouse model transgenic for the entire human dystrophin locus. See, for example: Bremmer-Bout et al. 2004 Mol. Ther. 10, 232-240.

(963) Additional tests which can be performed to evaluate the efficacy of DMO oligonucleotides include centrally nucleated fiber counts and dystrophin-positive fiber counts, and functional grip strength analysis. See, as non-limiting examples, experimental protocols reported in: Yin et al. 2009 Hum. Mol. Genet. 18: 4405-4414.

(964) Additional methods of testing DMD oligonucleotides include, as non-limiting example, methods reported in: Kinali et al. 2009 Lancet 8: 918; Bertoni et al. 2003 Hum. Mol. Gen. 12: 1087-1099.

(965) Certain Embodiments of Oligonucleotides and Compositions Thereof

(966) Among other things, the present disclosure provides oligonucleotides, and compositions and methods of use thereof, useful for targeting various genes, including products encoded thereby and/or conditions, diseases and/or disorders associated therewith. In some embodiments, the present disclosure provides oligonucleotides, and compositions and methods of use thereof, for DMD. In some embodiments, the present disclosure provides a DMD oligonucleotide, wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein. In some embodiments, the present disclosure provides a DMD oligonucleotide, wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein, and wherein the DMD oligonucleotide is less than about 50 bases long. In some embodiments, the present disclosure provides an oligonucleotide or an oligonucleotide composition which comprises a non-negatively charged internucleotidic linkage.

(967) In some embodiments, the present disclosure provides a chirally controlled composition of a DMD oligonucleotide (a plurality of DMD oligonucleotides), wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein. In some embodiments, the present disclosure provides a chirally controlled composition of a DMD oligonucleotide, wherein the base sequence of the DMD oligonucleotide is or comprises at least 15 contiguous bases of the sequence of any DMD oligonucleotide listed herein, and wherein the DMD oligonucleotide is less than about 50 bases long.

(968) In some embodiments, the present disclosure provides a chirally controlled oligonucleotide having a sequence consisting of or comprising a sequence or a 15 base portion thereof found in any oligonucleotide listed in Table A1, wherein one or more U may be optionally and independently replaced with T or vice versa.

(969) In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC(SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC(SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC(SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC(SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one chirally controlled internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC(SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC(SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one chirally controlled internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC(SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC(SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein each internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC (SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC(SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage has the structure of formula I-c or a salt form thereof. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC(SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC(SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage has the structure of formula I-c or a salt form thereof, and at least one internucleotidic linkage is a non-negatively charged internucleotidic linkage. In some embodiments, the present disclosure provides a chirally controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC(SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC (SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein at least one internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage, and at least one internucleotidic linkage is a non-negatively charged internucleotidic linkage having the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, the present disclosure provides chirally a controlled oligonucleotide comprising a sequence of UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 1), CUCCGGUUCUGAAGGUGUUC(SEQ ID NO: 18), or UUCUGAAGGUGUUCUUGUAC(SEQ ID NO: 40), or a portion thereof at least 15 bases long, wherein each U can be optionally and independently replaced by T, wherein each internucleotidic linkage is a phosphodiester.

(970) In some embodiments, an oligonucleotide comprises one or more internucleotidic linkages which comprise a phosphorus modification prone to autorelease under certain conditions. That is, under certain conditions, a particular phosphorus modification is designed such that it self-cleaves from the oligonucleotide to provide, e.g., a phosphate diester such as those found in naturally occurring DNA and RNA. In some embodiments, such a phosphorus modification has a structure of O-L-R.sup.1, wherein each of L and R.sup.1 is independently as described in the present disclosure.

(971) In some embodiments, a provided oligonucleotide of the present disclosure comprises chemical modifications and/or stereochemistry that delivers desirable properties, e.g., delivery to target cells/tissues/organs, pharmacodynamics, pharmacokinetics, etc.

(972) In some embodiments, an oligonucleotide comprises a modification at a linkage phosphorus which can be transformed to a natural phosphate linkage by one or more esterases, nucleases, and/or cytochrome P450 enzymes, including but not limited to: CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3A4, CYP3A5, CYP3A7, CYP3A43, CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1, CYP5A1, CYP7A1, CYP7B1, CYP8A1 (prostacyclin synthase), CYP8B1 (bile acid biosynthesis), CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP20A1, CYP21A2, CYP24A1, CYP26A1, CYP26B1, CYP26C1, CYP27A1 (bile acid biosynthesis), CYP27B1 (vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknown function), CYP39A1, CYP46A1, and CYP51A1 (lanosterol 14-alpha demethylase).

(973) In some embodiments, an oligonucleotide comprises a modification at a linkage phosphorus that is a pro-drug moiety, e.g., a P-modification moiety facilitates delivery of an oligonucleotide to a desired location prior to removal. For instance, in some embodiments, a P-modification moiety results from PEGylation at the linkage phosphorus. One of skill in the relevant arts will appreciate that various PEG chain lengths are useful and that the selection of chain length will be determined in part by the result that is sought to be achieved by PEGylation. For instance, in some embodiments, PEGylation is effected in order to reduce RES uptake and extend in vivo circulation lifetime of an oligonucleotide.

(974) In some embodiments, a PEGylation reagent for use in accordance with the present disclosure is of a molecular weight of about 300 g/mol to about 100,000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 300 g/mol to about 10,000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 300 g/mol to about 5,000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 500 g/mol. In some embodiments, a PEGylation reagent of a molecular weight of about 1000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 3000 g/mol. In some embodiments, a PEGylation reagent is of a molecular weight of about 5000 g/mol.

(975) In certain embodiments, a PEGylation reagent is PEG500. In certain embodiments, a PEGylation reagent is PEG1000. In certain embodiments, a PEGylation reagent is PEG3000. In certain embodiments, a PEGylation reagent is PEG5000.

(976) In some embodiments, an oligonucleotide comprises a P-modification moiety that acts as a PK enhancer, e.g., lipids, PEGylated lipids, etc.

(977) In some embodiments, oligonucleotides of the present disclosure, e.g., DMD oligonucleotides, comprise a P-modification moiety that promotes cell entry and/or endosomal escape, such as a membrane-disruptive lipid or peptide.

(978) In some embodiments, an oligonucleotide comprises a P-modification moiety that acts as a targeting moiety. In some embodiments, a P-modification moiety is or comprises a targeting moiety. In some embodiments, a target moiety is an entity that is associates with a payload of interest (e.g., with an oligonucleotide or oligonucleotide composition) and also interacts with a target site of interest so that the payload of interest is targeted to the target site of interest when associated with the targeting moiety to a materially greater extent than is observed under otherwise comparable conditions when the payload of interest is not associated with the targeting moiety. A targeting moiety may be, or comprise, any of a variety of chemical moieties, including, for example, small molecule moieties, nucleic acids, polypeptides, carbohydrates, etc. Targeting moieties are described, e.g., in Adarsh et al., Organelle Specific Targeted Drug DeliveryA Review, International Journal of Research in Pharmaceutical and Biomedical Sciences, 2011, p. 895.

(979) Examples of such targeting moieties include, but are not limited to, proteins (e.g. Transferrin), oligopeptides (e.g., cyclic and acyclic RGD-containing oligopeptides), antibodies (monoclonal and polyclonal antibodies, e.g. IgG, IgA, IgM, IgD, IgE antibodies), sugars/carbohydrates (e.g., monosaccharides and/or oligosaccharides (mannose, mannose-6-phosphate, galactose, and the like)), vitamins (e.g., folate), or other small biomolecules. In some embodiments, a targeting moiety is a steroid molecule (e.g., bile acids including cholic acid, deoxycholic acid, dehydrocholic acid; cortisone; digoxigenin; testosterone; cholesterol; cationic steroids such as cortisone having a trimethylaminomethyl hydrazide group attached via a double bond at the 3-position of the cortisone ring, etc.). In some embodiments, a targeting moiety is a lipophilic molecule (e.g., alicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes, terpenes, and polyalicyclic hydrocarbons such as adamantine and buckminsterfullerenes). In some embodiments, a lipophilic molecule is a terpenoid such as vitamin A, retinoic acid, retinal, or dehydroretinal. In some embodiments, a targeting moiety is a peptide.

(980) In some embodiments, a P-modification moiety is a targeting moiety having the structure of X-L-R.sup.1 wherein each of X, L, and R.sup.1 is independently as described in the present disclosure.

(981) In some embodiments, a P-modification moiety facilitates cell specific delivery.

(982) In some embodiments, a P-modification moiety may perform one or more than one functions. For instance, in some embodiments, a P-modification moiety acts as a PK enhancer and a targeting ligand. In some embodiments, a P-modification moiety acts as a pro-drug and an endosomal escape agent. Numerous other such combinations are possible and are included in the present disclosure.

(983) Certain Examples of Oligonucleotides and Compositions

(984) In some embodiments, the present disclosure provides oligonucleotides and/or oligonucleotide compositions that are useful for various purposes, e.g., modulating skipping, reducing levels of transcripts, improving levels of beneficial proteins, treating conditions, diseases and disorders, etc. In some embodiments, the present disclosure provides oligonucleotide compositions with improved properties, e.g., increased activities, reduced toxicities, etc. Among other things, oligonucleotides of the present disclosure comprise chemical modifications, stereochemistry, and/or combinations thereof which can improve various properties and activities of oligonucleotides. Non-limiting examples are listed in Table A1. In some embodiments, an oligonucleotide type is a type as defined by the base sequence, pattern of backbone linkages, pattern of backbone chiral centers and pattern of backbone phosphorus modifications of an oligonucleotide in Table A1, wherein the oligonucleotide comprises at least one chirally controlled internucleotidic linkage (at least one R or S in Stereochemistry/Linkage). In some embodiments, a plurality of oligonucleotides of a particular oligonucleotide type is a plurality of an oligonucleotide in Table A1 (e.g., a plurality of oligonucleotides is a plurality of WV-1095). In some embodiments, a plurality of oligonucleotides in a chirally controlled oligonucleotide composition is a plurality of an oligonucleotide in Table A1 (e.g., a plurality of oligonucleotides is a plurality of WV-1095), wherein the oligonucleotide comprises at least one chirally controlled internucleotidic linkage (at least one R or S in Stereochemistry/Linkage).

(985) Table A1 lists non-limiting examples of DMD oligonucleotides. All of the oligonucleotides in Table A1 are DMD oligonucleotides, except for WV-12915, WV-12914, WV-12913, WV-12912, WV-12911, WV-12910, WV-12909, WV-12908, WV-12907, WV-12906, WV-12905, WV-12904, WV-15887, WV-24100, WV-24101, WV-24102, WV-24103, WV-24104, WV-24105, WV-24106, WV-24107, WV-24108, WV-24109, WV-24110, WV-XBD108, WV-XBD 109, WV-XBD 110, WV-XKCD108, WV-XKCD 109, WV-XKCD 110, which all target Malat-1, which is a gene target different than DMD.

(986) In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases, with 1-3 mismatches, of the base sequence of a DMD oligonucleotide disclosed in Table A1. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases of the base sequence of a DMD oligonucleotide disclosed in Table A1. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises the base sequence of a DMD oligonucleotide disclosed in Table A1. In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide is the base sequence of a DMD oligonucleotide disclosed in Table A1.

(987) In some embodiments, the present disclosure pertains to an oligonucleotide or oligonucleotide composition, wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases, with 1-3 mismatches, of the base sequence of a DMD oligonucleotide disclosed in Table A1, or wherein the base sequence of the oligonucleotide comprises at least 15 contiguous bases of the base sequence of a DMD oligonucleotide disclosed in Table A1, or wherein the base sequence of the oligonucleotide comprises the base sequence of a DMD oligonucleotide disclosed in Table A1, or wherein the base sequence of the oligonucleotide is the base sequence of a DMD oligonucleotide disclosed in Table A1; and wherein the oligonucleotide is stereorandom (e.g., not chirally controlled), or the oligonucleotide is chirally controlled, and/or the oligonucleotide comprises at least one internucleotidic linkage which is chirally controlled, and/or the oligonucleotide optionally comprises a sugar modification which is a LNA, and/or the oligonucleotide comprises a sugar which is a natural deoxyribose, a 2-OMe or a 2-MOE. In some embodiments, the present disclosure pertains to an oligonucleotide capable of mediating skipping of a DMD exon, wherein the oligonucleotide comprises at least one LNA.

(988) In the following table ID indicates identification or oligonucleotide number; and Description indicates the modified sequence.

(989) TABLE-US-00001 TABLEA1 ExampleOligonucleotides ID Description SEQIDNO: NakedBaseSequence Linkage/Stereochemistry ONT mU*SmC*SmA*SmA*SmG*SmG*SmA*SmA*SmG*SmA*SmU*S 61 UCAAGGAAGAUGGCA SSSSSSSSSSSSSSS -395 mG*SmG*SmC*SmA*SmU*SmU*SmU*SmC*SmU UUUCU SSSS WV- G*G*C*C*A*A*A*C*C*T*C*G*G*C*T*T*A*C*C*T 62 GGCCAAACCTCGGCT XXXXXXXXXX 1093 TACCT XXXXXXXXX WV- mGmGmCmCmAmAmAmCmCmUmCmGmGmCmUmUmAmCmC 63 GGCCAAACCUCGGCU OOOOOOOOOO 1094 mU UACCU OOOOOOOOO WV- G*RG*RC*RC*RA*RA*RA*RC*RC*RT*RC*RG*RG* 64 GGCCAAACCUCGGCU RRRRRRRRRRRRR 1095 RC*RT*RT*RA*RC*RC*RT TACCT RRRRRR WV- G*SG*SC*SC*SA*SA*SA*SC*SC*ST*SC*SG*SG*SC*ST* 65 GGCCAAACCTCGGCT SSSSSSSSSSSSSSS 1096 ST*SA*SC*SC*ST TACCT SSSS WV- G*SG*SC*SC*SA*SmAmAmCmCmUmCmGmGmCT*ST*SA* 66 GGCCAAACCUCGGCT SSSSSOOOOOOOO 1097 SC*SC*ST TACCT OSSSSS WV- mGmGmCmCA*SA*SA*SmCC*ST*SC*SG*SmGC*ST*ST*S 67 GGCCAAACCUCGGCT OOOOSSSOSSSSOS 1098 mAmCmCmU TACCU SSOOO WV- G*SmGC*SmCA*SmAA*SmCC*SmUC*SmGG*SmCT*SmUA 68 GGCCAAACCUCGGCT SOSOSOSOSOSOS 1099 *SmCC*SmU UACCU OSOSOS WV- mGG*SmCC*SmAA*SmAC*SmCT*SmCG*SmGC*SmUT*S 69 GGCCAAACCTCGGCU OSOSOSOSOSOSO 1100 mAC*SmCmU TACCU SOSOSO WV- G*SG*SmCmCA*SA*SmAmCC*ST*SC*SmGmGC*ST*SmU 70 GGCCAAACCTCGGCT SSOOSSOOSSSOOS 1101 mAC*SC*SmU UACCU SOOSS WV- G*SG*SC*SmCmAmAA*SC*SmCmUmCG*SG*SmCmUmUA* 71 GGCCAAACCUCGGCU SSSOOOSSOOOSS 1102 SC*SC*SmU UACCU OOOSSS WV- G*SG*SC*SC*SmAmAmAmCC*ST*SC*SmGmGmCmUT*SA 72 GGCCAAACCTCGGCU SSSSOOOOSSSOO 1103 *SC*SC*SmU TACCU OOSSSS WV- G*SG*SC*SmCA*SA*SA*SmCC*ST*SC*SmGG*SC*ST*S 73 GGCCAAACCTCGGCT SSSOSSSOSSSOSS 1104 mUA*SC*SC*SmU UACCU SOSSS WV- mGmGmCmCA*SA*SA*SC*SC*SmUmCmGmGmCT*ST*SA* 74 GGCCAAACCUCGGCT OOOOSSSSSOOOO 1105 SC*SC*SmU TACCU OSSSSS WV- G*SG*SmCmCmAmAmAmCmCmUC*SmGmGC*SmUT*SA* 75 GGCCAAACCUCGGCU SSOOOOOOOOSO 1106 SC*SC*SmU TACCU OSOSSSS WV- T*C*A*A*G*G*A*A*G*A*T*G*G*C*A*T*T*T*C*T 76 TCAAGGAAGATGGCA XXXXXXXXXX 1107 TTTCT XXXXXXXXX WV- mUmCmAmAmGmGmAmAmGmAmUmGmGmCmAmUmUmU 77 UCAAGGAAGAU OOOOOOOOOOO 1108 mCmU GGCAUUUCU OOOOOOOO WV- T*RC*RA*RA*RG*RG*RA*RA*RG*RA*RT*RG*RG* 78 TCAAGGAAGATGGCA RRRRRRRRRRRRR 1109 RC*RA*RT*RT*RT*RC*RT TTTCT RRRRRR WV- T*SC*SA*SA*SG*SG*SA*SA*SG*SA*ST*SG*SG*SC*SA* 79 TCAAGGAAGATGGCA SSSSSSSSSSSSSSS 1110 ST*ST*ST*SC*ST TTTCT SSSS WV- T*SC*SA*SA*SG*SmGmAmAmGmAmUmGmGmCA*ST*ST* 80 TCAAGGAAGAUGGCA SSSSSOOOOOOOO 1111 ST*SC*ST TTTCT OSSSSS WV- mUmCmAmAG*SG*SA*SmAG*SA*ST*SG*SmGC*SA*ST*S 81 UCAAGGAAGATGGCA OOOOSSSOSSSSOSSS 1112 mUmUmCmU TUUCU OOO WV- T*SmCA*SmAG*SmGA*SmAG*SmAT*SmGG*SmCA*SmUT 82 TCAAGGAAGATGGCA SOSOSOSOSOSOS 1113 *SmUC*SmU UTUCU OSOSOS WV- mUC*SmAA*SmGG*SmAA*SmGA*SmUG*SmGC*SmAT*S 83 UCAAGGAAGAUGGCA OSOSOSOSOSOSO 1114 mUT*SmCmU TUTCU SOSOSO WV- T*SC*SmAmAG*SG*SmAmAG*SA*ST*SmGmGC*SA*SmU 84 TCAAGGAAGATGGCA SSOOSSOOSSSOOS 1115 mUT*SC*SmU UUTCU SOOSS WV- T*SC*SA*SmAmGmGA*SA*SmGmAmUG*SG*SmCmAmUT* 85 TCAAGGAAGAUGGCA SSSOOOSSOOOSS 1116 ST*SC*SmU UTTCU OOOSSS WV- T*SC*SA*SA*SmGmGmAmAG*SA*ST*SmGmGmCmAT*ST 86 TCAAGGAAGATGGCA SSSSOOOOSSSOO 1117 *ST*SC*SmU TTTCU OOSSSS WV- T*SC*SA*SmAG*SG*SA*SmAG*SA*ST*SmGG*SC*SA*S 87 TCAAGGAAGATGGCA SSSOSSSOSSSOSS 1118 mUT*ST*SC*SmU UTTCU SOSSS WV- mUmCmAmAG*SG*SA*SA*SG*SmAmUmGmGmCA*ST*ST* 88 UCAAGGAAGAUGGCA OOOOSSSSSOOOO 1119 ST*SC*SmU TTTCU OSSSSS WV- T*SC*SmAmAmGmGmAmAmGmAT*SmGmGC*SmAT*ST*ST 89 TCAAGGAAGATGGCA SSOOOOOOOOSO 1120 *SC*SmU TTTCU OSOSSSS WV- G*G*C*C*A*mAmAmCmCmUmCmGmGmCT*T*A*C*C*T 90 GGCCAAACCUCGGCT XXXXXOOOOOOO 1121 TACCT OOXXXXX WV- mGmGmCmCA*A*A*mCC*T*C*G*mGC*T*T*mAmCmC 91 GGCCAAACCTCGGCT OOOOXXXOXXXX 1122 mU TACCU OXXXOOO WV- G*mGC*mCA*mAA*mCC*mUC*mGG*mCT*mUA*mCC* 92 GGCCAAACCUCGGCT XOXOXOXOXOXO 1123 mU UACCU XOXOXOX WV- mGG*mCC*mAA*mAC*mCT*mCG*mGC*mUT*mAC*mC 93 GGCCAAACCTCGGCU OXOXOXOXOXOX 1124 mU TACCU OXOXOXO WV- G*G*mCmCA*A*mAmCmCT*C*mGmGC*T*mUmAC*C* 94 GGCCAAACCTCGGCT XXOOXXOOOXXO 1125 mU UACCU OXXOOXX WV- G*G*C*mCmAmAA*C*mCmUmCG*G*mCmUmUA*C*C* 95 GGCCAAACCUCGGCU XXXOOOXXOOOX 1126 mU UACCU XOOOXXX WV- G*G*C*C*mAmAmAmCC*T*C*mGmGmCmUT*A*C*C* 96 GGCCAAACCTCGGCU XXXXOOOOXXXO 1127 mU TACCU OOOXXXX WV- G*G*C*mCA*A*A*mCC*T*C*mGG*C*T*mUA*C*C* 97 GGCCAAACCTCGGCT XXXOXXXOXXXO 1128 mU UACCU XXXOXXX WV- mGmGmCmCA*A*A*C*C*mUmCmGmGmCT*T*A*C*C* 98 GGCCAAACCUCGGCT OOOOXXXXXOOO 1129 mU TACCU OOXXXXX WV- G*G*mCmCmAmAmAmCmCmUC*mGmGC*mUT*A*C*C* 99 GGCCAAACCUCGGCU XXOOOOOOOOXO 1130 mU TACCU OXOXXXX WV- T*C*A*A*G*mGmAmAmGmAmUmGmGmCA*T*T*T*C*T 100 TCAAGGAAGAUGGCA XXXXXOOOOOOO 1131 TTTCT OOXXXXX WV- mUmCmAmAG*G*A*mAG*A*T*G*mGC*A*T*mUmUmC 101 UCAAGGAAGATGGCA OOOOXXXOXXXX 1132 mU UUUCU OXXXOOO WV- T*mCA*mAG*mGA*mAG*mAT*mGG*mCA*mUT*mUC* 102 TCAAGGAAGATGGCA XOXOXOXOXOXO 1133 mU UTUCU XOXOXOX WV- mUC*mAA*mGG*mAA*mGA*mUG*mGC*mAT*mUT*mC 103 UCAAGGAAGAUGGCA OXOXOXOXOXOX 1134 mU TUTCU OXOXOXO WV- T*C*mAmAG*G*mAmAG*A*T*mGmGC*A*mUmUT*C* 104 TCAAGGAAGATGGCA XXOOXXOOXXXO 1135 mU UUTCU OXXOOXX WV- T*C*A*mAmGmGA*A*mGmAmUG*G*mCmAmUT*T*C* 105 TCAAGGAAGAUGGCA XXXOOOXXOOOX 1136 mU UTTCU XOOOXXX WV- T*C*A*A*mGmGmAmAG*A*T*mGmGmCmAT*T*T*C* 106 TCAAGGAAGATGGCA XXXXOOOOXXXO 1137 mU TTTCU OOOXXXX WV- T*C*A*mAG*G*A*mAG*A*T*mGG*C*A*mUT*T*C* 107 TCAAGGAAGATGGCA XXXOXXXOXXXO 1138 mU UTTCU XXXOXXX WV- mUmCmAmAG*G*A*A*G*mAmUmGmGmCA*T*T*T*C* 108 UCAAGGAAGAUGGCA OOOOXXXXXOOO 1139 mU TTTCU OOXXXXX WV- T*C*mAmAmGmGmAmAmGmAT*mGmGC*mAT*T*T*C* 109 TCAAGGAAGATGGCA XXOOOOOOOOXO 1140 mU TTTCU OXOXXXX WV- mG*mG*mC*mC*mA*mAmAmCmCmUmCmGmGmCmU* 110 GGCCAAACCUCGGCU XXXXXOOOOOOO 1141 mU*mA*mC*mC*mU UACCU OOXXXXX WV- mGmGmCmCmA*mA*mA*mCmC*mU*mC*mG*mGmC* 111 GGCCAAACCUCGGCU OOOOXXXOXXXX 1142 mU*mU*mAmCmCmU UACCU OXXXOOO WV- mG*mGmC*mCmA*mAmA*mCmC*mUmC*mGmG*mCmU 112 GGCCAAACCUCGGCU XOXOXOXOXOXO 1143 *mUmA*mCmC*mU UACCU XOXOXOX WV- mGmG*mCmC*mAmA*mAmC*mCmU*mCmG*mGmC*mU 113 GGCCAAACCUCGGCU OXOXOXOXOXOX 1144 mU*mAmC*mCmU UACCU OXOXOXO WV- mG*mG*mCmCmA*mA*mAmCmCmU*mC*mGmGmC*mU 114 GGCCAAACCUCGGCU XXOOXXOOOXXO 1145 *mUmAmC*mC*mU UACCU OXXOOXX WV- mG*mG*mC*mCmAmAmA*mC*mCmUmCmG*mG*mCmU 115 GGCCAAACCUCGGCU XXXOOOXXOOOX 1146 mUmA*mC*mC*mU UACCU XOOOXXX WV- mG*mG*mC*mC*mAmAmAmCmC*mU*mC*mGmGmCmU 116 GGCCAAACCUCGGCU XXXXOOOOXXXO 1147 mU*mA*mC*mC*mU UACCU OOOXXXX WV- mG*mG*mC*mCmA*mA*mA*mCmC*mU*mC*mGmG* 117 GGCCAAACCUCGGCU XXXOXXXOXXXO 1148 mC*mU*mUmA*mC*mC*mU UACCU XXXOXXX WV- mGmGmCmCmA*mA*mA*mC*mC*mUmCmGmGmCmU* 118 GGCCAAACCUCGGCU OOOOXXXXXOOO 1149 mU*mA*mC*mC*mU UACCU OOXXXXX WV- mG*mG*mCmCmAmAmAmCmCmUmC*mGmGmC*mUmU* 119 GGCCAAACCUCGGCU XXOOOOOOOOXO 1150 mA*mC*mC*mU UACCU OXOXXXX WV- mU*mC*mA*mA*mG*mGmAmAmGmAmUmGmGmCmA* 120 UCAAGGAAGAUGGCA XXXXXOOOOOOO 1151 mU*mU*mU*mC*mU UUUCU OOXXXXX WV- mUmCmAmAmG*mG*mA*mAmG*mA*mU*mG*mGmC* 121 UCAAGGAAGAUGGCA OOOOXXXOXXXX 1152 mA*mU*mUmUmCmU UUUCU OXXXOOO WV- mU*mCmA*mAmG*mGmA*mAmG*mAmU*mGmG*mC 122 UCAAGGAAGAUGGCA XOXOXOXOXOXO 1153 mA*mUmU*mUmC*mU UUUCU XOXOXOX WV- mUmC*mAmA*mGmG*mAmA*mGmA*mUmG*mGmC* 123 UCAAGGAAGAUGGCA OXOXOXOXOXOX 1154 mAmU*mUmU*mCmU UUUCU OXOXOXO WV- mU*mC*mAmAmG*mG*mAmAmG*mA*mU*mGmGmC* 124 UCAAGGAAGAUGGCA XXOOXXOOXXXO 1155 mA*mUmUmU*mC*mU UUUCU OXXOOXX WV- mU*mC*mA*mAmGmGmA*mA*mGmAmUmG*mG*mC 125 UCAAGGAAGAUGGCA XXXOOOXXOOOX 1156 mAmUmU*mU*mC*mU UUUCU XOOOXXX WV- mU*mC*mA*mA*mGmGmAmAmG*mA*mU*mGmGmC 126 UCAAGGAAGAUGGCA XXXXOOOOXXXO 1157 mAmU*mU*mU*mC*mU UUUCU OOOXXXX WV- mU*mC*mA*mAmG*mG*mA*mAmG*mA*mU*mGmG* 127 UCAAGGAAGAUGGCA XXXOXXXOXXXO 1158 mC*mA*mUmU*mU*mC*mU UUUCU XXXOXXX WV- mUmCmAmAmG*mG*mA*mA*mG*mAmUmGmGmCmA* 128 UCAAGGAAGAUGGCA OOOOXXXXXOOO 1159 mU*mU*mU*mC*mU UUUCU OOXXXXX WV- mU*mC*mAmAmGmGmAmAmGmAmU*mGmGmC*mAmU* 129 UCAAGGAAGAUGGCA XXOOOOOOOOXO 1160 mU*mU*mC*mU UUUCU OXOXXXX WV- fG*fG*fC*fC*fA*fA*fA*fC*fC*fU*fC*fG*fG*fC*fU*fU* 130 GGCCAAACCUCGGCU XXXXXXXXXX 1678 fA*fC*fC*fU UACCU XXXXXXXXX WV- mG*mG*fC*fC*mA*mA*mA*fC*fC*fU*fC*mG*mG*fC 131 GGCCAAACCUCGGCU XXXXXXXXXX 1679 *fU*fU*mA*fC*fC*fU UACCU XXXXXXXXX WV- fG*fG*mC*mC*fA*fA*fA*mC*mC*mU*mC*fG*fG*mC 132 GGCCAAACCUCGGCU XXXXXXXXXX 1680 *mU*mU*fA*mC*mC*mU UACCU XXXXXXXXX WV- mG*fG*mC*fC*mA*fA*mA*fC*mC*fU*mC*fG*mG*fC 133 GGCCAAACCUCGGCU XXXXXXXXXX 1681 *mU*fU*mA*fC*mC*fU UACCU XXXXXXXXX WV- mG*mG*mC*mC*mA*mA*fA*fC*fC*fU*fC*fG*fG*fC* 134 GGCCAAACCUCGGCU XXXXXXXXXX 1682 mU*mU*mA*mC*mC*mU UACCU XXXXXXXXX WV- fG*fG*fC*fC*fA*fA*mA*mC*mC*mU*mC*mG*mG*mC 135 GGCCAAACCUCGGCU XXXXXXXXXX 1683 *fU*fU*fA*fC*fC*fU UACCU XXXXXXXXX WV- fG*fU*fC*fC*mA*mA*mA*fC*fC*mU*fC*fG*fG*fC*mU 136 GGCCAAACCUCGGCU XXXXXXXXXX 1684 *mU*mA*fC*fC*mU UACCU XXXXXXXXX WV- mG*mG*mC*mC*fA*fA*fA*mC*mC*fu*mC*mG*mG* 137 GGCCAAACCUCGGCU XXXXXXXXXX 1685 mC*fU*fU*fA*mC*mC*fU UACCU XXXXXXXXX WV- rArGrArArArUrGrCrCrArUrCrUrUrCrCrUrUrGrA 138 AGAAAUGCCAUCUUC OOOOOOOOOO 1687 CUUGA OOOOOOOOO WV- fU*fC*fA*fA*fG*fG*fA*fA*fG*fA*fU*fG*fG*fC*fA*fU* 139 UCAAGGAAGAUGGCA XXXXXXXXXX 1709 fU*fU*fC*fU UUUCU XXXXXXXXX WV- fU*fC*mA*mA*mG*mG*mA*mA*mG*mA*fU*mG*mG 140 UCAAGGAAGAUGGCA XXXXXXXXXX 1710 *fC*mA*fU*fU*fU*fC*fU UUUCU XXXXXXXXX WV- mU*mC*fA*fA*fG*fG*fA*fA*fG*fA*mU*fG*fG*mC*fA 141 UCAAGGAAGAUGGCA XXXXXXXXXX 1711 *mU*mU*mU*mC*mU UUUCU XXXXXXXXX WV- mU*fC*mA*fA*mG*fG*mA*fA*mG*fA*mU*fG*mG*fC 142 UCAAGGAAGAUGGCA XXXXXXXXXX 1712 *mA*fU*mU*fU*mC*fU UUUCU XXXXXXXXX WV- mU*mC*mA*mA*mG*mG*fA*fA*fG*fA*fU*fG*fG*fC* 143 UCAAGGAAGAUGGCA XXXXXXXXXX 1713 mA*mU*mU*mU*mC*mU UUUCU XXXXXXXXX WV- fU*fC*fA*fA*fG*fG*mA*mA*mG*mA*mU*mG*mG* 144 UCAAGGAAGAUGGCA XXXXXXXXXX 1714 mC*fA*fU*fU*fU*fC*fU UUUCU XXXXXXXXX WV- mU*fC*mA*mA*fG*fG*mA*mA*fG*mA*mU*fG*fG*fC 145 UCAAGGAAGAUGGCA XXXXXXXXXX 1715 *mA*mU*mU*mU*fC*mU UUUCU XXXXXXXXX WV- fU*mC*fA*fA*mG*mG*fA*fA*mG*fA*fU*mG*mG*mC 146 UCAAGGAAGAUGGCA XXXXXXXXXX 1716 *fA*fU*fU*fU*mC*fU UUUCU XXXXXXXXX WV- fU*fC*fA*fA*fG*mG*mA*mA*mG*mA*mU*mG*mG* 147 UCAAGGAAGAUGGCA XXXXXXXXXX 2095 mC*mA*fU*fU*fU*fC*fU UUUCU XXXXXXXXX WV- fU*fC*fA*fA*mG*mG*mA*mA*mG*mA*mU*mG*mG* 148 UCAAGGAAGAUGGCA XXXXXXXXXX 2096 mC*mA*mU*fU*fU*fC*fU UUUCU XXXXXXXXX WV- fU*fC*fA*mA*mG*mG*mA*mA*mG*mA*mU*mG*mG 149 UCAAGGAAGAUGGCA XXXXXXXXXX 2097 *mC*mA*mU*mU*fU*fC*fU UUUCU XXXXXXXXX WV- fU*fC*mA*mA*mG*mG*mA*mA*mG*mA*mU*mG* 150 UCAAGGAAGAUGGCA XXXXXXXXXX 2098 mG*mC*mA*mU*mU*mU*fC*fU UUUCU XXXXXXXXX WV- fU*mC*mA*mA*mG*mG*mA*mA*mG*mA*mU*mG* 151 UCAAGGAAGAUGGCA XXXXXXXXXX 2099 mG*mC*mA*mU*mU*mU*mC*fU UUUCU XXXXXXXXX WV- fU*fC*fA*fA*fG*fGmA*mA*mG*mA*mU*mG*mG* 152 UCAAGGAAGAUGGCA XXXXXOXXXXXX 2100 mCfA*fU*fU*fU*fC*fU UUUCU XOXXXXX WV- fU*fC*fA*fA*fGfGmA*mA*mG*mA*mU*mG*mG* 153 UCAAGGAAGAUGGCA XXXXOOXXXXXX 2101 mCfAfU*fU*fU*fC*fU UUUCU XOOXXXX WV- fU*fC*fA*fAfGfGmA*mA*mG*mA*mU*mG*mG* 154 UCAAGGAAGAUGGCA XXXOOOXXXXXX 2102 mCfAfUfU*fU*fC*fU UUUCU XOOOXXX WV- fU*fC*fAfAfGfGmA*mA*mG*mA*mU*mG*mG* 155 UCAAGGAAGAUGGCA XXOOOOXXXXXX 2103 mCfAfUfUfU*fC*fU UUUCU XOOOOXX WV- fU*fCfAfAfGfGmA*mA*mG*mA*mU*mG*mG* 156 UCAAGGAAGAUGGCA XOOOOOXXXXXX 2104 mCfAfUfUfUfC*fU UUUCU XOOOOOX WV- fUfCfAfAfGfGmA*mA*mG*mA*mU*mG*mG* 157 UCAAGGAAGAUGGCA OOOOOOXXXXXX 2105 mCfAfUfUfUfCfU UUUCU XOOOOOO WV- fU*fC*fA*fA*fG*fG*fA*fA*fG*fA*mU*mG*mG*mC* 158 UCAAGGAAGAUGGCA XXXXXXXXXX 2106 mA*mU*mU*mU*mC*mU UUUCU XXXXXXXXX WV- mU*mC*mA*mA*mG*mG*mA*mA*mG*mA*fU*fG*fG 159 UCAAGGAAGAUGGCA XXXXXXXXXX 2107 *fC*fA*fU*fU*fU*fC*fU UUUCU XXXXXXXXX WV- fU*fC*fA*fA*fG*fG*mA*mA*mG*mA*mU*mG*mG* 160 UCAAGGAAGAUGGCA XXXXXXXXXX 2108 mC*mA*mU*mU*mU*mC*mU UUUCU XXXXXXXXX WV- mU*mC*mA*mA*mG*mG*mA*mA*mG*mA*mU*mG* 161 UCAAGGAAGAUGGCA XXXXXXXXXX 2109 mG*mC*fA*fU*fU*fU*fC*fU UUUCU XXXXXXXXX WV- mC*mU*mC*mC*mA*mA*mC*mA*mU*mC*mA*mA* 162 CUCCAACAUCAAGGA XXXXXXXXXX 2165 mG*mG*mA*mA*mG*mA*mU*mG*mG*mC*mA*mU* AG XXXXXXXXXX mU*mU*mC*mU*mA*mG AUGGCAUUUCUAG XXXXXXXXX WV- mA*mC*mC*mA*mG*mA*mG*mU*mA*mA*mC*mA* 163 ACCAGAGUAACAG XXXXXXXXXX 2179 mG*mU*mC*mU*mG*mA*mG*mU*mA*mG*mG*mA* UCUGAGUAGGAG XXXXXXXXXX mG XXXX WV- mC*mA*mC*mC*mA*mG*mA*mG*mU*mA*mA*mC* 164 CACCAGAGUAACAG XXXXXXXXXX 2180 mA*mG*mU*mC*mU*mG*mA*mG*mU*mA*mG*mG* UCUGAGUAGGA XXXXXXXXXX mA XXXX WV- mU*mC*mA*mC*mC*mA*mG*mA*mG*mU*mA*mA* 165 UCACCAGAGUAACA XXXXXXXXXX 2181 mC*mA*mG*mU*mC*mU*mG*mA*mG*mU*mA*mG* GUCUGAGUAGG XXXXXXXXXX mG XXXX WV- mG*mU*mC*mA*mC*mC*mA*mG*mA*mG*mU*mA* 166 GUCACCAGAGUAAC XXXXXXXXXX 2182 mA*mC*mA*mG*mU*mC*mU*mG*mA*mG*mU*mA* AGUCUGAGUAG XXXXXXXXXX mG XXXX WV- mG*mU*mU*mG*mU*mG*mU*mC*mA*mC*mC*mA* 167 GUUGUGUCACCAGA XXXXXXXXXX 2183 mG*mA*mG*mU*mA*mA*mC*mA*mG*mU*mC*mU* GUAACAGUCUG 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GCUACUCAGGAGGCU XXXXXXXXXX 10601 mU*mG*mA*mG*mG*mC*mA*fG*fG*fA*fG*fA*fA*fU* GAGGC XXXXXXXXXX fC*fA AGGAGAAUCA XXXXXXXXX WV- fA*fG*fC*fA*fC*fA*fC*fG*fC*mC*mU*mG*mU*mA* 1630 AGCACACGCCUGUAA XXXXXXXXXX 10602 mA*mU*mC*mC*mC*mA*mG*fC*fU*fA*fC*fU*fC*fA* UCCCA XXXXXXXXXX fG*fG GCUACUCAGG XXXXXXXXX WV- fA*fG*fC*fC*fU*fG*fA*fC*fC*mG*mA*mC*mA*mU* 1631 AGCCUGACCGACAUG XXXXXXXXXX 10603 mG*mC*mU*mG*mA*mA*mA*fC*fC*fC*fA*fG*fU*fC* CUGAA XXXXXXXXXX fU*fC ACCCAGUCUC XXXXXXXXX WV- fG*fU*fU*fC*fG*fA*fG*fA*fC*mC*mA*mG*mC*mC* 1632 GUUCGAGACCAGCCU XXXXXXXXXX 10604 mU*mG*mA*mC*mC*mG*mA*fC*fA*fU*fG*fC*fU*fG* GACCG XXXXXXXXXX fA*fA ACAUGCUGAA XXXXXXXXX WV- fG*fG*fU*fC*fU*fC*fU*fG*fG*mG*mA*mG*mG*mC* 1633 GGUCUCUGGGAGGCC XXXXXXXXXX 10605 mC*mA*mA*mA*mG*mC*mG*fG*fG*fU*fG*fG*fA*fU* AAAGC XXXXXXXXXX fC*fA GGGUGGAUCA XXXXXXXXX WV- fG*fC*fU*fC*fA*fC*fG*fC*fC*mU*mG*mU*mA*mA* 1634 GCUCACGCCUGUAAU XXXXXXXXXX 10606 mU*mC*mC*mC*mA*mG*mG*fU*fC*fU*fC*fU*fG*fG* CCCAG XXXXXXXXXX fG*fA GUCUCUGGGA XXXXXXXXX WV- 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RfA*SfU*SfU*SfU*SfC*SfU UUUCU ORSSSSS WV- fU*SfC*RfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 1673 UCAAGGAAGAUGGCA SRSSSSOSOSSO 10875 *SfA*SfU*SfU*SfU*SfC*SfU UUUCU OSSSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC* 1674 UCAAGGAAGAUGGCA SSSSSSOSOSSO 10876 SfA*RfU*SfU*SfU*SfC*SfU UUUCU OSRSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*RmGmA*SfU*SmGmGfC 1675 UCAAGGAAGAUGGCA SSSSSSOROSSO 10877 *SfA*SfU*SfU*SfU*SfC*SfU UUUCU OSSSSSS WV- fU*SfC*SfA*SfA*RfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 1676 UCAAGGAAGAUGGCA SSSRSSOSOSSO 10878 *SfA*SfU*SfU*SfU*SfC*SfU UUUCU OSSSSSS WV- fU*SfC*SfA*RfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 1677 UCAAGGAAGAUGGCA SSRSSSOSOSSO 10879 *SfA*SfU*SfU*SfU*SfC*SfU UUUCU OSSSSSS WV- fU*SfC*SfA*SfA*SfG*RfG*SmAfA*SmGmA*SfU*SmGmGfC 1678 UCAAGGAAGAUGGCA SSSSRSOSOSSO 10880 *SfA*SfU*SfU*SfU*SfC*SfU UUUCU OSSSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*RmAfA*SmGmA*SfU*SmGmGfC 1679 UCAAGGAAGAUGGCA SSSSSROSOSSO 10881 *SfA*SfU*SfU*SfU*SfC*SfU UUUCU OSSSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*RfU*SmGmGfC 1680 UCAAGGAAGAUGGCA SSSSSSOSORSO 10882 *SfA*SfU*SfU*SfU*SfC*SfU UUUCU OSSSSSS WV- Mod012L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU* 1681 UCAAGGAAGAUGGCA OSSSSSSOSOSS 10883 SmGmGfC*SfA*SfU*SfU*SfU*SfU*SfU UUUCU OOSSSSSS WV- Mod085L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU* 1682 UCAAGGAAGAUGGCA OSSSSSSOSOSS 10884 SmGmGfC*SfA*SfU*SfU*SfU*SfC*SfU UUUCU OOSSSSSS WV- Mod086L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU* 1683 UCAAGGAAGAUGGCA OSSSSSSOSOSS 10885 SmGmGfC*SfA*SfU*SfU*SfU*SfC*SfU UUUCU OOSSSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC* 1684 UCAAGGAAGAUGGCA SSSSSSOSOSSO 10886 SfA*SfU*SfU*SfU*SfC*SfUL004Mod012 UUUCU OSSSSSSO WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC* 1685 UCAAGGAAGAUGGCA SSSSSSOSOSSO 10887 SfA*SfU*SfU*SfU*SfC*SfUL004Mod085 UUUCU OSSSSSSO WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC* 1686 UCAAGGAAGAUGGCA SSSSSSOSOSSO 10888 SfA*SfU*SfU*SfU*SfC*SfUL004Mod086 UUUCU OSSSSSSO WV- fU*SfU*SfA*SfA*SfA*SfA*SmA*SmG*SmU*SmC*SmU* 1687 UUAAAAAGUCUGCUA SSSSSSSSS 11047 SmG*SmC*SmU*SfA*SfA*SfA*SfA*SfU*SfG AAAUG SSSSSSSSSS WV- fA*SfA*SfG*SfU*SfC*SfU*SmG*SmC*SmU*SmA*SmA* 1688 AAGUCUGCUAAAAUG SSSSSSSSS 11048 SmA*SmA*SmU*SfG*SfU*SfU*SfU*SfU*SfC UUUUC SSSSSSSSSS WV- fU*SfG*SfC*SfU*SfA*SfA*SmA*SmA*SmU*SmG*SmU* 1689 UGCUAAAAUGUUUUC SSSSSSSSS 11049 SmU*SmU*SmU*SfC*SfA*SfU*SfU*SfC*SfC AUUCC SSSSSSSSSS WV- fA*SfA*SfA*SfU*SfG*SfU*SmU*SmU*SmU*SmC*SmA* 1690 AAAUGUUUUCAUUCC SSSSSSSSS 11050 SmU*SmU*SmC*SfC*SfU*SfA*SfU*SfU*SfA UAUUA SSSSSSSSSS WV- fU*SfU*SfU*SfU*SfC*SfA*SmU*SmU*SmC*SmC*SmU* 1691 UUUUCAUUCCUAUUA SSSSSSSSS 11051 SmA*SmU*SmU*SfA*SfG*SfA*SfU*SfC*SfU GAUCU SSSSSSSSSS WV- fA*SfU*SfU*SfC*SfC*SfU*SmA*SmU*SmU*SmA*SmG* 1692 AUUCCUAUUAGAUCU SSSSSSSSS 11052 SmA*SmU*SmC*SfU*SfG*SfU*SfC*SfG*SfC GUCGC SSSSSSSSSS WV- fU*SfA*SfU*SfU*SfA*SfG*SmA*SmU*SmC*SmU*SmG* 1693 UAUUAGAUCUGUCGC SSSSSSSSS 11053 SmU*SmC*SmG*SfC*SfC*SfC*SfU*SfA*SfC CCUAC SSSSSSSSSS WV- fG*SfA*SfU*SfC*SfU*SfG*SmU*SmC*SmG*SmC*SmC* 1694 GAUCUGUCGCCCUAC SSSSSSSSS 11054 SmC*SmU*SmA*SfC*SfC*SfU*SfC*SfU*SfU CUCUU SSSSSSSSSS WV- fG*SfU*SfC*SfG*SfC*SfC*SmC*SmU*SmA*SmC*SmC* 1695 GUCGCCCUACCUCUU SSSSSSSSS 11055 SmU*SmC*SmU*SfU*SfU*SfU*SfU*SfU*SfC UUUUC SSSSSSSSSS WV- fC*SfC*SfU*SfA*SfC*SfC*SmU*SmC*SmU*SmU*SmU* 1696 CCUACCUCUUUUUUC SSSSSSSSS 11056 SmU*SmU*SmU*SfC*SfU*SfG*SfU*SfC*SfU UGUCU SSSSSSSSSS WV- fC*SfU*SfC*SfU*SfU*SfU*SmU*SmU*SmU*SmC*SmU* 1697 CUCUUUUUUCUGUCU SSSSSSSSS 11057 SmG*SmU*SmC*SfU*SfG*SfA*SfC*SfA*SfG GACAG SSSSSSSSSS WV- fU*SfU*SfU*SfU*SfC*SfU*SmG*SmU*SmC*SmU*SmG* 1698 UUUUCUGUCUGACAG SSSSSSSSS 11058 SmA*SmC*SmA*SfG*SfC*SfU*SfG*SfU*SfU CUGUU SSSSSSSSSS WV- fU*SfG*SfU*SfC*SfU*SfG*SmA*SmC*SmA*SmG*SmC* 1699 UGUCUGACAGCUGUU SSSSSSSSS 11059 SmU*SmG*SmU*SfU*SfU*SfG*SfC*SfA*SfG UGCAG SSSSSSSSSS WV- fG*SfA*SfC*SfA*SfG*SfC*SmU*SmG*SmU*SmU*SmU* 1700 GACAGCUGUUUGCAG SSSSSSSSS 11060 SmG*SmC*SmA*SfG*SfA*SfC*SfC*SfU*SfC ACCUC SSSSSSSSSS WV- fU*SfU*SfG*SfU*SfU*SfU*SmG*SmC*SmA*SmG*SmA* 1701 CUGUUUGCAGACCUC SSSSSSSSS 11061 SmC*SmC*SmU*SfC*SfC*SfU*SfG*SfC*SfC CUGCC SSSSSSSSSS WV- fU*SfG*SfC*SfA*SfG*SfA*SmC*SmC*SmU*SmC*SmC* 1702 UGCAGACCUCCUGCC SSSSSSSSS 11062 SmU*SmG*SmC*SfC*SfA*SfC*SfC*SfG*SfC ACCGC SSSSSSSSSS WV- fA*SfC*SfC*SfU*SfC*SfC*SmU*SmG*SmC*SmC*SmA* 1703 ACCUCCUGCCACCGC SSSSSSSSS 11063 SmC*SmC*SmG*SfC*SfA*SfG*SfA*SfU*SfU AGAUU SSSSSSSSSS WV- fC*SfU*SfG*SfC*SfC*SfA*SmC*SmC*SmG*SmC*SmA* 1704 CUGCCACCGCAGAUU SSSSSSSSS 11064 SmG*SmA*SmU*SfU*SfC*SfA*SfG*SfG*SfC CAGGC SSSSSSSSSS WV- fA*SfC*SfC*SfG*SfC*SfA*SmG*SmA*SmU*SmU*SmC* 1705 ACCGCAGAUUCAGGC SSSSSSSSS 11065 SmA*SmG*SmG*SfC*SfU*SfU*SfC*SfC*SfC UUCCC SSSSSSSSSS WV- fA*SfG*SfA*SfU*SfG*SfC*SmA*SmG*SmG*SmC*SmU* 1706 AGAUUCAGGCUUCCC SSSSSSSSS 11066 SmU*SmC*SmC*SfC*SfA*SfA*SfU*SfU*SfU AAUUU SSSSSSSSSS WV- fC*SfA*SfG*SfG*SfC*SfU*SmU*SmC*SmC*SmC*SmA* 1707 CAGGCUUCCCAAUUU SSSSSSSSS 11067 SmA*SmU*SmU*SfU*SfU*SfU*SfC*SfC*SfU UUCCU SSSSSSSSSS WV- fU*SfU*SfC*SfC*SfC*SfA*SmA*SmU*SmU*SmU*SmU* 1708 UUCCCAAUUUUUCCU SSSSSSSSS 11068 SmU*SmC*SmC*SfU*SfG*SfU*SfA*SfG*SfA GUAGA SSSSSSSSSS WV- fA*SfA*SfU*SfU*SfU*SfU*SmU*SmC*SmC*SmU*SmG* 1709 AAUUUUUCCUGUAGA SSSSSSSSS 11069 SmU*SmA*SmG*SfA*SfA*SfU*SfA*SfC*SfU AUACU SSSSSSSSSS WV- fU*SfU*SfC*SfC*SfU*SfG*SmU*SmA*SmG*SmA*SmA* 1710 UUCCUGUAGAAUACU SSSSSSSSS 11070 SmU*SmA*SmC*SfU*SfG*SfG*SfC*SfA*SfU GGCAU SSSSSSSSSS WV- fG*SfU*SfA*SfG*SfA*SfA*SmU*SmA*SmC*SmU*SmG* 1711 GUAGAAUACUGGCAU SSSSSSSSS 11071 SmG*SmC*SmA*SfU*SfC*SfU*SfG*SfU*SfU CUGUU SSSSSSSSSS WV- fA*SfG*SfA*SfC*SfU*SfG*SmG*SmC*SmA*SmU*SmC* 1712 AUACUGGCAUCUGUU SSSSSSSSS 11072 SmU*SmG*SmU*SfU*SfU*SfU*SfU*SfG*SfA UUUGA SSSSSSSSSS WV- fG*SfG*SfC*SfA*SfU*SfC*SmU*SmG*SmU*SmU*SmU* 1713 GGCAUCUGUUUUUGA SSSSSSSSS 11073 SmU*SmU*SmG*SfA*SfG*SfG*SfA*SfU*SfU GGAUU SSSSSSSSSS WV- fC*SfU*SfG*SfU*SfU*SfU*SmU*SmU*SmG*SmA*SmG* 1714 CUGUUUUUGAGGAU SSSSSSSSS 11074 SmG*SmA*SmU*SfU*SfG*SfC*SfU*SfG*SfA UGCUGA SSSSSSSSSS WV- fU*SfU*SfU*SfG*SfA*SfG*SmG*SmA*SmU*SmU*SmG* 1715 UUUGAGGAUUGCUG SSSSSSSSS 11075 SmC*SmU*SmG*SfA*SfA*SfU*SfU*SfA*SfU 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fA*SfU*SfG*SfU*SfU*SfC*SmU*SmG*SmA*SmC*SmA* 1723 AUGUUCUGACAACAG SSSSSSSSS 11083 SmA*SmC*SmA*SfG*SfU*SfU*SfU*SfG*SfC UUUGC SSSSSSSSSS WV- fC*SfU*SfG*SfA*SfC*SfA*SmA*SmC*SmA*SmG*SmU* 1724 CUGACAACAGUUUGC SSSSSSSSS 11084 SmU*SmU*SmG*SfC*SfC*SfG*SfC*SfU*SfG CGCUG SSSSSSSSSS WV- fA*SfA*SfC*SfA*SfG*SfU*SmU*SmU*SmG*SmC*SmC* 1725 AACAGUUUGCCGCUG SSSSSSSSS 11085 SmG*SmC*SmU*SfG*SfC*SfC*SfC*SfA*SfA CCCAA SSSSSSSSSS WV- fU*SfU*SfU*SfG*SfC*SfC*SmG*SmC*SmU*SmG*SmC* 1726 UUUGCCGCUGCCCAA SSSSSSSSS 11086 SmC*SmC*SmA*SfA*SfU*SfG*SfC*SfC*SfA UGCCA SSSSSSSSSS WV- fC*SfG*SfC*SfU*SfG*SfC*SmC*SmC*SmA*SmA*SmU* 1727 CGCUGCCCAAUGCCA SSSSSSSSS 11087 SmG*SmC*SmC*SfA*SfU*SfC*SfC*SfU*SfG UCCUG SSSSSSSSSS WV- fC*SfC*SfC*SfA*SfA*SfU*SmG*SmC*SmC*SmA*SmU* 1728 CCCAAUGCCAUCCUG SSSSSSSSS 11088 SmC*SmC*SmU*SfG*SfG*SfA*SfG*SfU*SfU GAGUU SSSSSSSSSS WV- fU*SfG*SfC*SfC*SfA*SfU*SmC*SmC*SmU*SmG*SmG* 1729 UGCCAUCCUGGAGUU SSSSSSSSS 11089 SmA*SmG*SmU*SfU*SfC*SfC*SfU*SfG*SfU CCUGU SSSSSSSSSS WV- 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SSSSSSnXnXnXS 12135 SfUn001mGn001mGn001fC*SfA*SfU*SfU*SfU*SfC*SfU UUUCU nXnXnXSSSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAn001fAn001mGn001mAn001fU* 1780 UCAAGGAAGAUGGCA SSSSSSnXnXnX 12136 SmGn001mGn001fC*SfA*SfU*SfU*SfU*SfC*SfU UUUCU nXSnXnXSSSSSS WV- rGrGrCrUrUrCrArArCrUrArUrCrUrGrArGrUrGrA 1781 GGCUUCAACUAUCUG OOOOOOOOOOOO 12422 AGUGA OOOOOOO WV- rGrArArCrArCrCrUrUrCrArGrArArCrCrGrGrArG 1782 GAACACCUUCAGAAC OOOOOOOOOO 12423 CGGAG OOOOOOOOO WV- fA*SfU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU* 1783 AUCAAGGAAGAUGGC SSSSSSSOSOS 12494 SmGmGfC*SfA*SfU*SfU*SfU*SfC*SfU AUUUCU SOOSSSSSS WV- fU*SfU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU* 1784 UUCAAGGAAGAUGGC SSSSSSSOSOS 12495 SmGmGfC*SfA*SfU*SfU*SfU*SfC*SfU AUUUCU SOOSSSSSS WV- fUfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC* 1785 UCAAGGAAGAUGGCA OSSSS 12496 SfA*SfU*SfU*SfU*SfC*SfU UUUCU SOSOSSOOSSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001fU*SmG*SfA* 1786 CUCCGGUUCUGAAGG SSSSSSSSnXS 12553 SmAn001mGfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnXOSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001RfU*SmG*SfA 1787 CUCCGGUUCUGAAGG SSSSSSSSnRS 12554 *SmAn001RmGfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnROSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001RfU*SmG*SfA 1788 CUCCGGUUCUGAAGG SSSSSSSSnRS 12555 *SmAn001RfG*SfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnRSSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001RfU*SmG*SfA 1789 CUCCGGUUCUGAAGG SSSSSSSSnRS 12556 *SmAn001RmG*SfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnRSSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001SfU*SmG*SfA 1790 CUCCGGUUCUGAAGG SSSSSSSSnSSS 12557 *SmAn001SmGfG*SfU*SfG*SfU*SfU*SfC UGUUC SnSOSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001SfU*SmG*SfA 1791 CUCCGGUUCUGAAGG SSSSSSSSnSS 12558 *SmAn001SfG*SfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnSSSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001SfU*SmG*SfA 1792 CUCCGGUUCUGAAGG SSSSSSSSnSS 12559 *SmAn001SmG*SfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnSSSSSSS WV- L001fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU*SfA* 1793 UCACUCAGAUAGUUG OSSSSSSOSSSS 12566 SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfC AAGCC OOSSSSSS WV- Mod092L001fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU* 1794 UCACUCAGAUAGUUG OSSSSSSOSSSS 12567 SfA*SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfC AAGCC OOSSSSSS WV- Mod093L001fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU* 1795 UCACUCAGAUAGUUG OSSSSSSOSSSS 12568 SfA*SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfC AAGCC OOSSSSSS WV- L001TTTfU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU*SfA* 1796 TTTUCACUCAGAUAG OOOOSSSS 12569 SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfC UUGAAGCC SSOSSSSOOSSSS SS WV- Mod020L001TTTfU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU 1797 TTTUCACUCAGAUAG OOOOSSSS 12570 *SfA*SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfC UUGAAGCC SSOSSSSOOSSSS SS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU*SfA* 1798 UCACUCAGAUAGUUG SSSSSSOSSSS 12571 SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfCTTTL005 AAGCCTTT OOSSSSSSOOOO WV- fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU*SfA* 1799 UCACUCAGAUAGUUG SSSSSSOSSSS 12572 SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfCTTTL005Mod020 AAGCCTTT OOSSSSSSOOOOO WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001RfU*SmG*SfA 1800 CUCCGGUUCUGAAGG SSSSSSSSnRS 12872 *SmAn001RmGn001RfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnRnRSSSSS WV- fU*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001SfU*SmG*SfA 1801 CUCCGGUUCUGAAGG SSSSSSSSnSS 12873 *SmAn001SmGn001SfG*SfU*SfG*SfU*SfU*SfC UGUUC SSnSnSSSSSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCn001fU*SmG* 1802 CUCCGGUUCUGAAGG SSnXSSnXSSnX 12876 SfA*SmAn001mGn001fG*SfU*SfGn001fU*SfU*SfC UGUUC SSSnXnXSSnXSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCn001fU*SmG* 1803 CUCCGGUUCUGAAGG SSnXSSnXSSnXS 12877 SfA*SmAn001fG*SfG*SfU*SfGn001fU*SfU*SfC UGUUC SSnXSSSnXSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCn001fU*SmG* 1804 CUCCGGUUCUGAAGG SSnXSSnXSSnXS 12878 SfA*SmAn001mG*SfG*SfU*SfGn001fU*SfU*SfC UGUUC SSnXSSSnXSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU*SmG*SfA* 1805 CUCCGGUUCUGAAGG SSnXSSnXSSOS 12879 SmAmGfG*SfU*SfGn001fU*SfU*SfC UGUUC SSOOSSnXSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU*SmG*SfA* 1806 CUCCGGUUCUGAAGG SSnXSSnXSSOS 12880 SmAfG*SfG*SfU*SfGn001fU*SfU*SfC UGUUC SSOSSSnXSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU*SmG*SfA* 1807 CUCCGGUUCUGAAGG SSnXSSnXSSOS 12881 SmAmG*SfG*SfU*SfGn001fU*SfU*SfC UGUUC SSOSSSnXSS WV- fC*SfU*SfC*SfC*SfG*SfG*SmUn001mU*SmCn001mU* 1808 CUCCGGUUCUGAAGG SSSSSSnXSnXS 12882 SmGn001mA*SmAn001mG*SfG*SfU*SfG*SfU*SfU*SfC UGUUC nXSnXSSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SmUn001mUn001mCn001mUn001 1809 CUCCGGUUCUGAAGG SSSSSSnXnXnXnXn 12883 mGn001mAn001mAn001mGn001fG*SfU*SfG*SfU*SfU*SfC UGUUC XnXnXnXSSSSS WV- fU*SfC*SfAn001fC*SfU*SfCn001mAn001fG*SfA*SmU*SfA* 1810 UCACUCAGAUAGUUG SSnXSSnXnXSSS 12884 SmGn001mUn001fU*SfG*SfA*SfAn001fG*SfC*SfC AAGCC SnXnXSSSnXSS WV- fU*SfC*SfAn001fC*SfU*SfCn001mAfG*SfA*SmU*SfA* 1811 UCACUCAGAUAGUUG SSnXSSnXOSSSS 12885 SmGmUfU*SfG*SfA*SfAn001fG*SfC*SfC AAGCC OOSSSnXSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmA*SmG*SmA*SmU*SmA* 1812 UCACUCAGAUAGUUG SSSSSSSSSSS 12886 SmG*SmU*SmU*SfG*SfA*SfA*SfG*SfC*SfC AAGCC SSSSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAn001mG*SmAn001mU* 1813 UCACUCAGAUAGUUG SSSSSSnXSnX 12887 SmAn001mG*SmUn001mU*SfG*SfA*SfA*SfG*SfC*SfC AAGCC SnXSnXSSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAn001mGn001mAn001mUn001 1814 UCACUCAGAUAGUUG SSSSSSnXnXnXnXn 12888 mAn001mGn001mUn001mUn001fG*SfA*SfA*SfG*SfC*SfC AAGCC XnXnXnXSSSSS WV- GCGTGGTACCACGCL012mU*Geom5Ceom5CeomA*G*G*C*T*G 1815 GCGTGGTACCACGCU OOOOOOOOOO 12904 *G*T*T*A*T*mG*mA*mC*mU*mC GCCA OOOOOXOOO GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- GCGTGG*T*A*CCACGCL012mU*Geom5Ceom5CeomA*G*G*C 1816 GCGTGGTACCACGCU OOOOOXXXOO 12905 *T*G*G*T*T*A*T*mG*mA*mC*mU*mC GCCA OOOOOXOOO GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- G*C*G*T*G*G*T*A*C*C*A*C*G*CL012mU* 1817 GCGTGGTACCACGCU XXXXXXXXXXXX 12906 Geom5Ceom5CeomA*G*G*C*T*G*G*T*T*A*T*mG*mA* GCCA XOOXOOOXXX mC*mU*mC GGCTGGTTATGACUC XXXXXXXXXXXX WV- GfCGfUGGTACfCAfCGfCL012mU*Geom5Ceom5CeomA*G*G*C*T 1818 GCGUGGTACCACGCU OOOOOOOOOOO 12907 *G*G*T*T*A*T*mG*mA*mC*mU*mC GCCA OOOOXOOO GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- G*fCG*fUG*G*T*A*CfCA*fCG*fCL012mU* 1819 GCGUGGTACCACGCU XOXOXXXXOOXO 12908 Geom5Ceom5CeomA*G*G*C*T*G*G*T*T*A*T*mG*mA* GCCA XOOXOOO mC*mU*mC GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- G*fC*G*fU*G*G*T*A*C*fC*A*fC*G*fCL012mU* 1820 GCGUGGTACCACGCU XXXXXXXXXXXX 12909 Geom5Ceom5CeomA*G*G*C*T*G*G*T*T*A*T*mG*mA* GCCA XOOXOOO mC*mU*mC GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- GCGTGGTACCACGCL012BrmU*Geom5Ceom5CeomA*G*G*C*T* 1821 GCGTGGTACCACGCU OOOOOOOOOOO 12910 G*G*T*T*A*T*mG*mA*mC*mU*mC GCCA OOOOXOOO GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- GCGTGG*T*A*CCACGCL012BrmU*Geom5Ceom5CeomA*G*G* 1822 GCGTGGTACCACGCU OOOOOXXXOOO 12911 C*T*G*G*T*T*A*T*mG*mA*mC*mU*mC GCCA OOOOXOOO GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- G*C*G*T*G*G*T*A*C*C*A*C*G*CL012BrmU* 1823 GCGTGGTACCACGCU XXXXXXXXXXXX 12912 Geom5Ceom5CeomA*G*G*C*T*G*G*T*T*A*T*mG*mA* GCCA XOOXOOO mC*mU*mC GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- GfCGfUGGTACfCAfCGfCL012BrmU*Geom5Ceom5CeomA*G*G*C 1824 GCGUGGTACCACGCU OOOOOOOOOOO 12913 *T*G*G*T*T*A*T*mG*mA*mC*mU*mC GCCA OOOOXOOO GGCTGGTTATGACUC XXXXXXXXXXXX XXX WV- G*fCG*fUG*G*T*A*CfCA*fCG*fCL012BrmU*Geom5Ceo 1825 GCGUGGTACCACGCU XOXOXXXXOOXO 12914 m5CeomA*G*G*C*T*G*G*T*T*A*T*mG*mA*mC*mU GCCA XOOXOOOXXX *mC GGCTGGTTATGACUC XXXXXXXXXXXX WV- G*fC*G*fU*G*G*T*A*C*fC*A*fC*G*fCL012BrmU* 1826 GCGUGGTACCACGCU XXXXXXXXXXXX 12915 Geom5Ceom5CeomA*G*G*C*T*G*G*T*T*A*T*mG*mA GCCA XOOXOOOXXXX mC*mU*mC GGCTGGTTATGACUC XXXXXXXXXXX WV- fC*SfU*SfC*SfC*SfU*SfG*SfU*SfU*SmCfU*SmG*SfC* 1827 CUCCUGUUCUG SSSSSSSSOSS 13319 SmAmGfC*SfU*SfG*SfU*SfU*SfC CAGCUGUUC SOOSSSSS WV- fC*SfU*SfC*SfC*SfU*SfG*SfU*SfU*SmCfU*SmG*SfC* 1828 CUCCUGUUCUG SSSSSSSSOSS 13320 SmAfG*SfC*SfU*SfG*SfU*SfU*SfC CAGCUGUUC SOSSSSSS WV- fC*SfU*SfC*SfC*SfU*SfG*SfU*SfU*SmCfU*SmG*SfC* 1829 CUCCUGUUCUG SSSSSSSSOSS 13321 SmAmG*SfC*SfU*SfG*SfU*SfU*SfC CAGCUGUUC SOSSSSSS WV- fC*SfU*SfC*SfC*SfU*SfG*SfU*SfU*SfC*SfU*SmG*SfC* 1830 CUCCUGUUCUG SSSSSSSSSSS 13322 SmAmGfC*SfU*SfG*SfU*SfU*SfC CAGCUGUUC SOOSSSSS WV- GTTGCCTCCGGTTCTGAAGGTGTTC+allPMO 1831 GTTGCCTCCGG OOOOOOOOOOO 13405 TTCTGAAGGTGTTC OOOOOOOOOOOOO WV- CTCCGGTTCTGAAGGTGTTC+allPMO 1832 CTCCGGTTCTG OOOOOOOOOOO 13406 AAGGTGTTC OOOOOOOO WV- TGCCTCCGGTTCTGAAGGTGTTCTTGTA+allPMO 1833 TGCCTCCGGTT OOOOOOOOOOO 13407 CTGAAGGTGTT OOOOOOOOOOO CTTGTA OOOOO WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001RfU*SmG*SfA 1834 CUCCGGUUC SSSSSSSSnRS 13408 *SmAn001RfGn001RfG*SfU*SfG*SfU*SfU*SfC UGAAGGUGUUC SSnRnRSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCn001RfU*SmG*SfA 1835 CUCCGGUUC SSSSSSSSnRSSS 13409 *SmAn001RfGfG*SfU*SfG*SfU*SfU*SfC UGAAGGUGUUC nROSSSSS WV- fU*fU*fG*fu*fA*fC*fU*mU*mC*mA*mU* 1836 UUGUACUUCAUCCCACUGAUUCUGA XXXXXXXXXXXXXX 13594 mC*mC*mC*mA*mC*mU*fG*fA* XXXXXnXnXnXnXnX fUn001fUn001fCn001fUn001fGn00fA WV- fC*fC*fG*fG*fU*fU*fC*mU*mG*mA*mA* 1837 CCGGUUCUGAAGGUGUUCUUGUACU XXXXXXXXXXXXXX 13595 mG*mG*mU*mG*mU*mU*fC*fU* XXXXXnXnXnXnXnX fUn001fGn001fUn001fAn001fCn001fU WV- fUn001fUn001fGn001fUn001fAn001fC*fU*mU*mC* 1838 UUGUACUUCAUCCCACUGAUUCUGA nXnXnXnXnXXXXXXX 13596 mA*mU*mC*mC*mC*mA*mC*mU*fG*fA*fU XXXXXXXXXXXXX *fU*fC*fU*fG*fA WV- fCn001fCn001fGn001fGn001fUn001fU*fC*mU*mG* 1839 CCGGUUCUGAAGGUGUUCUUGUACU nXnXnXnXnXXXXXXX 13597 mA*mA*mG*mG*mU*mG*mU*mU*fC*fU* XXXXXXXXXXXXX fU*fG*fU*fA*fC*fU WV- fU*SfG*SfA*SfC*SfU*SfU*SmG*SmC*SmU* 1840 UGACUUCUCAAGCUUUUCU SSSSSSSSSSSSSSS 13701 SmC*SmA*SmA*SmG*SmC*SfU*SfU*SfU*SfU SSSS *SfC*SfU WV- fC*SfA*SfA*SfG*SfC*SfU*SmU*SmU*SmU* 1841 CAAGCUUUUCUUUUAGUUGC SSSSSSSSSSSSSSS 13702 SmC*SmU*SmU*SmU*SmU*SfA*SfG*SfU*SfU SSSS *SfG*SfC WV- fC*SfU*SfU*SfU*SfU*SfA*SmG*SmU*SmU* 1842 CUUUUAGUUGCUGCUCUUUU SSSSSSSSSSSSSSS 13703 SmG*SmC*SmU*SmG*SmC*SfU*SfC*SfU*SfU SSSS *SfU*SfU WV- fG*SfC*SfU*SfG*SfC*SfU*SmC*SmU*SmU* 1843 GCUGCUCUUUUCCAGGUUCA SSSSSSSSSSSSSSS 13704 SmU*SmU*SmC*SmC*SmA*SfG*SfG*SfU*SfU SSSS *SfC*SfA WV- fU*SfU*SfC*SfC*SfA*SfG*SmG*SmU*SmU* 1844 UUCCAGGUUCAAGUGGGAUA SSSSSSSSSSSSSSS 13705 SmC*SmA*SmA*SmG*SmU*SfG*SfG*SfG*SfA SSSS *SfU*SfA WV- fC*SfA*SfA*SfG*SfU*SfG*SmG*SmG*SmA* 1845 CAAGUGGGAUACUAGCAAUG SSSSSSSSSSSSSSS 13706 SmU*SmA*SmC*SmU*SmA*SfG*SfC*SfA*SfA SSSS *SfU*SfG WV- fU*SfA*SfC*SfU*SfA*SfG*SmC*SmA*SmA* 1846 UACUAGCAAUGUUAUCUGCU SSSSSSSSSSSSSSS 13707 SmU*SmG*SmU*SmU*SmA*SfU*SfC*SfU*SfG SSSS *SfC*SfU WV- fU*SfG*SfU*SfU*SfA*SfU*SmC*SmU*SmG* 1847 UGUUAUCUGCUUCCUCCAAC SSSSSSSSSSSSSSS 13708 SmC*SmU*SmU*SmC*SmC*SfU*SfC*SfC*SfA SSSS *SfA*SfC WV- fC*SfU*SfU*SfC*SfC*SfU*SmC*SmC*SmA* 1848 CUUCCUCCAACCAUAAAACA SSSSSSSSSSSSSSS 13709 SmA*SmC*SmC*SmA*SmU*SfA*SfA*SfA*SfA SSSS *SfC*SfA WV- fC*SfC*SfA*SfU*SfA*SfA*SmA*SmA*SmC* 1849 CCAUAAAACAAAUUCAUUUA SSSSSSSSSSSSSSS 13710 SmA*SmA*SmA*SmU*SmU*SfC*SfA*SfU*SfU SSSS *SfU*SfA WV- fA*SfA*SfU*SfU*SfC*SfA*SmU*SmU*SmU* 1850 AAUUCAUUUAAAUCUCUUUG SSSSSSSSSSSSSSS 13711 SmA*SmA*SmA*SmU*SmC*SfU*SfC*SfU*SfU SSSS *SfU*SfG WV- fA*SfA*SfU*SfC*SfU*SfC*SmU*SmU*SmU* 1851 AAUCUCUUUGAAAUUCUGAC SSSSSSSSSSSSSSS 13712 SmG*SmA*SmA*SmA*SmU*SfU*SfC*SfU*SfG SSSS *SfA*SfC WV- fU*SfG*SfA*SfA*SfA*SfU*SmU*SmC*SmU* 1852 UGAAAUUCUGACAAGAUAUU SSSSSSSSSSSSSSS 13713 SmG*SmA*SmC*SmA*SmA*SfG*SfA*SfU*SfA SSSS *SfU*SfU WV- fA*SfC*SfA*SfA*SfG*SfA*SmU*SmA*SmU* 1853 ACAAGAUAUUCUUUUGUUCU SSSSSSSSSSSSSSS 13714 SmU*SmC*SmU*SmU*SmU*SfU*SfG*SfU*SfU SSSS *SfC*SfU WV- fU*SfA*SfU*SfU*SfC*SfU*SmU*SmU*SmU* 1854 UAUUCUUUUGUUCUUCUAGC SSSSSSSSSSSSSSS 13715 SmG*SmU*SmU*SmC*SmU*SfU*SfC*SfU*SfA SSSS *SfG*SfC WV- fU*SfU*SfC*SfU*SfU*SfU*SmU*SmG*SmU* 1855 UUCUUUUGUUCUUCUAGCCU SSSSSSSSSSSSSSS 13716 SmU*SmC*SmU*SmU*SmC*SfU*SfA*SfG*SfC SSSS *SfC*SfU WV- fA*SfU*SfC*SfC*SfA*SfC*SmU*SmG*SmG* 1856 AUCCACUGGAGAUUUGUCUG SSSSSSSSSSSSSSS 13717 SmA*SmG*SmA*SmU*SmU*SfU*SfG*SfU*SfC SSSS *SfU*SfG WV- fA*SfG*SfA*SfU*SfU*SfU*SmG*SmU*SmC* 1857 AGAUUUGUCUGCUUGAGCUU SSSSSSSSSSSSSSS 13718 SmU*SmG*SmC*SmU*SmU*SfG*SfA*SfG*SfC SSSS *SfU*SfU WV- fU*SfG*SfC*SfU*SfU*SfG*SmA*SmG*SmC* 1858 UGCUUGAGCUUAUUUUCAAG SSSSSSSSSSSSSSS 13719 SmU*SmU*SmA*SmU*SmU*SfU*SfU*SfC*SfA SSSS *SfA*SfG WV- fU*SfA*SfU*SfU*SfU*SfU*SmC*SmA*SmA* 1859 UAUUUUCAAGUUUAUCUUGC SSSSSSSSSSSSSSS 13720 SmG*SmU*SmU*SmU*SmA*SfU*SfC*SfU*SfU SSSS *SfG*SfC WV- fU*SfU*SfU*SfA*SfU*SfC*SmU*SmU*SmG* 1860 UUUAUCUUGCUCUUCUGGGC SSSSSSSSSSSSSSS 13721 SmC*SmU*SmC*SmU*SmU*SfC*SfU*SfG*SfG SSSS *SfG*SfC WV- fU*SfC*SfU*SfU*SfC*SfU*SmG*SmG*SmG* 1861 UCUUCUGGGCUUAUGGGAGC SSSSSSSSSSSSSSS 13722 SmC*SmU*SmU*SmA*SmU*SfG*SfG*SfG*SfA SSSS *SfG*SfC WV- fU*SfU*SfA*SfU*SfG*SfG*SmG*SmA*SmG* 1862 UUAUGGGAGCACUUACAAGC SSSSSSSSSSSSSSS 13723 SmC*SmA*SmC*SmU*SmU*SfA*SfC*SfA*SfA SSSS *SfG*SfC WV- fG*SfC*SfA*SfC*SfU*SfU*SmA*SmC*SmA* 1863 GCACUUACAAGCACGGGUCC SSSSSSSSSSSSSSS 13724 SmA*SmG*SmC*SmA*SmC*SfG*SfG*SfG*SfU SSSS *SfC*SfC WV- fG*SfC*SfA*SfC*SfG*SfG*SmG*SmU*SmC* 1864 GCACGGGUCCUCCAGUUUCA SSSSSSSSSSSSSSS 13725 SmC*SmU*SmC*SmC*SmA*SfG*SfU*SfU*SfU SSSS *SfC*SfA WV- fU*SfC*SfC*SfA*SfG*SfU*SmU*SmU*SmC* 1865 UCCAGUUUCAUUUAAUUGUU SSSSSSSSSSSSSSS 13726 SmA*SmU*SmU*SmU*SmA*SfA*SfU*SfU*SfG SSSS *SfU*SfU WV- fU*SfU*SfU*SfA*SfA*SfU*SmU*SmG*SmU* 1866 UUUAAUUGUUUGAGAAUUCC SSSSSSSSSSSSSSS 13727 SmU*SmU*SmG*SmA*SmG*SfA*SfA*SfU*SfU SSSS *SfC*SfC WV- fG*SfA*SfG*SfA*SfA*SfU*SmU*SmC*SmC* 1867 GAGAAUUCCCUGGCGCAGGG SSSSSSSSSSSSSSS 13728 SmC*SmU*SmG*SmG*SmC*SfG*SfC*SfA*SfG SSSS *SfG*SfG WV- fC*SfU*SfG*SfG*SfC*SfG*SmC*SmA*SmG* 1868 CUGGCGCAGGGGCAACUCUU SSSSSSSSSSSSSSS 13729 SmG*SmG*SmG*SmC*SmA*SfA*SfC*SfU*SfC SSSS *SfU*SfU WV- fG*SfC*SfA*SfG*SfG*SfG*SmG*SmC*SmA* 1869 GCAGGGGCAACUCUUCCACC SSSSSSSSSSSSSSS 13730 SmA*SmC*SmU*SmC*SmU*SfU*SfC*SfC*SfA SSSS *SfU*SfC WV- fG*SfG*SfC*SfA*SfA*SfC*SmU*SmC*SmU* 1870 GGCAACUCUUCCACCAGUAA SSSSSSSSSSSSSSS 13731 SmU*SmC*SmC*SmA*SmC*SfC*SfA*SfG*SfU SSSS *SfA*SfA WV- fC*SfU*SfC*SfU*SfU*SfC*SmC*SmA*SmC* 1871 CUCUUCCACCAGUAACUGAA SSSSSSSSSSSSSSS 13732 SmC*SmA*SmG*SmU*SmA*SfA*SfC*SfU*SfG SSSS *SfA*SfA WV- fU*SfU*SfC*SfG*SfA*SfU*SmC*SmC*SmG* 1872 UUCGAUCCGUAAUGAUUGUU SSSSSSSSSSSSSSS 13733 SmU*SmA*SmA*SmU*SmG*SfA*SfU*SfU*SfG SSSS *SfU*SfU WV- fA*SfA*SfU*SfG*SfA*SfU*SmU*SmG*SmU* 1873 AAUGAUUGUUCUAGCCUCUU SSSSSSSSSSSSSSS 13734 SmU*SmC*SmU*SmA*SmG*SfC*SfC*SfU*SfC SSSS *SfU*SfU WV- fC*SfU*SfA*SfG*SfC*SfC*SmU*SmC*SmU* 1874 CUAGCCUCUUGAUUGCUGGU SSSSSSSSSSSSSSS 13735 SmU*SmG*SmA*SmU*SmU*SfG*SfC*SfU*SfG SSSS *SfG*SfU WV- fG*SfA*SfU*SfU*SfG*SfC*SmU*SmG*SmG* 1875 GAUUGCUGGUCUUGUUUUUC SSSSSSSSSSSSSSS 13736 SmU*SmC*SmU*SmU*SmG*SfU*SfU*SfU*SfU SSSS *SfU*SfC WV- fC*SfU*SfU*SfG*SfU*SfU*SmU*SmU*SmU* 1876 CUUGUUUUUCAAAUUUUGGG SSSSSSSSSSSSSSS 13737 SmC*SmA*SmA*SmA*SmU*SfU*SfU*SfU*SfG SSSS *SfG*SfG WV- fA*SfA*SfA*SfU*SfU*SfU*SmU*SmG*SmG* 1877 AAAUUUUGGGCAGCGGUAAU SSSSSSSSSSSSSSS 13738 SmG*SmC*SmA*SmG*SmC*SfG*SfG*SfU*SfA SSSS *SfA*SfU WV- fC*SfA*SfG*SfC*SfG*SfG*SmU*SmA*SmA* 1878 CAGCGGUAAUGAGUUCUUCC SSSSSSSSSSSSSSS 13739 SmU*SmG*SmA*SmG*SmU*SfU*SfC*SfU*SfU SSSS *SfC*SfC WV- fG*SfA*SfG*SfU*SfU*SfC*SmU*SmU*SmC* 1879 GAGUUCUUCCAACUGGGGAC SSSSSSSSSSSSSSS 13740 SmC*SmA*SmA*SmC*SmU*SfG*SfG*SfG*SfG SSSS *SfA*SfC WV- fA*SfA*SfC*SfU*SfG*SfG*SmG*SmG*SmA* 1880 AACUGGGGACGCCUCUGUUC SSSSSSSSSSSSSSS 13741 SmC*SmG*SmC*SmC*SmU*SfC*SfU*SfG*SfU SSSS *SfU*SfC WV- fG*SfC*SfC*SfU*SfC*SfU*SmG*SmU*SmU* 1881 GCCUCUGUUCCAAAUCCUGC SSSSSSSSSSSSSSS 13742 SmC*SmC*SmA*SmA*SmA*SfU*SfC*SfC*SfU SSSS *SfG*SfC WV- fU*SfG*SfU*SfU*SfC*SfC*SmA*SmA*SmA* 1882 UGUUCAAAUCCUGCAUUGU SSSSSSSSSSSSSSS 13743 SmU*SmC*SmC*SmU*SmG*SfC*SfA*SfU*SfU SSSS *SfG*SfU WV- fC*SfA*SfA*SfA*SfU*SfC*SmC*SmU*SmG* 1883 CAAAUCCUGCAUUGUUGCCU SSSSSSSSSSSSSSS 13744 SmC*SmA*SmU*SmU*SmG*SfU*SfU*SfG*SfC SSSS *SfC*SfU WV- fC*SfU*SfU*SfU*SfU*SfA*SmU*SmG*SmA* 1884 CUUUUAUGAAUGCUUCUCCA SSSSSSSSSSSSSSS 13745 SmA*SmU*SmG*SmC*SmU*SfU*SfC*SfU*SfC SSSS *SfC*SfA WV- fA*SfU*SfG*SfC*SfU*SfU*SmC*SmU*SmC* 1885 AUGCUUCUCCAAGAGGCAUU SSSSSSSSSSSSSSS 13746 SmC*SmA*SmA*SmG*SmA*SfG*SfG*SfC*SfA SSSS *SfU*SfU WV- fA*SfA*SfG*SfA*SfG*SfG*SmC*SmA*SmU* 1886 AAGAGGCAUUGAUAUUCUCU SSSSSSSSSSSSSSS 13747 SmU*SmG*SmA*SmU*SmA*SfU*SfU*SfC*SfU SSSS *SfC*SfU WV- fG*SfA*SfU*SfA*SfU*SfU*SmC*SmU*SmC* 1887 GAUAUUCUCUGUUAUCAUGU SSSSSSSSSSSSSSS 13748 SmU*SmG*SmU*SmU*SmA*SfU*SfC*SfA*SfU SSSS *SfG*SfU WV- fG*SfU*SfU*SfA*SfU*SfC*SmA*SmU*SmG* 1888 GUUAUCAUGUGGACUUUUCU SSSSSSSSSSSSSSS 13749 SmU*SmG*SmG*SmA*SmC*SfU*SfU*SfU*SfU SSSS *SfC*SfU WV- fG*SfG*SfA*SfC*SfU*SfU*SmU*SmU*SmC* 1889 GGACUUUUCUGGUAUCAUCU SSSSSSSSSSSSSSS 13750 SmU*SmG*SmG*SmU*SmA*SfU*SfC*SfA*SfU SSSS *SfC*SfU WV- fG*SfG*SfU*SfA*SfU*SfC*SmA*SmU*SmC* 1890 GGUAUCAUCUGCAGAAUAAU SSSSSSSSSSSSSSS 13751 SmU*SmG*SmC*SmA*SmG*SfA*SfA*SfU*SfA SSSS *SfA*SfU WV- fG*SfC*SfA*SfG*SfA*SfA*SmU*SmA*SmA* 1891 GCAGAAUAAUCCCGGAGAAG SSSSSSSSSSSSSSS 13752 SmU*SmC*SmC*SmC*SmG*SfG*SfA*SfG*SfA SSSS *SfA*SfG WV- fC*SfC*SfG*SfG*SfA*SmG*SmA*SmA*SmG* 1892 CCGGAGAAGUUUCAGGGCCA SSSSSSSSSSSSSSS 13753 SmU*SmU*SmU*SmC*SfA*SfG*SfG*SfG*SfC* SSSS SfC*SfA WV- fU*SfU*SfU*SfC*SfA*SfG*SmG*SmG*SmC* 1893 UUUCAGGGCCAAGUCAUUUG SSSSSSSSSSSSSSS 13754 SmC*SmA*SmA*SmG*SmU*SfC*SfA*SfU*SfU SSSS *SfU*SfG WV- fA*SfA*SfG*SfU*SfC*SfA*SmU*SmU*SmU* 1894 AAGUCAUUUGCCACAUCUAC SSSSSSSSSSSSSSS 13755 SmG*SmC*SmC*SmA*SmC*SfA*SfU*SfC*SfU SSSS *SfA*SfC WV- fC*SfC*SfA*SfC*SfA*SfU*SmC*SmU*SmA* 1895 CCACAUCUACAUUUGUCUGC SSSSSSSSSSSSSSS 13756 SmC*SmA*SmU*SmU*SmU*SfG*SfU*SfC*SfU SSSS *SfG*SfC WV- fA*SfU*SfU*SfU*SfG*SfU*SmC*SmU*SmG* 1896 AUUUGUCUGCCACUGGCGGA SSSSSSSSSSSSSSS 13757 SmC*SmC*SmA*SmC*SmU*SfG*SfG*SfC*SfG SSSS *SfG*SfA WV- fC*SfA*SfC*SfU*SfG*SfG*SmC*SmG*SmG* 1897 CACUGGCGGAGGUCUUUGGC SSSSSSSSSSSSSSS 13758 SmA*SmG*SmG*SmU*SmC*SfU*SfU*SfU*SfG SSSS *SfG*SfC WV- fG*SfC*SfG*SfG*SfA*SfG*SmG*SmU*SmC* 1898 GCGGAGGUCUUUGGCCAACU SSSSSSSSSSSSSSS 13759 SmU*SmU*SmU*SmG*SmG*SfC*SfC*SfA*SfA SSSS *SfC*SfU WV- fG*SfG*SfU*SfC*SfU*SfU*SmU*SmG*SmG* 1899 GGUCUUUGGCCAACUGCUAU SSSSSSSSSSSSSSS 13760 SmC*SmC*SmA*SmA*SmC*SfU*SfG*SfC*SfU SSSS *SfA*SfU WV- fU*SfU*SfG*SfC*SfC*SfA*SmU*SmU*SmG* 1900 UUGCCAUUGUUUCAUCAGCU SSSSSSSSSSSSSSS 13761 SmU*SmU*SmU*SmC*SmA*SfU*SfC*SfA*SfG SSSS *SfC*SfU WV- fU*SfU*SfU*SfC*SfA*SfU*SmC*SmA*SmG* 1901 UUUCAUCAGCUCUUUUACUC SSSSSSSSSSSSSSS 13762 SmC*SmU*SmC*SmU*SmU*SfU*SfU*SfA*SfC SSSS *SfU*SfC WV- fU*SfC*SfU*SfU*SfU*SfU*SmA*SmC*SmU* 1902 UCUUUUACUCCCUUGGAGUC SSSSSSSSSSSSSSS 13763 SmC*SmC*SmC*SmU*SmU*SfG*SfG*SfA*SfG SSSS *SfU*SfC WV- fC*SfC*SfU*SfU*SfG*SfG*SmA*SmG*SmU* 1903 CCUUGGAGUCUUCUAGGAGC SSSSSSSSSSSSSSS 13764 SmC*SmU*SmU*SmC*SmU*SfA*SfG*SfG*SfA SSSS *SfG*SfC WV- fU*SfU*SfC*SfU*SfA*SfG*SmG*SmA*SmG* 1904 UUCUAGGAGCCUUUCCUUAC SSSSSSSSSSSSSSS 13765 SmC*SmC*SmU*SmU*SmU*SfC*SfC*SfU*SfU SSSS *SfA*SfC WV- fC*SfU*SfU*SfU*SfC*SfC*SmU*SmU*SmA* 1905 CUUUCCUUACGGGUAGCAUC SSSSSSSSSSSSSSS 13766 SmC*SmG*SmG*SmG*SmU*SfA*SfG*SfC*SfA SSSS *SfU*SfC WV- fG*SfG*SfG*SfU*SfA*SfG*SmC*SmA*SmU* 1906 GGGUAGCAUCCUGUAGGACA SSSSSSSSSSSSSSS 13767 SmC*SmC*SmU*SmG*SmU*SfA*SfG*SfG*SfA SSSS *SfC*SfA WV- fC*SfU*SfG*SfU*SfA*SfG*SmG*SmA*SmC* 1907 CUGUAGGACAUUGGCAGUUG SSSSSSSSSSSSSSS 13768 SmA*SmU*SmU*SmG*SmG*SfC*SfA*SfG*SfU SSSS *SfU*SfG WV- fU*SfU*SfG*SfG*SfC*SfA*SmG*SmU*SmU* 1908 UUGGCAGUUGUUUCAGCUUC SSSSSSSSSSSSSSS 13769 SmG*SmU*SmU*SmU*SmC*SfA*SfG*SfC*SfU SSSS *SfU*SfC WV- fU*SfU*SfU*SfC*SfA*SfG*SmC*SmU*SmU* 1909 UUUCAGCUUCUGUAAGCCAG SSSSSSSSSSSSSSS 13770 SmC*SmU*SmG*SmU*SmA*SfA*SfG*SfC*SfC SSSS *SfA*SfG WV- fU*SfG*SfU*SfA*SfA*SfG*SmC*SmC*SmA* 1910 UGUAAGCCAGGCAAGAAACU SSSSSSSSSSSSSSS 13771 SmG*SmG*SmC*SmA*SmA*SfG*SfA*SfA*SfA SSSS *SfC*SfU WV- fG*SfC*SfA*SfA*SfG*SfA*SmA*SmA*SmC* 1911 GCAAGAAACUUUUCCAGGUC SSSSSSSSSSSSSSS 13772 SmU*SmU*SmU*SmU*SmC*SfC*SfA*SfG*SfG SSSS *SfU*SfC WV- fU*SfU*SfU*SfC*SfC*SfA*SmG*SmG*SmU* 1912 UUUCCAGGUCCAGGGGGAAC SSSSSSSSSSSSSSS 13773 SmC*SmC*SmA*SmG*SmG*SfG*SfG*SfG*SfA SSSS *SfA*SfC WV- fC*SfA*SfG*SfG*SfG*SfG*SmG*SmA*SmA* 1913 CAGGGGGAACUGUUGCAGUA SSSSSSSSSSSSSSS 13774 SmC*SmU*SmG*SmU*SmU*SfG*SfC*SfA*SfG SSSS *SfU*SfA WV- fU*SfG*SfU*SfU*SfG*SfC*SmA*SmG*SmU* 1914 UGUUGCAGUAAUCUAUGAGU SSSSSSSSSSSSSSS 13775 SmA*SmA*SmU*SmC*SmU*SfA*SfU*SfG*SfA SSSS *SfG*SfA WV- fA*SfU*SfC*SfU*SfA*SfU*SmG*SmA*SmG* 1915 AUCUAUGAGUUUCUUCCAAA SSSSSSSSSSSSSSS 13776 SmU*SmU*SmU*SmC*SmU*SfU*SfC*SfC*SfA SSSS *SfA*SfA WV- fU*SfG*SfC*SfU*SfU*SfC*SmC*SmA*SmA* 1916 UUCUUCCAAAGCAGCCUCUC SSSSSSSSSSSSSSS 13777 SmA*SmG*SmC*SmA*SmG*SfC*SfC*SfU*SfC SSSS *SfU*SfC WV- fG*SfC*SfA*SfG*SfC*SfC*SmU*SmC*SmU* 1917 GCAGCCUCUCGCUCACUCAC SSSSSSSSSSSSSSS 13778 SmC*SmG*SmC*SmU*SmC*SfA*SfC*SfU*SfC SSSS *SfA*SfC WV- fC*SfU*SfC*SfU*SfC*SfG*SmC*SmU*SmC* 1918 CUCUCGCUCACUCACCCUGC SSSSSSSSSSSSSSS 13779 SmA*SmC*SmU*SmC*SmA*SfC*SfC*SfC*SfU SSSS *SfG*SfC WV- fA*SfG*SfG*SfU*SfU*SfC*SmA*SmA*SmG* 1919 AGGUUCAAGUGGGAUACUAG SSSSSSSSSSSSSSS 13780 SmU*SmG*SmG*SmG*SmA*SfU*SfA*SfC*SfU SSSS *SfA*SfG WV- fU*SfC*SfC*SfA*SfG*SfG*SmU*SmU*SmC* 1920 UCCAGGUUCAAGUGGGAUAC SSSSSSSSSSSSSSS 13781 SmA*SmA*SmG*SmU*SmG*SfG*SfG*SfA*SfU SSSS *SfA*SfC WV- fU*SfU*SfG*SfC*SfU*SfG*SmG*SmU*SmC* 1921 UUGCUGGUCUUGUUUUUCAA SSSSSSSSSSSSSSS 13782 SmU*SmU*SmG*SmU*SmU*SfU*SfU*SfU*SfC SSSS *SfA*SfA WV- fA*SfC*SfU*SfG*SfG*SfG*SmG*SmA*SmC* 1922 ACUGGGGACGCCUCUGUUCC SSSSSSSSSSSSSSS 13783 SmG*SmC*SmC*SmU*SmC*SfU*SfG*SfU*SfU SSSS *SfC*SfC WV- fU*SfA*SfC*SfA*SfU*SfU*SmU*SmG*SmU* 1923 UACAUUUGUCUGCCACUGGC SSSSSSSSSSSSSSS 13784 SmC*SmU*SmG*SmC*SmC*SfA*SfC*SfU*SfG SSSS *SfG*SfC WV- fC*SfC*SfC*SfG*SfG*SfA*SmG*SmA*SmA* 1924 CCCGGAGAAGUUUCAGGGCC SSSSSSSSSSSSSSS 13785 SmG*SmU*SmU*SmU*SmC*SfA*SfG*SfG*SfG SSSS *SfC*SfC WV- fU*SfC*SfC*SfU*SfG*SfU*SmA*SmG*SmG* 1925 UCCUGUAGGACAUUGGCAGU SSSSSSSSSSSSSSS 13786 SmA*SmC*SmA*SmU*SmU*SfG*SfG*SfC*SfA SSSS *SfG*SfU WV- fG*SfA*SfG*SfU*SfC*SfU*SmU*SmC*SmU* 1926 GAGUCUUCUAGGAGCCUUUC SSSSSSSSSSSSSSS 13787 SmA*SmG*SmG*SmA*SmG*SfC*SfC*SfU*SfU SSSS *SfU*SfC WV- fC*SfU*SfU*SfG*SfA*SfG*SmC*SmU*SmU* 1927 CUUGAGCUUAUUUUCAAGUU SSSSSSSSSSSSSSS 13788 SmA*SmU*SmU*SmU*SmU*SfC*SfA*SfA*SfG SSSS *SfU*SfU WV- fA*SfG*SfC*SfA*SfC*SfU*SmU*SmA*SmC* 1928 AGCACUUACAAGCACGGGUC SSSSSSSSSSSSSSS 13789 SmA*SmA*SmG*SmC*SmA*SfC*SfG*SfG*SfG SSSS *SfU*SfC WV- fU*SfU*SfG*SfU*SfA*SfC*SfU*SmU*SmC* 1929 UUGUACUUCAUCCCACUGAUUCUGA SSSSSSSSSSSSSSS 13790 SmA*SmU*SmC*SmC*SmC*SmA*SmC*SmU* SSSSSSSSS SfG*SfA*SfU*SfU*SfC*SfU*SfG*SfA WV- fU*SfU*SfU*SfU*SfA*SfC*SfU*SfU*SfC* 1930 UUGUACUUCAUCCCACUGAUUCUGA SSSSSSSSSOSSSS 13791 SmAfU*SfC*SfC*SfC*SmAfC*SfU*SmGfA*SfU* OSSOSSSSSS SfU*SfC*SfU*SfG*SfA WV- fU*SfU*SfG*SfU*SfA*SfC*SfU*SmUmCfA* 1931 UUGUACUUCAUCCCACUGAUUCUGA SSSSSSSOOSOOO 13792 SmUmCmCmCfA*SmCmUfG*SfA*SfU*SfU*SfC* OSOOSSSSSSS SfU*SfG*SfA WV- fU*SfU*SfG*SfU*SfA*SfC*SfU*SmUfC*SmAfU 1932 UUGUACUUCAUCCCACUGAUUCUGA SSSSSSSOSOSOSO 13793 *SmCfC*SmCfA*SmCfU*SmGfA*SfU*SfU*SfC* SOSOSSSSSS SfU*SfG*SfA WV- fU*SfU*SfG*SfU*SfA*SfC*SfU*SfU*SmCfA* 1933 UUGUACUUCAUCCCACUGAUUCUGA SSSSSSSSOSOSOS 13794 SmUfC*SmCfC*SmAfC*SmUfG*SfA*SfU*SfU* OSOSSSSSSS SfC*SfU*SfG*SfA WV- fC*SfC*SfG*SfG*SfU*SfG*SfC*SmU*SmG* 1934 CCGGUUCUGAAGGUGUUCUUGUACU SSSSSSSSSSSSSSS 13795 SmA*SmA*SmG*SmG*SmU*SmG*SmU*SmU* SSSSSSSSS SfC*SfU*SfU*SfG*SfU*SfA*SfC*SfC WV- fC*SfC*SfG*SfG*SfU*SfU*SfC*SfU* 1935 CCGGUUCUGAAGGUGUUCUUGUACU SSSSSSSSOOOOO 13796 SmGmAmAmGmGfU*SmGfU*SfU*SfC*SfU*SfU* SOSSSSSSSSS SfG*SfU*SfA*SfC*SfU WV- fC*SfC*SfG*SfG*SfU*SfU*SfC*SmUfG*SfA* 1936 CCGGUUCUGAAGGUGUUCUUGUACU SSSSSSSOSSSSSO 13797 SfA*SfG*SfG*SmUfG*SmUmUmCfU*SfU*SfG* SOOOSSSSSS SfU*SfA*SfC*SfU WV- fC*SfC*SfG*SfG*SfU*SfU*SfC*SmUfG*SmAfA 1937 CCGGUUCUGAAGGUGUUCUUGUACU SSSSSSSOSOSOS 13798 *SmGfG*SmUfG*SmUfU*SmCfU*SfU*SfG*SfU* OSOSOSSSSSS SfA*SfC*SfU WV- fC*SfC*SfG*SfG*SfU*SfU*SfC*SfU*SmGfA* 1938 CCGGUUCUGAAGGUGUUCUUGUACU SSSSSSSSOSOSO 13799 SmAfG*SmGfU*SmGfU*SmU*SfC*SfU*SfU*SfG SOSSSSSSSSS SfU*SfA*SfC*SfU WV- fU*SfU*SfU*SfG*SfC*SfC*SfG*SfC*SmUfG* 1939 UUUGCCGCUGCCCAAUGCCA SSSSSSSSOSSS 13810 SmC*SfC*SmCmAfA*SfU*SfG*SfC*SfC*SfA OOSSSSS WV- fU*SfU*SfU*SfG*SfC*SfC*SfG*SfC*SmUfG* 1940 UUUGCCGCUGCCCAAUGCCA SSSSSSSSOSSS 13811 SmC*SfC*SmCfA*SfA*SfU*SfG*SfC*SfC*SfA OSSSSSS WV- fU*SfU*SfU*SfG*SfC*SfC*SfG*SfC* 1941 UUUGCCGCUGCCCAAUGCCA SSSSSSSSnXSSS 13812 SmUn001fG*SmC*SfC*SmCn001mAn001fA*SfU* nXnXSSSSS SfG*SfC*SfC*SfA WV- fU*SfU*SfU*SfG*SfC*SfC*SfG*SfC* 1942 UUUGCCGCUGCCCAAUGCCA SSSSSSSSnXSSS 13813 SmUn001fG*SmC*SfC*SmCn001fA*SfA*SfU*SfG nXSSSSSS *SfC*SfC*SfA WV- fU*SfU*SfUn001fG*SfC*SfCn001fG*SfC*SmUfG* 1943 UUUGCCGCUGCCCAAUGCCA SSnXSSnXSSOSS 13814 SmC*SfC*SmCmAfA*SfU*SfGn001fC*SfC*SfA SOOSSnXSS WV- fU*SfU*SfUn001fG*SfC*SfCn001fG*SfC*SmUfG* 1944 UUUGCCGCUGCCCAAUGCCA SSnXSSnXSSOSS 13815 SmC*SfC*SmCfA*SfA*SfU*SfGn001fC*SfC*SfA SOSSSnXSS WV- fU*SfU*SfUn001fG*SfC*SfCn001fG*SfC* 1945 UUUGCCGCUGCCCAAUGCCA SSnXSSnXSSnXSSS 13816 SmUn001fG*SmC*SfC*SmCn001mAn001fA*SfU* nXnXSSnXSS SfGn001fC*SfC*SfA WV- fU*SfU*SfUn001fG*SfC*SfCn001fG*SfC* 1946 UUUGCCGCUGCCCAAUGCCA SSnXSSnXSSnXSSS 13817 SmUn001fG*SmC*SfC*SmCn001fA*SfA*SfU * nXSSSnXSS SfGn001fC*SfC*SfA WV- fU*SfG*SfC*SfC*SfA*SfU*SfC*SfC*SmUfG* 1947 UGCCAUCCUGGAGUUCCUGU SSSSSSSSOSSS 13818 SmG*SfA*SmGmUfU*SfC*SfC*SfU*SfG*SfU OOSSSSS WV- fU*SfG*SfC*SfC*SfA*SfU*SfC*SfC*SmUfG* 1948 UGCCAUCCUGGAGUUCCUGU SSSSSSSSOSSS 13819 SmG*SfA*SmGfU*SfU*SfU*SfC*SfU*SfG*SfU OSSSSSS WV fU*SfG*SfC*SfC*SfA*SfU*SfC*SfC* 1949 UGCCAUCCUGGAGUUCCUGU SSSSSSSSnXSSS 13820 SmUn001fG*SmG*SfA*SmGn001mUn001fU*SfC* nXnXSSSSS SfC*SfU*SfG*SfU WV- fU*SfG*SfC*SfC*SfA*SfU*SfC*SfC* 1950 UGCCAUCCUGGAGUUCCUGU SSSSSSSSnXSSS 13821 SmUn001fG*SmG*SfA*SmGn001fU*SfU*SfC*SfC nXSSSSSS *SfU*SfG*SfU WV- fU*SfG*SfCn001fC*SfA*SfUn001fC*SfC*SmUfG* 1951 UGCCAUCCUGGAGUUCCUGU SSnXSSnXSSOSSSO 13822 SmG*SfA*SmGmUfU*SfC*SfCn001fU*SfG*SfU OSSnXSS WV- fU*SfG*SfCn001fC*SfA*SfUn001fC*SfC*SmUfG* 1952 UGCCAUCCUGGAGUUCCUGU SSnXSSnXSSOSSSO 13823 SmG*SfA*SmGfU*SfU*SfC*SfCn001fU*SfG*SfU SSSnXSS WV- fU*SfG*SfCn001fC*SfA*SfUn001fC*SfC* 1953 UGCCAUCCUGGAGUUCCUGU SSnXSSnXSSnXSSS 13824 SmUn001fG*SmG*SfA*SmGn001mUn001fU*SfC* nXnXSSnXSS SfCn001fU*SfG*Sfu WV- fU*SfG*SfCn001fC*SfA*SfUn001fC*SfC* 1954 UGCCAUCCUGGAGUUCCUGU SSnXSSnXSSnXSSS 13825 SmUn001fG*SmG*SfA*SmGn001fU*SfU*SfC* nXSSSnXSS SfCn001fU*SfG*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU*SmG* 1955 UCCGGUUCUGAAGGUGUUC SSSSSSSOSSS 13826 SfA*SmAmGfG*SfU*SfG*SfU*SfU*SfC OOSSSSS WV- fC*SfU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 1956 CUCCGGUUCUGAAGGUGUU SSSSSSSSOSSS 13827 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU OOSSSS WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU*SmG* 1957 UCCGGUUCUGAAGGUGUU SSSSSSSOSSSOOSSSS 13828 SfA*SmAmGfG*SfU*SfG*SfU*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU*SmG* 1958 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSS 13835 SfA*SmAmGfG*SfU*SfG*SfU*SfU*SfC*SfU OOSSSSSS WV- fC*SfC*SfU*SfC*SfC*SfG*SfG*SfU*SfU* 1959 CCUCCGGUUCUGAAGGUGUU SSSSSSSSSOSSS 13836 SmCfU*SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU OOSSSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU* 1960 CUCCGGUUCUGAAGGUGUUC SSnXSSnXSSOS 13857 SmGn001fA*SmAfG*SfG*SfU*SfGn001fU*SfU* nXSOSSSnXSS SfC WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU* 1961 CUCCGGUUCUGAAGGUGUU SSnXSSnXSSOSS 13858 SmG*SfA*SmAfG*SfG*SfU*SfGn001fU*SfU SOSSSnXS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU* 1962 CUCCGGUUCUGAAGGUGUU SSnXSSnXSSOS 13859 SmGn001fA*SmAfG*SfG*SfU*SfGn001fU*SfU nXSOSSSnXS WV- fU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU*SmG 1963 UCCGGUUCUGAAGGUGUUC SnXSSnXSSOSSSO 13860 *SfA*SmAfG*SfG*SfU*SfGn001fU*SfU*SfC SSSnXSS WV- fU*SfCn001fC*SfG*SfGn001fU*SffU*SmCfU* 1964 UCCGGUUCUGAAGGUGUUC SnXSSnXSSOSnX 13861 SmGn001fA*SmAfG*SfG*SfU*SfGn001fU*SfU* SOSSSnXSS SfC WV- fU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU*SmG 1965 UCCGGUUCUGAAGGUGUU SnXSSnXSSOSSS 13862 *SfA*SmAfG*SfG*SfU*SfGn001fU*SfU OSSSnXS WV- fU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU* 1966 UCCGGUUCUGAAGGUGUU SnXSSnXSSOSnX 13863 SmGn001fA*SmAfG*SfG*SfU*SfGn001fU*SfU SOSSSnXS WV- fC*SfG*SfCn001RfC*SfG*SfGn001RfU*SfU* 1967 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 13864 SmCfU*SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU SOSSSnRSS *SfU*SfC WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU* 1968 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOS 13865 SmCfU*SmGn001RfA*SmAfG*SfG*SfU* nRSOSSSnRSS SfGn001RfU*SfU*SfC WV- fA*SfC*SfA*SfA*SfG*SfU*SmU*SmC*SmU* 1969 ACAAGUUCUCCUUCUGGAAA SSSSSSSSSSSSSSS 13963 SmC*SmC*SmU*SmU*SmC*SfU*SfG*SfG*SfA SSSS *SfA*SfA WV- fC*SfU*SfU*SfC*SfU*SfG*SmG*SmA*SmA* 1970 CUUCUGGAAAGGUUCCAACA SSSSSSSSSSSSSSS 13964 SmA*SmG*SmG*SmU*SmU*SfC*SfC*SfA*SfA SSSS *SfC*SfA WV- fG*SfG*SfU*SfU*SfC*SfC*SmA*SmA*SmC* 1971 GGUUCCAACAUAAAGCCGAA SSSSSSSSSSSSSSS 13965 SmA*SmU*SmA*SmA*SmA*SfG*SfC*SfC*SfG SSSS *SfA*SfA WV- fA*SfA*SfA*SfG*SfC*SfC*SmG*SmA*SmA* 1972 AAAGCCGAAAUACACACUGC SSSSSSSSSSSSSSS 13966 SmA*SmU*SmA*SmC*SmA*SfC*SfA*SfC*SfU SSSS *SfG*SfC WV- fA*SfC*SfA*SfC*SfA*SfC*SmU*SmG*SmC* 1973 ACACACUGCCCCAAAGCCAC SSSSSSSSSSSSSSS 13967 SmC*SmC*SmC*SmA*SmA*SfA*SfG*SfC*SfC SSSS *SfA*SfC WV- fC*SfA*SfA*SfA*SfG*SfC*SmC*SmA*SmC* 1974 CAAAGCCACAAAACACCUUG SSSSSSSSSSSSSSS 13968 SmA*SmA*SmA*SmA*SmC*SfA*SfC*SfC*SfU SSSS *SfU*SfG WV- fA*SfA*SfC*SfA*SfC*SfC*SmU*SmU*SmG* 1975 AACACCUUGCUGUUACGAUG SSSSSSSSSSSSSSS 13969 SmC*SmU*SmG*SmU*SmU*SfA*SfC*SfG*SfA SSSS *SfG*SfG WV- fG*SfU*SfU*SfA*SfC*SfG*SmA*SmU*SmG* 1976 GUUACGAUGCUUCCCUCUGU SSSSSSSSSSSSSSS 13970 SmC*SmU*SmU*SmC*SmC*SfC*SfU*SfC*SfU SSSS *SfG*SfU WV- fU*SfC*SfC*SfC*SfU*SfC*SmU*SmG*SmU* 1977 UCCCUCUGUCACAGAUUCAA SSSSSSSSSSSSSSS 13971 SmC*SmA*SmC*SmA*SmG*SfA*SfU*SfU*SfC SSSS *SfA*SfA WV- fC*SfA*SfG*SfA*SfU*SfU*SmC*SmA*SmA* 1978 CAGAUUCAAUUAUAUUUUGC SSSSSSSSSSSSSSS 13972 SmU*SmU*SmA*SmU*SmA*SfU*SfU*SfU*SfU SSSS *SfA*SfC WV- fA*SfU*SfA*SfU*SfU*SfU*SmU*SmG*SmC* 1979 AUAUUUUGCAGUUUAUCAGA SSSSSSSSSSSSSSS 13973 SmA*SmG*SmU*SmU*SmU*SfA*SfU*SfC*SfA SSSS *SfG*SfA WV- fU*SfU*SfU*SfA*SfU*SfC*SmA*SmG*SmA* 1980 UUUAUCAGAUAAACCAGCUC SSSSSSSSSSSSSSS 13974 SmU*SmA*SmA*SmA*SmC*SfC*SfA*SfG*SfC SSSS *SfU*SfC WV- fA*SfA*SfC*SfC*SfA*SfG*SmC*SmU*SmC* 1981 AACCAGCUCCGUCCAGGCAA SSSSSSSSSSSSSSS 13975 SmC*SmG*SmU*SmC*SmC*SfA*SfG*SfG*SfC SSSS *SfA*SfA WV- fU*SfC*SfC*SfA*SfG*SfG*SmC*SmA*SmA* 1982 UCCAGGCAAACUCUCUCAUC SSSSSSSSSSSSSSS 13976 SmA*SmC*SmU*SmC*SmU*SfC*SfU*SfC*SfA SSSS *SfU*SfC WV- fU*SfC*SfU*SfC*SfU*SfC*SmA*SmU*SmC* 1983 UCUCUCAUCCUGACACAAAA SSSSSSSSSSSSSSS 13977 SmC*SmU*SmG*SmA*SmC*SfA*SfC*SfA*SfA SSSS *SfA*SfA WV- fG*SfA*SfC*SfA*SfC*SfA*SmA*SmA*SmA* 1984 GACACAAAAAGUCCAUAGCA SSSSSSSSSSSSSSS 13978 SmA*SmG*SmU*SmC*SmC*SfA*SfU*SfA*SfG SSSS *SfC*SfA WV- fU*SfC*SfC*SfA*SfU*SfA*SmG*SmC*SmA* 1985 UCCAUAGCACCGUGCUCUAA SSSSSSSSSSSSSSS 13979 SmC*SmC*SmG*SmU*SmG*SfC*SfU*SfC*SfU SSSS *SfA*SfA WV- fG*SfU*SfG*SfC*SfU*SfC*SmU*SmA*SmA* 1986 GUGCUCUAAUAUUAUCAUUA SSSSSSSSSSSSSSS 13980 SmU*SmA*SmU*SmU*SmA*SfU*SfC*SfA*SfU SSSS *SfU*SfA WV- fU*SfU*SfA*SfU*SfC*SfA*SmU*SmU*SmA* 1987 UUAUCAUUAUGAUAAUUUUC SSSSSSSSSSSSSSS 13981 SmU*SmG*SmA*SmU*SmA*SfA*SfU*SfU*SfU SSSS *SfU*SfC WV- fA*SfU*SfA*SfA*SfU*SfU*SmU*SmU*SmC* 1988 AUAAUUUUCUUUCUAGUAAU SSSSSSSSSSSSSSS 13982 SmU*SmU*SmU*SmC*SmU*SfA*SfG*SfU*SfA SSSS *SfA*SfU WV- fA*SfA*SfU*SfG*SfA*SfU*SmG*SmA*SmC* 1989 AAUGAUGACAACAACAGUCA SSSSSSSSSSSSSSS 13983 SmA*SmA*SmC*SmA*SmA*SfC*SfA*SfG*SfU SSSS *SfC*SfA WV- fC*SfA*SfA*SfC*SfA*SfG*SmU*SmC*SmA* 1990 CAACAGUCAAAAGUAAUUUC SSSSSSSSSSSSSSS 13984 SmA*SmA*SmA*SmG*SmU*SfA*SfA*SfU*SfU SSSS *SfU*SfC WV- fA*SfG*SfU*SfA*SfA*SfU*SmU*SmU*SmC* 1991 AGUAAUUUCCAUCACCCUUC SSSSSSSSSSSSSSS 13985 SmC*SmA*SmU*SmC*SmA*SfC*SfC*SfC*SfU SSSS *SfU*SfC WV- fU*SfC*SfA*SfC*SfC*SfC*SmU*SmU*SmC* 1992 UCACCCUUCAGAACCUGAUC SSSSSSSSSSSSSSS 13986 SmA*SmG*SmA*SmA*SmC*SfC*SfU*SfG*SfA SSSS *SfU*SfC WV- fA*SfA*SfC*SfC*SfU*SfG*SmA*SmU*SmC* 1993 AACCUGAUCUUUAAGAAGUU SSSSSSSSSSSSSSS 13987 SmU*SmU*SmU*SmA*SmA*SfG*SfA*SfA*SfG SSSS *SfU*SfU WV- fU*SfA*SfA*SfG*SfA*SfA*SmG*SmU*SmU* 1994 UAAGAAGUUAAAGAGUCCAG SSSSSSSSSSSSSSS 13988 SmA*SmA*SmA*SmG*SmA*SfG*SfU*SfC*SfC SSSS *SfA*SfG WV- fA*SfG*SfA*SfG*SfU*SfC*SmC*SmA*SmG* 1995 AGAGUCCAGAUGUGCUGAAG SSSSSSSSSSSSSSS 13989 SmA*SmU*SmG*SmU*SmG*SfC*SfU*SfG*SfA SSSS *SfA*SfG WV- fG*SfU*SfG*SfC*SfU*SfG*SmA*SmA*SmG* 1996 GUGCUGAAGAUAAAUACAAU SSSSSSSSSSSSSSS 13990 SmA*SmU*SmA*SmA*SmA*SfU*SfA*SfC*SfA SSSS *SfA*SfU WV- fU*SfA*SfA*SfA*SfU*SfA*SmC*SmA*SmA* 1997 UAAAUACAAUUUCGAAAAAA SSSSSSSSSSSSSSS 13991 SmU*SmU*SmU*SmC*SmG*SfA*SfA*SfA*SfA SSSS *SfA*SfA WV- fA*SfC*SfA*SfA*SfU*SfU*SmU*SmC*SmG* 1998 ACAAUUUCGAAAAAACAAAU SSSSSSSSSSSSSSS 13992 SmA*SmA*SmA*SmA*SmA*SfA*SfC*SfA*SfA SSSS *SfA*SfU WV- fU*SfC*SfG*SfA*SfA*SfA*SmA*SmA*SmA* 1999 UCGAAAAAACAAAUCAAAGA SSSSSSSSSSSSSSS 13993 SmC*SmA*SmA*SmA*SmU*SfC*SfA*SfA*SfA SSSS *SfG*SfA WV- fA*SfA*SfA*SfC*SfA*SfA*SmA*SmU*SmC* 2000 AAACAAAUCAAAGACUUACC SSSSSSSSSSSSSSS 13994 SmA*SmA*SmA*SmG*SmA*SfC*SfU*SfU*SfA SSSS *SfC*SfC WV- fA*SfU*SfC*SfA*SfA*SfA*SmG*SmA*SmC* 2001 AUCAAAGACUUACCUUAAGA SSSSSSSSSSSSSSS 13995 SmU*SmU*SmA*SmC*SmC*SfU*SfU*SfA*SfA SSSS *SfG*SfA WV- fG*SfA*SfC*SfU*SfU*SfA*SmC*SmC*SmU* 2002 GACUUACCUUAAGAUACCAU SSSSSSSSSSSSSSS 13996 SmU*SmA*SmA*SmG*SmA*SfU*SfA*SfC*SfC SSSS *SfA*SfU WV- fU*SfU*SfA*SfC*SfC*SfU*SmU*SmA*SmA* 2003 UUACCUUAAGAUACCAUUUG SSSSSSSSSSSSSSS 13997 SmG*SmA*SmU*SmA*SmC*SfC*SfA*SfU*SfU SSSS *SfU*SfG WV- fU*SfA*SfC*SfC*SfU*SfU*SmA*SmA*SmG* 2004 UACCUUAAGAUACCAUUUGU SSSSSSSSSSSSSSS 13998 SmA*SmU*SmA*SmC*SmC*SfA*SfU*SfU*SfU SSSS *SfG*SfU WV- fA*SfC*SfC*SfU*SfU*SfA*SmA*SmG*SmA* 2005 ACCUUAAGAUACCAUUUGUA SSSSSSSSSSSSSSS 13999 SmU*SmA*SmC*SmC*SmA*SfU*SfU*SfU*SfG SSSS *SfU*SfA WV- fC*SfC*SfU*SfU*SfA*SfA*SmG*SmA*SmU* 2006 CCUUAAGAUACCAUUUGUAU SSSSSSSSSSSSSSS 14000 SmA*SmC*SmC*SmA*SmU*SfU*SfU*SfG*SfU SSSS *SfA*SfU WV- fG*SfA*SfU*SfA*SfC*SfC*SmA*SmU*SmU* 2007 GAUACCAUUUGUAUUUAGCA SSSSSSSSSSSSSSS 14001 SmU*SmG*SmU*SmA*SmU*SfU*SfU*SfA*SfG SSSS *SfC*SfA WV- fA*SfU*SfU*SfU*SfG*SfU*SmA*SmU*SmU* 2008 AUUUGUAUUUAGCAUGUUCC SSSSSSSSSSSSSSS 14002 SmU*SmA*SmG*SmC*SmA*SfU*SfG*SfU*SfU SSSS *SfC*SfC WV- fA*SfU*SfU*SfU*SfA*SfG*SmC*SmA*SmU* 2009 AUUUAGCAUGUUCCCAAUUC SSSSSSSSSSSSSSS 14003 SmG*SmU*SmU*SmC*SmC*SfC*SfA*SfA*SfU SSSS *SfU*SfC WV- fC*SfA*SfU*SfG*SfU*SfU*SmC*SmC*SmC* 2010 CAUGUUCCCAAUUCUCAGGA SSSSSSSSSSSSSSS 14004 SmA*SmA*SmU*SmU*SmC*SfU*SfC*SfA*SfG SSSS *SfG*SfA WV- fC*SfC*SfC*SfA*SfA*SfU*SmU*SmC*SmU* 2011 CCCAAUUCUCAGGAAUUUGU SSSSSSSSSSSSSSS 14005 SmC*SmA*SmG*SmG*SmA*SfA*SfU*SfU*SfU SSSS *SfG*SfU WV- fU*SfC*SfU*SfC*SfA*SfG*SmG*SmA*SmA* 2012 UCUCAGGAAUUUGUGUCUUU SSSSSSSSSSSSSSS 14006 SmU*SmU*SmU*SmG*SmU*SfG*SfU*SfC*SfU SSSS *SfU*SfU WV- fG*SfA*SfA*SfU*SfU*SfU*SmG*SmU*SmG* 2013 GAAUUUGUGUCUUUCUGAGA SSSSSSSSSSSSSSS 14007 SmU*SmC*SmU*SmU*SmU*SfC*SfU*SfG*SfA SSSS *SfG*SfA WV- fG*SfU*SfG*SfU*SfC*SfU*SmU*SmU*SmC* 2014 GUGUCUUUCUGAGAAACUGU SSSSSSSSSSSSSSS 14008 SmU*SmG*SmA*SmG*SmA*SfA*SfA*SfC*SfU SSSS *SfG*SfU WV- fU*SfU*SfC*SfU*SfG*SfA*SmG*SmA*SmA* 2015 UUCUGAGAAACUGUUCAGCU SSSSSSSSSSSSSSS 14009 SmA*SmC*SmU*SmG*SmU*SfU*SfC*SfA*SfG SSSS *SfC*SfU WV- fG*SfA*SfA*SfA*SfC*SfU*SmG*SmU*SmU* 2016 GAAACUGUUCAGCUUCUGUU SSSSSSSSSSSSSSS 14010 SmC*SmA*SmG*SmC*SmU*SfU*SfC*SfU*SfG SSSS *SfU*SfU WV- fG*SfU*SfU*SfC*SfA*SfG*SmC*SmU*SmU* 2017 GUUCAGCUUCUGUUAGCCAC SSSSSSSSSSSSSSS 14011 SmC*SmU*SmG*SmU*SmU*SfA*SfG*SfC*SfC SSSS *SfA*SfC WV- fC*SfU*SfU*SfC*SfU*SfG*SmU*SmU*SmA* 2018 CUUCUGUUAGCCACUGAUUA SSSSSSSSSSSSSSS 14012 SmG*SmC*SmC*SmA*SmC*SfU*SfG*SfA*SfU SSSS *SfU*SfA WV- fU*SfU*SfA*SfG*SfC*SfC*SmA*SmC*SmU* 2019 UUAGCCACUGAUUAAAUAUC SSSSSSSSSSSSSSS 14013 SmG*SmA*SmU*SmU*SmA*SfA*SfA*SfU*SfA SSSS *SfU*SfC WV- fA*SfC*SfU*SfG*SfA*SfU*SmU*SmA*SmA* 2020 ACUGAUUAAAUAUCUUUAUA SSSSSSSSSSSSSSS 14014 SmA*SmU*SmA*SmU*SmC*SfU*SfU*SfU*SfA SSSS *SfU*SfA WV- fA*SfU*SfC*SfU*SfU*SfU*SmA*SmU*SmA* 2021 AUCUUUAUAUCAUAAUGAAA SSSSSSSSSSSSSSS 14015 SmU*SmC*SmA*SmU*SmA*SfA*SfU*SfG*SfA SSSS *SfA*SfA WV- fA*SfU*SfA*SfA*SfU*SfG*SmA*SmA*SmA* 2022 AUAAUGAAAACGCCGCCAUU SSSSSSSSSSSSSSS 14016 SmA*SmC*SmG*SmC*SmC*SfG*SfC*SfC*SfA SSSS *SfU*SfU WV- fG*SfC*SfC*SfG*SfC*SfC*SmA*SmU*SmU* 2023 GCCGCCAUUUCUCAACAGAU SSSSSSSSSSSSSSS 14017 SmU*SmC*SmU*SmC*SmA*SfA*SfC*SfA*SfG SSSS *SfA*SfU WV- fU*SfC*SfA*SfA*SfC*SfA*SmG*SmA*SmU* 2024 UCAACAGAUCUGUCAAAUCG SSSSSSSSSSSSSSS 14018 SmC*SmU*SmG*SmU*SmC*SfA*SfA*SfA*SfU SSSS *SfC*SfG WV- fU*SfG*SfA*SfA*SfG*SfA*SmU*SmA*SmA* 2025 UGAAGAUAAAUACAAUUUCG SSSSSSSSSSSSSSS 14019 SmA*SmU*SmA*SmC*SmA*SfA*SfU*SfU*SfU SSSS *SfC*SfG WV- fA*SfU*SfU*SfU*SfC*SfG*SmA*SmA*SmA* 2026 AUUUCGAAAAAACAAAUCAA SSSSSSSSSSSSSSS 14020 SmA*SmA*SmA*SmC*SmA*SfA*SfA*SfU*SfC SSSS *SfA*SfA WV- fA*SfA*SfA*SfA*SfA*SfA*SmC*SmA*SmA* 2027 AAAAAACAAAUCAAAGACUU SSSSSSSSSSSSSSS 14021 SmA*SmU*SmC*SmA*SmA*SfA*SfG*SfA*SfC SSSS *SfU*SfU WV- fC*SfA*SfA*SfA*SfU*SfC*SmA*SmA*SmA* 2028 CAAAUCAAAGACUUACCUUA SSSSSSSSSSSSSSS 14022 SmG*SmA*SmC*SmU*SmU*SfA*SfC*SfC*SfU SSSS *SfU*SfA WV- fA*SfA*SfA*SfG*SfA*SfC*SmU*SmU*SmA* 2029 AAAGACUUACCUUAAGAUAC SSSSSSSSSSSSSSS 14023 SmC*SmC*SmU*SmU*SmA*SfA*SfG*SfA*SfU SSSS *SfA*SfC WV- fU*SfA*SfA*SfG*SfA*SfU*SmA*SmC*SmC* 2030 UAAGAUACCAUUUGUAUUUA SSSSSSSSSSSSSSS 14024 SmA*SmU*SmU*SmU*SmG*SfU*SfA*SfU*SfU SSSS *SfU*SfA WV- fA*SfC*SfC*SfA*SfU*SfU*SmU*SmG*SmU* 2031 ACCAUUUGUAUUUAGCAUGU SSSSSSSSSSSSSSS 14025 SmA*SmU*SmU*SmU*SmA*SfG*SfC*SfA*SfU SSSS *SfG*SfU WV- fU*SfG*SfU*SfA*SfU*SfU*SmU*SmA*SmG* 2032 UGUAUUUAGCAUGUUCCCAA SSSSSSSSSSSSSSS 14026 SmC*SmA*SmU*SmG*SmU*SfU*SfC*SfC*SfC SSSS *SfA*SfA WV- fU*SfG*SfC*SfU*SfG*SfA*SmA*SmG*SmA* 2033 UGCUGAAGAUAAAUACAA SSSSSSSSSSSSSSSSS 14027 SmU*SmA*SmA*SfA*SfU*SfA*SfC*SfA*SfA WV- fA*SfA*SfA*SfU*SfA*SfC*SmA*SmA*SmU* 2034 AAAUACAAUUUCGAAAAA SSSSSSSSSSSSSSSSS 14028 SmU*SmU*SmC*SfG*SfA*SfA*SfA*SfA*SfA WV- fC*SfA*SfA*SfU*SfU*SfU*SmC*SmG*SmA* 2035 CAAUUUCGAAAAAACAAA SSSSSSSSSSSSSSSSS 14029 SmA*SmA*SmA*SfA*SfA*SfC*SfA*SfA*SfA WV- fC*SfG*SfA*SfA*SfA*SfA*SmA*SmA*SmC* 2036 CGAAAAAACAAAUCAAAG SSSSSSSSSSSSSSSSS 14030 SmA*SmA*SmA*SfU*SfC*SfA*SfA*SfA*SfG WV- fA*SfA*SfC*SfA*SfA*SfA*SmU*SmC*SmA* 2037 AACAAAUCAAAGACUUAC SSSSSSSSSSSSSSSSS 14031 SmA*SmA*SmG*SfA*SfC*SfU*SfU*SfA*SfC WV- fU*SfC*SfA*SfA*SfA*SfG*SmA*SmC*SmU* 2038 UCAAAGACUUACCUUAAG SSSSSSSSSSSSSSSSS 14032 SmU*SmA*SmC*SfC*SfU*SfU*SfA*SfA*SfG WV- fA*SfC*SfU*SfU*SfA*SfC*SmC*SmU*SmU* 2039 ACUUACCUUAAGAUACCA SSSSSSSSSSSSSSSSS 14033 SmA*SmA*SmG*SfA*SfU*SfA*SfC*SfC*SfA WV- fU*SfA*SfC*SfC*SfU*SfU*SmA*SmA*SmG* 2040 UACCUUAAGAUACCAUUU SSSSSSSSSSSSSSSSS 14034 SmA*SmU*SmA*SfC*SfC*SfA*SfU*SfU*SfU WV- fA*SfC*SfC*SfU*SfU*SfA*SmA*SmG*SmA* 2041 ACCUUAAGAUACCAUUUG SSSSSSSSSSSSSSSSS 14035 SmU*SmA*SmC*SfC*SfA*SfU*SfU*SfU*SfG WV- fC*SfC*SfU*SfU*SfA*SfA*SmG*SmA*SmU* 2042 CCUUAAGAUACCAUUUGU SSSSSSSSSSSSSSSSS 14036 SmA*SmC*SmC*SfA*SfU*SfU*SfU*SfG*SfU WV- fC*SfU*SfU*SfA*SfA*SfG*SmA*SmU*SmA* 2043 CUUAAGAUACCAUUUGUA SSSSSSSSSSSSSSSSS 14037 SmC*SmC*SmA*SfU*SfU*SfU*SfG*SfU*SfA WV- fA*SfU*SfA*SfC*SfC*SfA*SmU*SmU*SmU* 2044 AUACCAUUUGUAUUUAGC SSSSSSSSSSSSSSSSS 14038 SmG*SmU*SmA*SfU*SfU*SfU*SfA*SfG*SfC WV- fU*SfU*SfU*SfG*SfU*SfA*SmU*SmU*SmU* 2045 UUUGUAUUUAGCAUGUUC SSSSSSSSSSSSSSSSS 14039 SmA*SmG*SmC*SfA*SfU*SfG*SfU*SfU*SfC WV- fU*SfU*SfU*SfA*SfG*SfC*SmA*SmU*SmG* 2046 UUUAGCAUGUUCCCAAUU SSSSSSSSSSSSSSSSS 14040 SmU*SmU*SmC*SfC*SfC*SfA*SfA*SfU*SfU WV- fA*SfU*SfG*SfU*SfU*SfC*SmC*SmC*SmA* 2047 AUGUUCCCAAUUCUCAGG SSSSSSSSSSSSSSSSS 14041 SmA*SmU*SmU*SfC*SfU*SfC*SfA*SfG*SfG WV- fC*SfC*SfA*SfA*SfU*SfU*SmC*SmU*SmC* 2048 CCAAUUCUCAGGAAUUUG SSSSSSSSSSSSSSSSS 14042 SmA*SmG*SmG*SfA*SfA*SfU*SfU*SfU*SfG WV- fC*SfU*SfC*SfA*SfG*SfG*SmA*SmA*SmU* 2049 CUCAGGAAUUUGUGUCUU SSSSSSSSSSSSSSSSS 14043 SmU*SmU*SmG*SfU*SfG*SfU*SfC*SfU*SfU WV- fA*SfA*SfU*SfU*SfU*SfG*SmU*SmG*SmU* 2050 AAUUUGUGUCUUUCUGAG SSSSSSSSSSSSSSSSS 14044 SmC*SmU*SmU*SfU*SfC*SfU*SfG*SfA*SfG WV- fU*SfG*SfU*SfC*SfU*SfU*SmU*SmC*SmU* 2051 UGUCUUUCUGAGAAACUG 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fA*SfU*SfU*SfU*SfA*SfG*SfC*SfA*SmU*SfG*SmU* 2180 AUUUAGCAUGUU SSSSSSSSSSSSnX 15935 SfU*SmCn001fC*SfC*SfA*SfAn001fU*SfU*SfC CCCAAUUC SSSnXSS WV- mA*SmU*SmU*SmU*SmA*SmG*SmC*SmA*SmU*SmG* 2181 AUUUAGCAUGUU SSSSSSSSSSSS 15936 SmU*SmU*SmC*SmC*SmC*SmA*SmA*SmU*SmU*SmC CCCAAUUC SSSSSSS WV- mA*SmU*SmUn001mU*SmA*SmGn001mC*SmA*SmUn001 2182 AUUUAGCAUGUU SSnXSSnXSSnXSSSnX 15937 mG*SmU*SmU*SmCn001mC*SmC*SmA*SmAn001mU* CCCAAUUC SSSnXSS SmU*SmC WV- Aeo*STeo*STeo*STeo*SAeo*SGeo*Sm5Ceo*SAeo*STeo* 2183 ATTTAGCATGTT SSSSSSSSSSSS 15938 SGeo*STeo*STeo*Sm5Ceo*Sm5Ceo*Sm5Ceo*SAeo*SAeo* CCCAATTC SSSSSSS STeo*STeo*Sm5Ceo WV- Aeo*STeo*STeon001Teo*SAeo*SGeon001m5Ceo*SAeo* 2184 ATTTAGCATGTT SSnXSSnXSSnXSSSnX 15939 STeon001Geo*STeo*STeo*Sm5Ceon001m5Ceo*Sm5Ceo*SAeo CCCAATTC SSSnXSS *SAeon001Teo*STeo*Sm5Ceo WV- fG*SfC*SfAn001fU*SfG*SfUn001fU*SfC*SmCn001fC*SmA 2185 GCAUGUUCCC SSnXSSnXSSnXSSSnX 15940 *SfA*SmUn001fU*SfC*SfU*SfCn001fA*SfG*SfG AAUUCUCAGG SSSnXSS WV- fA*SfG*SfCn001fA*SfU*SfGn001fU*SfU*SmCn001fC*SmC 2186 AGCAUGUUCC SSnXSSnXSSnXSSSnX 15941 *SfA*SmAn001fU*SfU*SfC*SfUn001fC*SfA*SfG CAAUUCUCAG SSSnXSS WV- fU*SfA*SfGn001fC*SfA*SfUn001fG*SfU*SmUn001fC*SmC 2187 UAGCAUGUU SSnXSSnXSSnXSSSnX 15942 *SfC*SmAn001fA*SfU*SfU*SfCn001fU*SfC*SfA CCCAAUUCUCA SSSnXSS WV- fU*SfU*SfAn001fG*SfC*SfAn001fU*SfG*SmUn001fU*SmC 2188 UUAGCAUGUU SSnXSSnXSSnXSSSnX 15943 *SfC*SmCn001fA*SfA*SfU*SfUn001fC*SfU*SfC CCCAAUUCUC SSSnXSS WV- fU*SfU*SfUn001fA*SfG*SfCn001fA*SfU*SmGn001fU*SmU 2189 UUUAGCAUGUU SSnXSSnXSSnXSSSnX 15944 *SfC*SmCn001fC*SfA*SfA*SfUn001fU*SfC*SfU CCCAAUUCU SSSnXSS WV- fU*SfA*SfUn001fU*SfU*SfAn001fG*SfC*SmAn001fU*SmG 2190 UAUUUAGCAUGUU SSnXSSnXSSnXSSSnX 15945 *SfU*SmUn001fC*SfC*SfC*SfAn001fA*SfU*SfU CCCAAUU SSSnXSS WV- fG*SfG*SfAn001fU*SfU*SfUn001fA*SfG*SmCn001fA*SmU 2191 GUAUUUAGCAUGUU SSnXSSnXSSnXSSSnX 15946 *SfC*SmUn001fU*SfC*SfC*SfCn001fA*SfA*SfU CCCAAU SSSnXSS WV- fU*SfG*SfUn001fA*SfU*SfUn001fU*SfA*SmGn001fC*SmA 2192 UGUAUUUAGCA SSnXSSnXSSnXSSSnX 15947 *SfU*SmGn001fU*SfU*SfC*SfCn001fC*SfA*SfA UGUUCCCAA SSSnXSS WV- fU*SfU*SfGn001fU*SfA*SfUn001fU*SfU*SmAn001fG*SmC 2193 UUGUAUUUAGCAUGU SSnXSSnXSSnXSSSnX 15948 *SfA*SmUn001fG*SfU*SfU*SfCn001fC*SfC*SfA UCCCA SSSnXSS WV- fU*SfU*SfUn001fG*SfU*SfAn001fU*SfU*SmUn001fA* 2194 UUUGUAUUU SSnXSSnXSSnXSSSnX 15949 SmG*SfC*SmAn001fU*SfG*SfU*SfUn001fC*SfC*SfC AGCAUGUUCCC SSSnXSS WV- fG*SfC*SfU*SfG*SfC*SfU*SfC*SfU*SmU*SfU*SmU* 2195 GCUGCUCUUU SSSSSSSSSSSS 15950 SfC*SmC*SfA*SfG*SfG*SfU*SfU*SfC*SfA UCCAGGUUCA SSSSSSS WV- fC*SfU*SfU*SfC*SfC*SfU*SfC*SfC*SmA*SfA*SmC* 2196 CUUCCUCCAACCA SSSSSSSSSSSS 15951 SfC*SmA*SfU*SfA*SfA*SfA*SfA*SfC*SfA UAAAACA SSSSSSS WV- fA*SfG*SfG*SfU*SfU*SfC*SfA*SfA*SmG*SfU*SmG* 2197 AGGUUCAAGU SSSSSSSSSSSS 15952 SfG*SmG*SfA*SfU*SfA*SfC*SfU*SfA*SfG GGGAUACUAG SSSSSSS WV- fG*SfC*SfA*SfC*SfU*SfU*SfA*SfC*SmA*SfA*SmG* 2198 GCACUUACAAG SSSSSSSSSSSS 15953 SfC*SmA*SfC*SfG*SfG*SfG*SfU*SfC*SfC CACGGGUCC SSSSSSS WV- fG*SfG*SfC*SfA*SfA*SfC*SfU*SfC*SmU*SfU*SmC* 2199 GGCAACUCUU SSSSSSSSSSSS 15954 SfC*SmA*SfC*SfC*SfA*SfG*SfU*SfA*SfA CCACCAGUAA SSSSSSS WV- fG*SfA*SfG*SfU*SfU*SfC*SfU*SfU*SmC*SfC*SmA* 2200 GAGUUCUUCC SSSSSSSSSSSS 15955 SfA*SmC*SfU*SfG*SfG*SfG*SfG*SfA*SfC AACUGGGGAC SSSSSSS WV- fG*SfG*SfU*SfA*SfU*SfC*SfA*SfU*SmC*SfU*SmG* 2201 GGUAUCAUCU SSSSSSSSSSSS 15956 SfC*SmA*SfG*SfA*SfA*SfU*SfA*SfA*SfU GCAGAAUAAU SSSSSSS WV- fU*SfU*SfU*SfC*SfA*SfG*SfG*SfG*SmC*SfC*SmA* 2202 UUUCAGGGCCA SSSSSSSSSSSS 15957 SfA*SmG*SfU*SfC*SfA*SfU*SfU*SfU*SfG AGUCAUUUG SSSSSSS WV- fC*SfC*SfA*SfC*SfA*SfU*SfC*SfU*SmA*SfC*SmA* 2203 CCACAUCUACAU SSSSSSSSSSSS 15958 SfU*SmU*SfU*SfG*SfU*SfC*SfU*SfG*SfC UUGUCUGC SSSSSSS WV- fC*SfU*SfU*SfU*SfC*SfC*SfU*SfU*SmA*SfC*SmG* 2204 CUUUCCUUACG SSSSSSSSSSSS 15959 SfG*SmG*SfU*SfA*SfG*SfC*SfA*SfU*SfC GGUAGCAUC SSSSSSS WV- fU*SfU*SfC*SfU*SfU*SfC*SfC*SfA*SmA*SfA*SmG* 2205 UUCUUCC SSSSSSSSSSSS 15960 SfC*SmA*SfG*SfC*SfC*SfU*SfC*SfU*SfC AAAGCAGCCUCUC SSSSSSS WV- fU*SfC*SfC*SfU*SfG*SfU*SfA*SfG*SmG*SfA*SmC* 2206 UCCUGUAGGA SSSSSSSSSSSS 15961 SfA*SmU*SfU*SfG*SfG*SfC*SfA*SfG*SfU CAUUGGCAGU SSSSSSS WV- fG*SfC*SfUn001fG*SfC*SfUn001fC*SfU*SmUn001fU*SmU 2207 GCUGCUCUUU SSnXSSnXSSnXSSSnX 15962 *SfC*SmCn001fA*SfG*SfG*SfUn001fU*SfC*SfA UCCAGGUUCA SSSnXSS WV- fC*SfU*SfUn001fC*SfC*SfUn001fC*SfC*SmAn001fA*SmC 2208 CUUCCUCCAACCA SSnXSSnXSSnXSSSnX 15963 *SfC*SmAn001fU*SfA*SfA*SfAn001fA*SfC*SfA UAAAACA SSSnXSS WV- fA*SfG*SfGn001fU*SfU*SfCn001fA*SfA*SmGn001fU*SmG 2209 AGGUUCAAGU SSnXSSnXSSnXSSSnX 15964 *SfG*SmGn001fA*SfU*SfA*SfCn001fU*SfA*SfG GGGAUACUAG SSSnXSS WV- fG*SfC*SfAn001fC*SfU*SfUn001fA*SfC*SmAn001fA*SmG 2210 GCACUUACAAG SSnXSSnXSSnXSSSnX 15965 *SfC*SmAn001fC*SfG*SfG*SfGn001fU*SfC*SfC CACGGGUCC SSSnXSS WV- fG*SfG*SfCn001fA*SfA*SfCn001fU*SfC*SmUn001fU*SmC 2211 GGCAACUCUU SSnXSSnXSSnXSSSnX 15966 *SfC*SmAn001fC*SfC*SfA*SfGn001fU*SfA*SfA CCACCAGUAA SSSnXSS WV- fG*SfA*SfGn001fU*SfU*SfCn001fU*SfU*SmCn001fC*SmA 2212 GAGUUCUUCC SSnXSSnXSSnXSSSnX 15967 *SfA*SmCn001fU*SfG*SfG*SfGn001fG*SfA*SfC AACUGGGGAC SSSnXSS WV- fG*SfG*SfUn001fA*SfU*SfCn001fA*SfU*SmCn001fU*SmG 2213 GGUAUCAUCU SSnXSSnXSSnXSSSnX 15968 *SfC*SmAn001fG*SfA*SfA*SfUn001fA*SfA*SfU GCAGAAUAAU SSSnXSS WV- fU*SfU*SfUn001fC*SfA*SfGn001fG*SfG*SmCn001fC*SmA 2214 UUUCAGGGCCA SSnXSSnXSSnXSSSnX 15969 *SfA*SmGn001fU*SfC*SfA*SfUn001fU*SfU*SfG AGUCAUUUG SSSnXSS WV- fC*SfC*SfAn001fC*SfA*SfUn001fC*SfU*SmAn001fC*SmA 2215 CCACAUCUACAU SSnXSSnXSSnXSSSnX 15970 *SfU*SmUn001fU*SfG*SfU*SfCn001fU*SfG*SfC UUGUCUGC SSSnXSS WV- fC*SfU*SfUn001fU*SfC*SfCn001fU*SfU*SmAn001fC*SmG 2216 CUUUCCUUACG SSnXSSnXSSnXSSSnX 15971 *SfG*SmGn001fU*SfA*SfG*SfCn001fA*SfU*SfC GGUAGCAUC SSSnXSS WV- fU*SfU*SfCn001fU*SfU*SfCn001fC*SfA*SmAn001fA*SmG 2217 UUCUUCC SSnXSSnXSSnXSSSnX 15972 *SfC*SmAn001fG*SfC*SfC*SfUn001fC*SfU*SfC AAAGCAGCCUCUC SSSnXSS WV- fU*SfC*SfCn001fU*SfG*SfUn001fA*SfG*SmGn001fA*SmC 2218 UCCUGUAGGA SSnXSSnXSSnXSSSnX 15973 *SfA*SmUn001fU*SfG*SfG*SfCn001fA*SfG*SfU CAUUGGCAGU SSSnXSS WV- L00lfC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2219 CUCCGGUUCUGAAG OSSnRSSnR 16004 SmG*SfA*SmAfGfG*SfU*SfGn001RfU*SfU*SfC GUGUUC SSOSSSOOSSnRSS WV- Mod071L001fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU* 2220 CUCCGGUUCUGAAG OSSnRSSnR 16005 SmCfU*SmG*SfA*SmAfGfG*SfU*SfCn001RfU*SfU*SfC GUGUC SSOSSSOOSSnRSS WV- fC*SfU*SfCn003RfC*SfG*SfGn003RfU*SfU*SmCfU*SmG* 2221 CUCCGGUUCUGAAG SSnRSSnR 16006 SfA*SmAfGfG*SfU*SfGn003RfU*SfU*SfC GUGUUC SSOSSSOOSSnRSS WV- fC*SfU*SfCn004RfC*SfG*SfGn004RfU*SfU*SmCfU*SmG* 2222 CUCCGGUUCUGAAG SSnRSSnR 16007 SfA*SmAfGfG*SfU*SfGn004RfU*SfU*SfC GUGUUC SSOSSSOOSSnRSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAn003fG*SfA*SmU*SfA* 2223 UCACUCAGAUA SSSSSSnXSSSSnXnX 16008 SmGn003mUn003fU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC SSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAn004fG*SfA*SmU*SfA* 2224 UCACUCAGAUA SSSSSSnXSSSSnXnX 16009 SmGn004mUn004fU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC SSSSSS WV- L001L005fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU* 2225 CUCCGGUUCUGAAG OOSSnRSSnR 16010 SmCfU*SmG*SfA*SmAfGfG*SfU*SfGn001RfU*SfU*SfC GUGUUC SSOSSSOOSSnRSS WV- Mod107fC*SfU*SfCn001RfC*SfG*SfUn001RfU*SfU*SmCfU* 2226 CUCCGGUUCUGAAG SSnRSSnR 16011 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2786 CAUGUUCCCAAUUCUCA SSSSSSSSSSSSSSSSSS fGfG GG WV-20388 fCfCfCfAfAfUmUmCmUmCmAmGmGmAfAfUfU 2787 CCCAAUUCUCAGGAAUU SSSSSSSSSSSSSSSSSS fUfG UG WV-20389 fCfUfUfUfCfUmGmAmGmAmAmAmCmUfGfUfU 2788 CUUUCUGAGAAACUGUU SSSSSSSSSSSSSSSSSSS fCfAfG CAG WV-20390 fAfGfGfAfAfUmUmUmGmUmGmUmCmUfUfU 2789 AGGAAUUUGUGUCUUUC SSSSSSSSSSSSSSSSSSS fCfUfGfA UGA WV-20391 fUfGfUfGfUfCmUmUmUmCmUmGmAmGfAfA 2790 UGUGUCUUUCUGAGAAA SSSSSSSSSSSSSSSSSSS fAfCfUfG CUG WV-20392 fCfUfUfUfAfUmAmUmCmAmUmAmAmUfGfA 2791 CUUUAUAUCAUAAUGAA SSSSSSSSSSSSSSSSSSS fAfAfAfC AAC WV-20393 fCfAfCfUfGfAmUmUmAmAmAmUmAmUfCfUfU 2792 CACUGAUUAAAUAUCUU SSSSSSSSSSSSSSSSSSS fUfAfU UAU WV-20789 L001fUfCfAfAfGfGmAfAmGfAmUfGmGfCfAfU 2793 UCAAGGAAGAUGGCAUU ORRRRRROROROROR fUfUfCfU UCU RRRRR WV-20790 Mod012L001fUfCfAfAfGfGmAfAmGfAmUfGmG 2794 UCAAGGAAGAUGGCAUU ORRRRRROROROROR fCfAfUfUfUfCfU UCU RRRRR WV-21210 Mod118L001fUfCfAfCfUfCmAn001fGfAmUfA 2795 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-21211 Mod119L001fUfCfAfCfUfCmAn001fGfAmUfA 2796 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-21212 Mod120L001fUfCfAfCfUfCmAn001fGfAmUfA 2797 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-21217 fCfUfCn001RfCfGfGn001RfUfUmC CUCCGGUUC SSnRSSnRSS WV-21218 fUfCfAn001RfCfUfCn001RmAfGfAmUfAmGmU 2798 UCACUCAGAUAGUUGAA SSnRSSnROSSSSOSSS fUfGfAfAn001RfGfCfC GCC SnRSS WV-21245 fUfCfAn001RfCfUfCn001RmAfGfAmUfAmGmU 2799 UCACUCAGAUAGUUGAA SSnRSSnROSSSSSOSS fUfGfAfAn001RfGfCfC GCC SnRSS WV-21257 fCfGfGn001RfUfUmCfUmGfAmAfGfGfUfGn001R 2800 CGGUUCUGAAGGUGUUC SSnRSSOSSSOSSSnRSS fUfUfC WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA 2801 UCAAGGAAGAUGGCAUUUCG SSSSSSOSOSSOOSSSSSS 24310 *SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC* SmG WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA 2802 UCAAGGAAGAUGGCACCCCG SSSSSSOSOSSOOSSSSSS 24311 *SfU*SmGmGfC*SfA*SfC*SfC*SfC*SfC* SfG WV- fU*SfC*SfG*SfA*SfG*SfA*SmAfA*SmGmA 2803 UCGAGAAAGAUGGCAUUUCU SSSSSSOSOSSOOSSSSSS 24463 *SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC* SfU WV- fU*SfU*SfA*SfA*SfG*SfG*SmAfA*SmGmA 2804 UUAAGGAAGAUGGCAUUCCU SSSSSSOSOSSOOSSSSSS 24464 *SfU*SmGmGfC*SfA*SfU*SfU*SfC*SfC* SfU WV- fU*RfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2805 UCCGGUUCUGAAGGUGUUCU RSSSSSSOSSSOOSSSSSS 25439 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*RfC*SfG*SfG*SfU*SfU*SmCfU* 2806 UCCGGUUCUGAAGGUGUUCU SRSSSSSOSSSOOSSSSSS 25440 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*RfG*SfG*SfU*SfU*SmCfU* 2807 UCCGGUUCUGAAGGUGUUCU SSRSSSSOSSSOOSSSSSS 25441 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*RfG*SfU*SfU*SmCfU* 2808 UCCGGUUCUGAAGGUGUUCU SSSRSSSOSSSOOSSSSSS 25442 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*RfU*SfU*SmCfU* 2809 UCCGGUUCUGAAGGUGUUCU SSSSRSSOSSSOOSSSSSS 25443 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*RfU*SmCfU* 2810 UCCGGUUCUGAAGGUGUUCU SSSSSRSOSSSOOSSSSSS 25444 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*RmCfU* 2811 UCCGGUUCUGAAGGUGUUCU SSSSSSROSSSOOSSSSSS 25445 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2812 UCCGGUUCUGAAGGUGUUCU SSSSSSSORSSOOSSSSSS 25446 RmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2813 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSRSOOSSSSSS 25447 SmG*RfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2814 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSROOSSSSSS 25448 SmG*SfA*RmAmGfG*SfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2815 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSSOORSSSSS 25449 SmG*SfA*SmAmGfG*RfU*SfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2816 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSSOOSRSSSS 25450 SmG*SfA*SmAmGfG*SfU*RfG*SfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2817 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSSOOSSRSSS 25451 SmG*SfA*SmAmGfG*SfU*SfG*RfU*SfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2818 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSSOOSSSRSS 25452 SmG*SfA*SmAmGfG*SfU*SfG*SfU*RfU* SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2819 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSSOOSSSSRS 25453 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* RfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2820 UCCGGUUCUGAAGGUGUUCU SSSSSSSOSSSOOSSSSSR 25454 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfC*RfU WV- fC*SfG*SfG*SfU*SfU*SmCfU*SmG*SfA* 2821 CGGUUCUGAAGGUGUUCU SSSSSOSSSOOSSSSSS 25455 SmAmGfG*SfU*SfG*SfU*SfU*SfC*SfU WV- fU*SfU*SfC*SfC*SfG*SfG*SfU*SfU* 2822 UUCCGGUUCUGAAGGUGUUCU SSSSSSSSOSSSOOSSSSSS 25456 SmCfU*SmG*SfA*SmAmGfG*SfU*SfG*SfU* SfU*SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SfU* 2823 UCCGGUUUCUGAAGGUGUUCU SSSSSSSSOSSSOOSSSSSS 25457 SmCfU*SmG*SfA*SmAmGfG*SfU*SfG*SfU* SfU*SfC*SfU WV- fU*SfC*SfC*SfG*SfG*SfU*SfU*SmCfU* 2824 UCCGGUUCUGAAGGUGUUUCU SSSSSSSOSSSOOSSSSSSS 25458 SmG*SfA*SmAmGfG*SfU*SfG*SfU*SfU* SfU*SfC*SfU WV fU*SfC*SfC*SfG*SfG*SfU*SmCfU*SmG* 2825 UCCGGUCUGAAGGUGUUCU SSSSSSOSSSOOSSSSSS 25459 SfA*SmAmGfG*SfU*SfG*SfU*SfU*SfC*SfU WV- lT*SfC*SlA*SfC*SfU*SfC*SmAfG*SfA* 2826 TCACUCAGAUAGUUGAAGCC SSSSSSOSSSSOOSSSSSS 25536 SmU*SfA*SmGmUfU*SfG*SfA*SfA*SfG* SfC*SfC WV- fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA* 2827 UCACUCAGAUAGUUGAAGCC SSSSSSOSSSSOOSSSSSS 25537 SmU*SfA*SmGmUfU*SfG*SfA*SfA*SlG*SfC *SfC WV- lT*SfC*SlA*SfC*SfU*SfC*SmAfG*SfA* 2828 TCACUCAGAUAGUUGAAGCC SSSSSSOSSSSOOSSSSSS 25538 SmU*SfA*SmGmUfU*SfG*SfA*SfA*SlG*SfC *SfC WV- fU*SfC*SfA*SfC*SfU*SfC*SlAfG*SfA*SmU 2829 UCACUCAGAUAGTUGAAGCC SSSSSSOSSSSOOSSSSSS 25539 *SfA*SfGlTfU*SfG*SfA*SfA*SfG*SfC*SfC WV- fU*SfC*SfA*SfC*SfU*SfC*SlAfG*SfA*SmU 2830 UCACUCAGAUAGTTGAAGCC SSSSSSOSSSSOOSSSSSS 25540 *SfA*SlGlTlT*SfG*SfA*SfA*SfG*SfC*SfC WV- fU*SfC*SfA*SfC*SfU*SfC*S1An001RfG*SfA 2831 UCACUCAGAUAGTTGAAGCC SSSSSSnRSSSSnRnRSSSSSS 25541 *SmU*SfA*SlGn001RlTn001RlT*SfG*SfA*SfA *SfG*SfC*SfC WV- lT*SfC*SlA*SfC*SfU*SfC*SmAn001RfG*SfA 2832 TCACUCAGAUAGUUGAAGCC SSSSSSnRSSSSnRnRSSSSSS 25542 *SmU*SfA*SmGn001RmUn001RfU*SfG*SfA* SfA*SfG*SfC*SfC WV- fU*SfC*SfA*SfC*SfU*SfC*SmAn001RfG* 2833 UCACUCAGAUAGUUGAAGCC SSSSSSnRSSSSnRnRSSSSSS 25543 SfA*SmU*SfA*SmGn001RmUn001RfU*SfG* SfA*SfA*SlG*SfC*SfC WV- lT*SfC*SlA*SfC*SfU*SfC*SmAn001RfG*SfA 2834 TCACUCAGAUAGUUGAAGCC SSSSSSnRSSSSnRnRSSSSSS 25544 *SmU*SfA*SmGn001RmUn001RfU*SfG*SfA* SfA*SlG*SfC*SfC WV- L001fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA 2835 UCACUCAGAUAGUUGAAGCC OSSSSSSOSSSSOSSSSSSS 27163 *SmU*SfA*SmGmU*SfU*SfG*SfA*SfA*SfG *SfC*SfC WV- L001fU*SfC*SfAn001RfC*SfU*SfCn001RmAfG* 2836 UCACUCAGAUAGUUGAAGCC OSSnRSSnROSSSSOSSSSnRSS 27164 SfA*SmU*SfA*SmGmU*SfU*SfG*SfA* SfAn001RfG*SfC*SfC WV-19790 Mod020L001fUfCfAfCfUfCmAn001fGfAmUfA 2837 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19791 Mod015L001fUfCfAfCfUfCmAn001fGfAmUfA 2838 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19792 Mod109L001fUfCfAfCfUfCmAn00lfGfAmUfA 2839 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19793 Mod110L001fUfCfAfCfUfCmAn001fGfAmUfA 2840 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19794 Mod111L001fUfCfAfCfUfCmAn001fGfAmUfA 2841 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19795 Mod112L001fUfCfAfCfUfCmAn00lfGfAmUfA 2842 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19796 Mod113L001fUfCfAfCfUfCmAn001fGfAmUfA 2843 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19797 Mod114L001fUfCfAfCfUfCmAn001fGfAmUfA 2844 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-19798 Mod115L001fUfCfAfCfUfCmAn001fGfAmUfA 2845 UCACUCAGAUAGUUGAA OSSSSSSnXSSSSnXnXS mGn001mUn001fUfGfAfAfGfCfC GCC SSSSS WV-15883 fC*SfU*SfCn002RfC*SfG*SfGn002RfU*SfU*SmCfU 2846 CUCCGGUUCUGAAGGUG SSnRSSnRSSOSSSOOSSnR *SmC*SfA*SmAfGfG*SfU*SfGn002RfU*SfU*SfC UUC SS WV-15884 mU*SGeon002m5Ceon002m5Ceon002mA*SG*SG*RC 2847 UGCCAGGCTGGTTATGAC SnXnXnXSSRSSR *ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC* UC SSRSSSSSS SmU*SmC WV-15885 mU*SGeon002Rm5Ceon002Rm5Ceon002RmA*SG*SG* 2848 UGCCAGGCTGGTTATGAC SnRnRnRSSRSSR RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC SSRSSSSSS SmC*SmU*SmC WV-15886 fC*SfU*SfCn002fC*SfG*SfUn002fU*SfU*SmCfU* 2849 CUCCGGUUCUGAAGGUG SSnXSSnXSSOSSSOOSSnX SmG*SfA*SmAfGfG*SfU*SfUn002fU*SfU*SfC UUC SS WV-15887 mU*SGeon002Sm5Ceon002Sm5Ceon002SmA*SG*SG* 2850 UGCCAGGCTGGTTATGAC SnSnSnSSSRSSR RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC SSRSSSSSS SmC*SmU*SmC WV-16006 fCfUfCn003RfCfGfGn003RfUfUmCfUmGfAmAfGfGfUfGn0 2851 CUCCGGUUCUGAAGGUG SSnRSSnRSSOSSS 03RfUfUfC UUC OOSSnRSS WV-16008 fUfCfAfCfUfCmAn003fGfAmUfAmGn003mUn003fUfGfAfA 2852 UCACUCAGAUAGUUGAA SSSSSSnXSSSSnX fGfCfC GCC nXSSSSSS WV-16007 fCfUfCn004RfCfGfGn004RfUfUmCfU 2853 CUCCGGUUCUGAAGGUG SSnRSSnRSSOSSS mGfAmAfGfGfUGn004RfUfUfC UUC OOSSnRSS WV-16009 fUfCfAfCfUfCmAn004fGfAmUfAmG 2854 UCACUCAGAUAGUUGAA SSSSSSnXSSSSnX n004mUn004fUfGfAfAfGfCfC GCC nXSSSSSS WV-24088 fU*SfC*SfA*SfC*SfU*SfC*SmAn005fG*SfA* 2855 UCACUCAGAUAGUUGAA SSSSSSnXSSSS SmU*SfA*SmGn005mUn005fU*SfG*SfA*SfA*SfG* GCC nXnX SfC*SfC SSSSSS WV-24089 fU*SfC*SfA*SfC*SfU*SfC*SmAn005RfG*SfA* 2856 UCACUCAGAUAGUUGAA SSSSSSnRSSSS SmU*SfA*SmGn005RmUn005RfU*SfG*SfA*SfA* GCC nRnR SfG*SfC*SfC SSSSSS WV-24090 fU*SfU*SfA*SfC*SfU*SfC*SmAn005SfG*SfA* 2857 UCACUCAGAUAGUUGAA SSSSSSnSSSSS SmU*SfA*SmGn005SmUn005SfU*SfG*SfA*SfA* GCC nSnS SfG*SfC*SfC SSSSSS WV-24100 mU*SGeon005m5Ceon005m5Ceon005mA*SG*SG*RC 2858 UGCCAGGCTGGTTATGAC SnXnXnXSSRSS *ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC* UC RSSRSS SmU*SmC SSSS WV-24101 mU*SGeon005Rm5Ceon005Rm5Ceon005RmA*SG*SG* 2859 UGCCAGGCTGGTTATGAC SnRnRnRSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV-24102 mU*SGeon005Sm5Ceon005Sm5Ceon005SmA*SG*SG* 2860 UGCCAGGCTGGTTATGAC SnSnSnSSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV-24091 fU*SfC*SfA*SfC*SfU*SfC*SmAn006fG*SfA* 2861 UCACUCAGAUAGUUGAA SSSSSSnXSSSS SmU*SfA*SmGn006mUn006fU*SfG*SfA*SfA*SfG* GCC nXnX SfC*SfC SSSSSS WV-24092 fU*SfC*SfA*SfC*SfU*SfC*SmAn006RfG*SfA* 2862 UCACUCAGAUAGUUGAA SSSSSSnRSSSS SmU*SfA*SmGn006RmUn006RfU*SfG*SfA*SfA* GCC nRnR SfG*SfC*SfC SSSSSS WV-24093 fU*SfC*SfA*SfC*SfU*SfC*SmAn006SfG*SfA* 2863 UCACUCAGAUAGUUGAA SSSSSSnSSSSS SmU*SfA*SmGn006SmUn006SfU*SfG*SfA*SfA* GCC nSnS SfG*SfC*SfC SSSSSS WV-24103 mU*SGeon006m5Ceon006m5Ceon006mA*SG*SG*RC 2864 UGCCAGGCTGGTTATGAC SnXnXnXSSRSS *ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC* UC RSSRSS SmU*SmC SSSS WV-24104 mU*SGeon006Rm5Ceon006Rm5Ceon006RmA*SG*SG* 2865 UGCCAGGCTGGTTATGAC SnRnRnRSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV-24105 mU*SGeon006Sm5Ceon006Sm5Ceon006SmA*SG*SG* 2866 UGCCAGGCTGGTTATGAC SnSnSnSSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV-24094 fU*SfC*SfA*SfC*SfU*SfC*SmAn007fG*SfA* 2867 UCACUCAGAUAGUUGAA SSSSSSnXSSSS SmU*SfA*SmGn007mUn007fU*SfG*SfA*SfA*SfG* GCC nXnX SfC*SfC SSSSSS WV-24095 fU*SfC*SfA*SfC*SfU*SfC*SmAn007RfG*SfA* 2868 UCACUCAGAUAGUUGAA SSSSSSnRSSSS SmU*SfA*SmGn007RmUn0071RfU*SfG*SfA*SfA* GCC nRnR SfG*SfC*SfC SSSSSS WV-24096 fU*SfC*SfA*SfC*SfU*SfC*SmAn007SfG*SfA* 2869 UCACUCAGAUAGUUGAA SSSSSSnSSSSS SmU*SfA*SmGn007SmUn007SfU*SfG*SfA*SfA* GCC nSnS SfG*SfU*SfC SSSSSS WV-24106 mU*SGeon007Rm5Ceon007Rm5Ceon007RmA*SG*SG* 2870 UGCCAGGCTGGTTATGAC SnRnRnRSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV-24107 mU*SGeon007Sm5Ceon007Sm5Ceon007SmA*SG*SG* 2871 UGCCAGGCTGGTTATGAC SnSnSnSSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV-24097 fU*SfC*SfA*SfC*SfU*SfC*SmAn008fG*SfA* 2872 UCACUCAGAUAGUUGAA SSSSSSnXSSSS SmU*SfA*SmGn008mUn008fU*SfG*SfA*SfA*SfG* GCC nXnX SfC*SfC SSSSSS WV-24098 fU*SfC*SfA*SfC*SfU*SfC*SmAn008RfG*SfA* 2873 UCACUCAGAUAGUUGAA SSSSSSnRSSSS SmU*SfA*SmGn008RmUn008RfU*SfG*SfA*SfA* GCC nRnR SfG*SfC*SfC SSSSSS WV-24099 fU*SfC*SfA*SfC*SfU*SfC*SmAn008SfG*SfA* 2874 UCACUCAGAUAGUUGAA SSSSSSnSSSSS SmU*SfA*SmGn008SmUn008SfU*SfG*SfA*SfA* GCC nSnS SfG*SfC*SfC SSSSSS WV-24108 mU*SGeon008m5Ceon008m5Ceon008mA*SG*SG*RC 2875 UGCCAGGCTGGTTATGAC SnXnXnXSSRSS *ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC* UC RSSRSS SmU*SmC SSSS WV-24109 mU*SGeon008Rm5Ceon008Rm5Ceon008RmA*SG*SG* 2876 UGCCAGGCTGGTTATGAC SnRnRnRSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV-24110 mU*SGeon008Sm5Ceon008Sm5Ceon008SmA*SG*SG* 2877 UGCCAGGCTGGTTATGAC SnSnSnSSSRSS RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* UC RSSRSS SmC*SmU*SmC SSSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU*SmG 2878 CUCCGGUUCUGAAGGUGUUC SSnXSSnXSSOSS 12880 *SfA*SmAfG*SfG*SfU*SfGn001fU*SfU*SfC SOSSSnXSS WV- fC*SfU*SfCn001fC*SfG*SfGn001fU*SfU*SmCfU*SmG 2879 CUCCGGUUCUGAAGGUGUUC SSnXSSnXSSOSS 12880 *SfA*SmAfG*SfG*SfU*SfGn001fU*SfU*SfC SOSSSnXSS WV- fGn001RfU GU nR 21219 WV- fCn001RfC CC nR 21226 WV- fGn001SfU GU nS 21252 WV- fCn001SfC CC nS 21253 WV- fGn001RmA GA nR 21258 WV- fC*RfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2880 CUCCGGUUCUGAAGGUGUUC RSnRSSnRSSOSS 21374 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SOSSSnRSS WV- fC*SfU*RfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2881 CUCCGGUUCUGAAGGUGUUC SRnRSSnRSSOSS 21375 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SOSSSnRSS WV- fC*SfU*SfCn001SfC*SfG*SfGn001RfU*SfU*SmCfU* 2882 CUCCGGUUCUGAAGGUGUUC SSnSSSnRSSOSS 21376 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SOSSSnRSS WV- fC*SfU*SfCn001RfC*RfG*SfGn001RfU*SfU*SmCfU* 2883 CUCCGGUUCUGAAGGUGUUC SSnRRSnRSSOSS 21377 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SOSSSnRSS WV- fC*SfU*SfCn001RfC*SfG*RfGn001RfU*SfU*SmCfU* 2884 CUCCGGUUCUGAAGGUGUUC SSnRSSRnRSSOSS 21378 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SOSSSnRSS WV- fC*SfU*SfCn001RfC*SfG*SfGn001SfU*SfU*SmCfU* 2885 CUCCGGUUCUGAAGGUGUUC SSnRSSnSSSOSS 21379 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SOSSSnRSS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*RfU*SmCfU* 2886 CUCCGGUUCUGAAGGUGUUC SSnRSSnR 21380 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC RSOSSSOSSSnR SS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*RmCfU* 2887 CUCCGGUUCUGAAGGUGUUC SSnRSSnR 21381 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SROSSSOSSSnR SS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2888 CUCCGGUUCUGAAGGUGUUC SSnRSSnR 21382 RmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SSORSSOSSSnR SS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2889 CUCCGGUUCUGAAGGUGUUC SSnRSSnR 21383 SmG*RfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*SfC SSOSRSOSSSnRSS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2890 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 21384 SmG*SfA*RmAfG*SfG*SfU*SfGn001RfU*SfU*SfC ROSSSnRSS WV fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2891 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 21385 SmG*SfA*SmAfG*RfG*SfU*SfGn001RfU*SfU*SfC SORSSnRSS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2892 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 21386 SmG*SfA*SmAfG*SfG*RfU*SfGn001RfU*SfU*SfC SOSRSnRSS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2893 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 21387 SmG*SfA*SmAfG*SfG*SfU*RfGn001RfU*SfU*SfC SOSSRnRSS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2894 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 21388 SmG*SfA*SmAfG*SfG*SfU*SfGn001SfU*SfU*SfC SOSSSnSSS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2895 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 21389 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*RFU*SfC SOSSSnRRS WV- fC*SfU*SfCn001RfC*SfG*SfGn001RfU*SfU*SmCfU* 2896 CUCCGGUUCUGAAGGUGUUC SSnRSSnRSSOSS 21390 SmG*SfA*SmAfG*SfG*SfU*SfGn001RfU*SfU*RfC SOSSSnRSR WV- fC*SfU*SfUn001fA*SfA*SfGn001fA*SfU*SmA*SfC* 2897 CUUAAGAUACCAUUUGUAUU SSnXSSnXSSSSS 21578 SmC*SfA*SmU*SfU*SfU*SfG*SfUn001fA*SfU*SfU SSSSSnXSS WV- fU*SfU*SfAn001fA*SfG*SfAn001fU*SfA*SmC*SfC* 2898 UUAAGAUACCAUUUGUAUUU SSnXSSnXSSSSS 21579 SmA*SfU*SmU*SfU*SfG*SfU*SfAn001fU*SfU*SfU SSSSSnXSS WV- fU*SfA*SfAn001fG*SfA*SfUn001fA*SfC*SmC*SfA* 2899 UAAGAUACCAUUUGUAUUUA SSnXSSnXSSSSS 21580 SmU*SfU*SmU*SfG*SfU*SfA*SfUn001fU*SfU*SfA SSSSSnXSS WV- fA*SfA*SfGn001fA*SfU*SfAn001fC*SfC*SmA*SfU* 2900 AAGAUACCAUUUGUAUUUAG SSnXSSnXSSSSS 21581 SmU*SfU*SmG*SfU*SfA*SfU*SfUn001fU*SfA*SfG SSSSSnXSS WV- fA*SfG*SfAn001fU*SfA*SfCn001fC*SfA*SmU*SfU* 2901 AGAUACCAUUUGUAUUUAGC SSnXSSnXSSSSS 21582 SmU*SfG*SmU*SfA*SfU*SfU*SfUn001fA*SfG*SfC SSSSSnXSS WV- fG*SfA*SfUn001fA*SfC*SfCn001fA*SfU*SmU*SfU* 2902 GAUACCAUUUGUAUUUAGCA SSnXSSnXSSSSS 21583 SmG*SfU*SmA*SfU*SfU*SfU*SfAn001fG*SfC*SfA SSSSSnXSS WV- fA*SfU*SfAn001fC*SfC*SfAn001fU*SfU*SmU*SfG* 2903 AUACCAUUUGUAUUUAGCAU SSnXSSnXSSSSS 21584 SmU*SfA*SmU*SfU*SfU*SfA*SfGn001fC*SfA*SfU SSSSSnXSS WV- fU*SfA*SfCn001fC*SfA*SfUn001fU*SfU*SmG*SfU* 2904 UACCAUUUGUAUUUAGCAUG SSnXSSnXSSSSS 21585 SmA*SfU*SmU*SfU*SfA*SfG*SfCn001fA*SfU*SfG SSSSSnXSS WV- fA*SfC*SfCn001fA*SfU*SfUn001fU*SfG*SmU*SfA* 2905 ACCAUUUGUAUUUAGCAUGU SSnXSSnXSSSSS 21586 SmU*SfU*SmU*SfA*SfG*SfC*SfAn001fU*SfG*SfU SSSSSnXSS WV- fC*SfC*SfAn001fU*SfU*SfUn001fG*SfU*SmA*SfU* 2906 CCAUUUGUAUUUAGCAUGUU SSnXSSnXSSSSS 21587 SmU*SfU*SmA*SfG*SfC*SfA*SfUn001fG*SfU*SfU SSSSSnXSS WV- fC*SfA*SfUn001fU*SfU*SfGn001fU*SfA*SmU*SfU* 2907 CAUUUGUAUUUAGCAUGUUC SSnXSSnXSSSSS 21588 SmU*SfA*SmG*SfC*SfA*SfU*SfGn001fU*SfU*SfC SSSSSnXSS WV- fA*SfU*SfUn001fU*SfG*SfUn001fA*SfU*SmU*SfU* 2908 AUUUGUAUUUAGCAUGUUCC SSnXSSnXSSSSS 21589 SmA*SfG*SmC*SfA*SfU*SfG*SfUn001fU*SfC*SfC SSSSSnXSS WV- fU*SfU*SfUn001fG*SfU*SfAn001fU*SfU*SmU*SfA* 2909 UUUGUAUUUAGCAUGUUCCC SSnXSSnXSSSSS 21590 SmG*SfC*SmA*SfU*SfG*SfU*SfUn001fC*SfC*SfC SSSSSnXSS WV- fU*SfU*SfGn001fU*SfA*SfUn001fU*SfU*SmA*SfG* 2910 UUGUAUUUAGCAUGUUCCCA SSnXSSnXSSSSS 21591 SmC*SfA*SmU*SfG*SfU*SfU*SfCn001fC*SfC*SfA SSSSSnXSS WV- fU*SfG*SfUn001fA*SfU*SfUn001fU*SfA*SmG*SfC* 2911 UGUAUUUAGCAUGUUCCCAA SSnXSSnXSSSSS 21592 SmA*SfU*SmG*SfU*SfU*SfC*SfCn001fC*SfA*SfA SSSSSnXSS WV- fG*SfU*SfAn001fU*SfU*SfUn001fA*SfG*SmC*SfA* 2912 GUAUUUAGCAUGUUCCCAAU SSnXSSnXSSSSS 21593 SmU*SfG*SmU*SfU*SfC*SfC*SfCn001fA*SfA*SfU SSSSSnXSS WV- fU*SfA*SfUn001fU*SfU*SfAn001fG*SfC*SmA*SfU* 2913 UAUUUAGCAUGUUCCCAAUU SSnXSSnXSSSSS 21594 SmG*SfU*SmU*SfC*SfC*SfC*SfAn001fA*SfU*SfU SSSSSnXSS WV- fU*SfU*SfUn001fA*SfG*SfCn001fA*SfU*SmG*SfU* 2914 UUUAGCAUGUUCCCAAUUCU SSnXSSnXSSSSS 21595 SmU*SfC*SmC*SfC*SfA*SfA*SfUn001fU*SfC*SfU SSSSSnXSS WV- fU*SfU*SfAn001fG*SfC*SfAn001fU*SfG*SmU*SfU* 2915 UUAGCAUGUUCCCAAUUCUC SSnXSSnXSSSSS 21596 SmC*SfC*SmC*SfA*SfA*SfU*SfUn001fC*SfU*SfC SSSSSnXSS WV- fU*SfA*SfGn001fC*SfA*SfUn001fG*SfU*SmU*SfC* 2916 UAGCAUGUUCCCAAUUCUCA SSnXSSnXSSSSS 21597 SmC*SfC*SmA*SfA*SfU*SfU*SfCn001fU*SfU*SfA SSSSSnXSS WV- fA*SfG*SfCn001fA*SfU*SfGn001fU*SfG*SmC*SfC* 2917 AGCAUGUUCCCAAUUCUCAG SSnXSSnXSSSSS 21598 SmC*SfA*SmA*SfU*SfU*SfC*SfUn001fC*SfA*SfG SSSSSnXSS WV- fG*SfC*SfAn001fU*SfG*SfUn001fU*SfC*SmC*SfC* 2918 GCAUGUUCCCAAUUCUCAGG SSnXSSnXSSSSS 21599 SmA*SfA*SmU*SfU*SfC*SfU*SfCn001fA*SfG*SfG SSSSSnXSS WV- fC*SfA*SfUn001fG*SfU*SfUn001fC*SfC*SmC*SfA* 2919 CAUGUUCCCAAUUCUCAGGA SSnXSSnXSSSSS 21600 SmA*SfU*SmU*SfC*SfU*SfC*SfAn001fG*SfG*SfA SSSSSnXSS WV- fA*SfU*SfGn001fU*SfU*SfCn001fC*SfC*SmA*SfA* 2920 AUGUUCCCAAUUCUCAGGAA SSnXSSnXSSSSS 21601 SmU*SfU*SmC*SfU*SfC*SfA*SfGn001fG*SfA*SfA SSSSSnXSS WV- fU*SfG*SfUn001fU*SfC*SfCn001fC*SfA*SmA*SfU* 2921 UGUUCCCAAUUCUCAGGAAU SSnXSSnXSSSSS 21602 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ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU RSSRSSSSSS *SmC WV- mU*SGeon010m5Ceon010m5Ceon010mA*SG*SG*RC*ST 3101 UGCCAGGCTGGTTATGACUC SnXnXnXSSRSS XKCD108 *SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU* RSSRSSSSSS SmC WV- mU*SGeon010Rm5Ceon010Rm5Ceon010RmA*SG*SG*RC 3102 UGCCAGGCTGGTTATGACUC SnRnRnRSSRSS XKCD *ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC* RSSRSSSSSS 109 SmU*SmC WV- mU*SGeon010Sm5Ceon010Sm5Ceon010SmA*SG*SG*RC* 3103 UGCCAGGCTGGTTATGACUC SnSnSnSSSRSS XKCD ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU RSSRSSSSSS 110 *SmC WV-3519 Mod032fU*fC*fA*fA*fG*fG*mAfA*mGfA*mUfG*mGfC*fA 3104 UCAAGGAAGA OXXXXXXOXOX *fU*fU*fU*fC*fU UGGCAUUUCU OXOXXXXXX WV-3518 Mod031fU*fC*fA*fA*fG*fG*mAfA*mGfA*mUfG*mGfC*fA 3105 UCAAGGAAGA OXXXXXXOXOX *fu*fU*fU*fC*fU UGGCAUUUCU OXOXXXXXX WV-3517 Mod030fU*fC*fA*fA*fG*fG*mAfA*mGfA*mUfG*mGfC*fA 3106 UCAAGGAAGA OXXXXXXOXOX *fU*fU*fU*fC*fU UGGCAUUUCU OXOXXXXXX WV-3516 fU*fC*fA*fA*fG*fG*mAfA*mGfA*mUfG*mGfC*fA*fU* 3107 UCAAGGAAGA XXXXXXOXOX fU*fU*fC*fU UGGCAUUUCU OXOXXXXXX WV-3515 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmAfU*SmGmGfC* 3108 UCAAGGAAGA SSSSSSOSOO SfAfU*SfU*SfU*SfC*SfU UGGCAUUUCU SOOSOSSSS WV-3514 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGfAfU*SmGmGfC* 3109 UCAAGGAAGA SSSSSSOSOO SfAfU*SfU*SfU*SfC*SfU UGGCAUUUCU SOOSOSSSS WV-3513 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmAfU*SmGmGfC* 3110 UCAAGGAAGA SSSSSSOSOO SmAfU*SfU*SfU*SfC*SfU UGGCAUUUCU SOOSOSSSS WV-3512 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGfAU*SmGmGfC* 3111 UCAAGGAAGA SSSSSSOSOO SmAfU*SfU*SfU*SfC*SfU UGGCAUUUCU SOOSOSSSS WV-3511 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmAfU*SmGmGfC* 3112 UCAAGGAAGA SSSSSSOSOOSOO SmA*SfU*SfU*SfU*SfC*SfU UGGCAUUUCU SSSSSS WV-3510 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGfAfU*SmGmGfC* 3113 UCAAGGAAGA SSSSSSOSOOSOO SmA*SfU*SfU*SfU*SfC*SfU UGGCAUUUCU SSSSSS WV-3509 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 3114 UCAAGGAAGA SSSSSSOSOS *SfAfU*SfU*SfU*SfC*SfU UGGCAUUUCU SOOSOSSSS WV-3508 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGfA*SfU*SmGmGfC* 3115 UCAAGGAAGA SSSSSSOSOS SfAfU*SfU*SfU*SfC*SfU UGGCAUUUCU SOOSOSSSS WV-3507 fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmAfU*SmGmGfC* 3116 UCAAGGAAGA SSSSSSOSOOSOO SfA*SfU*SfU*SfU*SfC*SfU UGGCAUUUCU SSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAn011fG*SfA*SmU*SfA* 3117 UCACUCAGAUA SSSSSSnXSSSS 27250 SmGn011mUn011fU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC nXnXSSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAn010fG*SfA*SmU*SfA* 3118 UCACUCAGAUA SSSSS 27249 SmGn010mUn010fU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC SnXSSSSnXnXSSSSS S WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 3119 UCAAGGAAGA SSSSSSOSOSSOO 24086 *SfA*SfU*SfU*SfU*SfC*SfG UGGCAUUUCG SSSSSS WV- fG*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 3120 GCAAGGAAGAU SSSSSSOSOSSOO 24085 *SfA*SfU*SfU*SfU*SfC*SfU GGCAUUUCU SSSSSS WV- fU*SfG*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmG* 3121 UCAAGGAAGA SSSSSSOSOSSO 22919 SfC*SfA*SfU*SfU*SfU*SfC*SfU UGGCAUUUCU SSSSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmG* 3122 UCAAGGAAGA SSSSSSOSOSSSO 22918 SmGfC*SfA*SfU*SfU*SfU*SfC*SfU UGGCAUUUCU SSSSSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmG 3123 UCAAGGAAGAUG SSSSSSOSOSS 22765 WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 3124 UCAAGGAAGA SSSSSSOSOSSOOS 22764 *SfA UGGCA WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 3125 UCAAGGAAGA SSSSSSOSOS 22763 *SfA*SfU UGGCAU SOOSS WV- fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC 3126 UCAAGGAAGA SSSSSSOSOS 22762 *SfA*SfU*SfU UGGCAUU SOOSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmA*SfG*SfA*SmU*SfA*SmG 3127 UCACUCAGAUA SSSSSSSSSS 22752 *SmU*SfU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC SSSSSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmA*SfG*SfA*SmU*SfA* 3128 UCACUCAGAUA SSSSSSSSSSSOO 22751 SmGmUfU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC SSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU*SfA*SmG* 3129 UCACUCAGAUA SSSSSSOSSSSSO 22750 SmUfU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC SSSSSS WV- fU*SfC*SfA*SfC*SfU*SfC*SmAfG*SfA*SmU*SfA*SmGmU 3130 UCACUCAGAUA SSSSSSOSSSSO 22749 *SfU*SfG*SfA*SfA*SfG*SfC*SfC GUUGAAGCC SSSSSSS WV- fA*SfU*SfC*SfA*SfU*SfU*SfU*SfU*SmU*SfU*SmC*SfU* 3131 AUCAUUUUUU SSSSSSSSSS 21502 SmC*SfA*SfU*SfA*SfC*SfC*SfU*SfU CUCAUACCUU SSSSSSSSS WV- fU*SfA*SfU*SfC*SfA*SfU*SfU*SfU*SmU*SfU*SmU*SfC* 3132 UAUCAUUUUU SSSSSSSSSS 21501 SmU*SfC*SfA*SfU*SfA*SfC*SfC*SfU UCUCAUACCU SSSSSSSSS WV- fU*SfU*SfA*SfU*SfC*SfA*SfU*SfU*SmU*SfU*SmU*SfU* 3133 UUAUCAUUUUU SSSSSSSSSS 21500 SmC*SfU*SfC*SfA*SfU*SfA*SfC*SfC UCUCAUACC SSSSSSSSS WV- fU*SfU*SfU*SfA*SfU*SfC*SfA*SfU*SmU*SfU*SmU*SfU* 3134 UUUAUCAUUUU SSSSSSSSSS 21499 SmU*SfC*SfU*SfC*SfA*SfU*SfA*SfC UUCUCAUAC SSSSSSSSS WV- fU*SfU*SfU*SfU*SfA*SfU*SfC*SfA*SmU*SfU*SmU*SfU* 3135 UUUUAUCAUUUU SSSSSSSSSS 21498 SmU*SfU*SfC*SfU*SfC*SfA*SfU*SfA UUCUCAUA SSSSSSSSS WV- fC*SfU*SfU*SfU*SfU*SfA*SfU*SfC*SmA*SfU*SmU*SfU* 3136 CUUUUAUCAUUU SSSSSSSSSS 21497 SmU*SfU*SfU*SfC*SfU*SfC*SfA*SfU UUUCUCAU SSSSSSSSS WV- fA*SfC*SfU*SfU*SfU*SfU*SfA*SfU*SmC*SfA*SmU*SfU* 3137 ACUUUUAUCAUU SSSSSSSSSS 21496 SmU*SfU*SfU*SfU*SfC*SfU*SfC*SfA UUUUCUCA SSSSSSSSS WV- fA*SfA*SfC*SfU*SfU*SfU*SfU*SfA*SmU*SfC*SmA*SfU* 3138 AACUUUUAUCAU SSSSSSSSSS 21495 SmU*SfU*SfU*SfU*SfU*SfC*SfU*SfC UUUUUCUC SSSSSSSSS WV- fC*SfA*SfA*SfC*SfU*SfU*SfU*SfU*SmA*SfU*SmC*SfA* 3139 CAACUUUUAUCAU SSSSSSSSSS 21494 SmU*SfU*SfU*SfU*SfU*SfU*SfC*SfU UUUUUCU SSSSSSSSS WV- fC*SfC*SfA*SfA*SfC*SfU*SfU*SfU*SmU*SfA*SmU*SfC* 3140 CCAACUUUUAU SSSSSSSSSS 21493 SmA*SfU*SfU*SfU*SfU*SfU*SfU*SfU CAUUUUUUC SSSSSSSSS WV- fG*SfC*SfC*SfA*SfA*SfC*SfU*SfU*SmU*SfU*SmA*SfU* 3141 GCCAACUUUUA SSSSSSSSSS 21492 SmC*SfA*SfU*SfU*SfU*SfU*SfU*SfU UCAUUUUUU SSSSSSSSS WV- fU*SfG*SfC*SfC*SfA*SfA*SfC*SfU*SmU*SfU*SmU*SfA* 3142 UGCCAACUUUU SSSSSSSSSS 21491 SmU*SfC*SfA*SfU*SfU*SfU*SfU*SfU AUCAUUUUU SSSSSSSSS WV- fC*SfU*SfG*SfC*SfC*SfA*SfA*SfC*SmU*SfU*SmU*SfU* 3143 CUGCCAACUUUU SSSSSSSSSS 21490 SmA*SfU*SfC*SfA*SfU*SfU*SfU*SfU AUCAUUUU SSSSSSSSS WV- fU*SfC*SfU*SfG*SfC*SfC*SfA*SfA*SmC*SfU*SmU*SfU* 3144 UCUGCCAACUUU SSSSSSSSSS 21489 SmU*SfA*SfU*SfC*SfA*SfU*SfU*SfU UAUCAUUU SSSSSSSSS WV- fU*SfU*SfC*SfU*SfG*SfC*SfC*SfA*SmA*SfC*SmU*SfU* 3145 UUCUGCCAACUU SSSSSSSSSS 21488 SmU*SfU*SfA*SfU*SfC*SfA*SfU*SfU UUAUCAUU SSSSSSSSS WV- fC*SfU*SfU*SfC*SfU*SfG*SfC*SfC*SmA*SfA*SmC*SfU* 3146 CUUCUGCCAACU SSSSSSSSSS 21487 SmU*SfU*SfU*SfA*SfU*SfC*SfA*SfU UUUAUCAU SSSSSSSSS WV- fC*SfU*SfCfC*SfG*SfGfU*SfU*SmCfU*SmG*SfA*SmAfG* 3147 CUCCGGUUCUGA SSOSSOSSOSSSOSS 21373 SfG*SfU*SfGfU*SfU*SfC AGGUGUUC SOSS
In Table A1 (including Table A1.1., Table A1.2, Table A1.3, etc.):
Spaces in Table A1 are utilized for formatting and readability, e.g., OXXXXX XXXXX XXXXX XXXX illustrates the same stereochemistry as OXXXXXXXXXXXXXXX; *S and *S both indicate phosphorothioate internucleotidic linkage wherein the linkage phosphorus has Sp configuration; etc.
All oligonucleotides listed in Tables A1 are single-stranded. As described in the present application, they may be used as a single strand, or as a strand to form complexes with one or more other strands.
Some sequences, due to their length, are divided into multiple lines.
ID: Identification number for an oligonucleotide.
WV-8806, WV-13405, WV-13406 and WV-13407 are fully PMO(morpholino oligonucleotides; [all PMO] in Table).
Abbreviations in Tables: m5Ceo: 5-Methyl 2-Methoxyethyl C

(990) ##STR00376## 5MS: 5-(S)CH.sub.3 modification of sugar moieties; 5MSfC: 2-F-5-(S)-methyl C (in oligonucleotides,

(991) ##STR00377##
wherein in BA is nucleobase C and R.sup.2s is F, and the 5 and 3 positions independently connect to OH, internucleotidic linkages, linkers/linkages-H, linkers/linkages-Mod, etc. Nucleoside form is

(992) ##STR00378##
wherein in BA is nucleobase C and R.sup.2s is F); C6: C6 amino linker (L001, NH(CH.sub.2).sub.6 wherein NH is connected to Mod (e.g., through C(O) in Mod) or H, and (CH.sub.2).sub.6 is connected to the 5-end (or 3-end if indicated) of oligonucleotide chain through, e.g., phosphodiester (OP(O)(OH)O. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (OP(O)(SH)O. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled; *S, S, or Sp, if chirally controlled and has an Sp configuration, and *R, R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (OP(S)(SH)O. May exist as a salt form. May be illustrated in the Tables as PS2 or : or D) linkage. May also be referred to as C6 linker or C6 amine linker); : or D: Phosphodithioate (Phosphorodithioate), represented by D or a colon (:); n001: non-negatively charged linkage

(993) ##STR00379##
(which is stereorandom unless otherwise indicated (e.g., as n001R, or n001S)); n002: non-negatively charged linkage

(994) ##STR00380##
(which is stereorandom unless otherwise indicated (e.g., as n002R, or n002S)); n003: non-negatively charged linkage

(995) ##STR00381##
(which is stereorandom unless otherwise indicated (e.g., as n003R, or n003S)); n004: non-negatively charged linkage

(996) ##STR00382##
(which is stereorandom unless otherwise indicated (e.g., as n004R, or n004S); n005: non-negatively charged linkage

(997) ##STR00383##
(which is stereorandom unless otherwise indicated (e.g., as n005R, or n005S)); n006: non-negatively charged linkage

(998) ##STR00384##
(which is stereorandom unless otherwise indicated (e.g., as n006R, or n006S)); n007: non-negatively charged linkage

(999) ##STR00385##
(which is stereorandom at linkage phosphorus unless otherwise indicated (e.g., as n007R, or n007S)); n008: non-negatively charged linkage

(1000) ##STR00386##
(which is stereorandom unless otherwise indicated (e.g., as n008R, or n008S)); n009: non-negatively charged linkage

(1001) ##STR00387##
(which is stereorandom unless otherwise indicated (e.g., as n009R, or n009S)); n010: non-negatively charged linkage

(1002) ##STR00388##
(which is stereorandom unless otherwise indicated (e.g., as n010R, or n010S)); n001R: n001 being chirally controlled and having the Rp configuration; n002R: n002 being chirally controlled and having the Rp configuration; n003R: n003 being chirally controlled and having the Rp configuration; n004R: n004 being chirally controlled and having the Rp configuration; n005R: n005 being chirally controlled and having the Rp configuration; n006R: n006 being chirally controlled and having the Rp configuration; n007R: n007 being chirally controlled and having the Rp configuration; n008R: n008 being chirally controlled and having the Rp configuration; n009R: n009 being chirally controlled and having the Rp configuration; n010R: n010 being chirally controlled and having the Rp configuration; n001S: n001 being chirally controlled and having the Sp configuration; n002S: n002 being chirally controlled and having the Sp configuration; n003S: n003 being chirally controlled and having the Sp configuration; n004S: n004 being chirally controlled and having the Sp configuration; n005S: n005 being chirally controlled and having the Sp configuration; n006S: n006 being chirally controlled and having the Sp configuration; n007S: n007 being chirally controlled and having the Sp configuration; n008S: n008 being chirally controlled and having the Sp configuration; n009S: n009 being chirally controlled and having the Sp configuration; n010S: n010 being chirally controlled and having the Sp configuration; nO, nX: in Linkage/Stereochemistry, nO or nX indicates a stereorandom n001; nR: in Linkage/Stereochemistry, nR indicates a linkage, e.g., n001, n002, n003, n004, n005, n006, n007, n008, n009, etc., being chirally controlled and having the Rp configuration (e.g., for n001, n001R in Description); nS: in Linkage/Stereochemistry, nS indicates a linkage, e.g., n001, n002, n003, n004, n005, n006, n007, n008, n009, etc., being chirally controlled and having the Sp configuration (e.g., for n001, n001R in Description); BrfU: a nucleoside unit wherein the nucleobase is BrU

(1003) ##STR00389##
and wherein the sugar has a 2-F (f) modification

(1004) ##STR00390## BrmU: a nucleoside unit wherein the nucleobase is BrU

(1005) ##STR00391##
and wherein the sugar has a 2-OMe (m) modification

(1006) ##STR00392## BrdU: a nucleoside unit wherein the nucleobase is BrU

(1007) ##STR00393##
and wherein the sugar is 2-deoxyribose (as widely found in natural DNA; 2-deoxy (d))

(1008) ##STR00394## L004: linker having the structure of NH(CH.sub.2).sub.4CH(CH.sub.2OH)CH.sub.2, wherein NH is connected to Mod (e.g., through C(O) in Mod) or H, and the CH.sub.2 connecting site is connected to a linkage, e.g., phosphodiester (OP(O)(OH)O. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (OP(O)(SH)O. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled; *S, S, or Sp, if chirally controlled and has an Sp configuration, and *R, R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (OP(S)(SH)O. May exist as a salt form. May be illustrated in the Tables as PS2 or : or D) linkage, at the 5- or 3-end of an oligonucleotide chain as indicated. For example, an asterisk immediately preceding a L004 (e.g., *L004) indicates that the linkage is a phosphorothioate linkage, and the absence of the indication of any other linkage immediately preceding L004 indicates that the linkage is a phosphodiester linkage. For example, in WV-9858, which terminates in fUL004, the linker L004 is connected (via the CH.sub.2 site) to the phosphodiester linkage at the 3 position at the 3-terminal sugar (which is 2-F and connected to the nucleobase U), and the L004 linker is connected via NH to H; similarly, in WV-10886, WV-10887, and WV-10888, the L004 linker is connected (via the CH.sub.2 site) to the phosphodiester linkage at the 3 position of the 3-terminal sugar, and the L004 is connected via NH to Mod012 (WV-10886), Mod085 (WV-10887) or Mod086 (WV-10888); L005: linker having the structure of NH(CH.sub.2).sub.5C(O)N(CH.sub.2CH.sub.2OH) CH.sub.2CH.sub.2, wherein NH is connected to Mod (e.g., through C(O) in Mod) or H, and the CH.sub.2 connecting site is connected to a linkage, e.g., phosphodiester (OP(O)(OH)O. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (OP(O)(SH)O. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled; *S, S, or Sp, if chirally controlled and has an Sp configuration, and *R, R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (OP(S)(SH)O. May exist as a salt form. May be illustrated in the Tables as PS2 or : or D) linkage, at the 5- or 3-end of an oligonucleotide chain as indicated. For example, an asterisk immediately preceding a L005 (e.g., *L005) indicates that the linkage is a phosphorothioate linkage, and the absence of the indication of any other linkage immediately preceding L005 indicates that the linkage is a phosphodiester linkage. For example, in WV-12571, L005 is connected to H (no Mod following L005; via the NH site) and the phosphodiester linkage at the 3 position of the 3-terminal sugar (via the CH.sub.2 site); and in WV-12572, L005 is connected to Mod020 (via the NH site) and the phosphodiester linkage at the 3 position of the 3-terminal sugar (via the CH.sub.2 site); L001L005: linker having the structure of NH(CH.sub.2).sub.5C(O)N(CH.sub.2CH.sub.2OP(O)(OH)O(CH.sub.2).sub.6NH)CH.sub.2CH.sub.2, wherein each of the two NH is independently connected to Mod (e.g., through C(O)) or H, and the CH.sub.2 connecting site is connected to a linkage, e.g., phosphodiester (OP(O)(OH)O. May exist as a salt form. May be illustrated in the Tables as O or PO), phosphorothioate (OP(O)(SH)O. May exist as a salt form. May be illustrated in the Tables as * if the phosphorothioate not chirally controlled; *S, S, or Sp, if chirally controlled and has an Sp configuration, and *R, R, or Rp, if chirally controlled and has an Rp configuration), or phosphorodithioate (OP(S)(SH)O. May exist as a salt form. May be illustrated in the Tables as PS2 or: or D) linkage at the 5- or 3-end of an oligonucleotide chain as indicated. eo: 2-MOE (2-OCH.sub.2CH.sub.2OCH.sub.3) modification on the preceding nucleoside (e.g., Aeo (

(1009) ##STR00395##
wherein BA is nucleobase A)); F, f: 2-F modification on the following nucleoside (e.g., fA

(1010) ##STR00396##
wherein BA is nucleobase A)), m: 2-OMe modification on the following nucleoside (e.g., mA

(1011) ##STR00397##
wherein BA is nucleobase A)); r: 2-OH on the following nucleoside (e.g., rA

(1012) ##STR00398##
wherein BA is nucleobase A, as existed in natural RNA));

(1013) L012: internucleotidic linkage having the structure of OP(O)[O(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2OH]O. May be illustrated as OO in the Tables; *, PS: Phosphorothioate; PS2,: D: phosphorodithioate (e.g., WV-3078, wherein a colon (:) indicates a phosphorodithioate); *R, R, Rp: Phosphorothioate in Rp conformation; *S, S, Sp: Phosphorothioate in Sp conformation; X: Phosphorothioate stereorandom;

(1014) ##STR00399## NA: Not Applicable; O, PO: phosphodiester (phosphate). When no internucleotidic linkage is specified between two nucleoside units, the internucleotidic linkage is a phosphodiester linkage (natural phosphate linkage).

(1015) When used to indicate linkage between Mod and a linker, e.g., L001, O may indicate C(O) (connecting Mod and L001, for example: Mod013L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC *SfU (SEQ ID NO: 3148) (Description), OOSSSSSSOSOSSOOSSSSSS (Linkage/Stereochemistry). Note the second O in OOSSSSSSOSOSSOOSSSSSS (Linkage/Stereochemistry) represents phosphodiester linkage connecting L001 and the 5-O of the 5-terminal sugar of the oligonucleotide chain (see illustrations below. Alternatively, the 5-O may be considered part of the phosphodiester linkage (or another type of linkage such as a phosphorothioate linkage), in which case the phosphodiester linkage (or another type of linkage such as phosphorothioate linkage) is connected to the 5 position of the 5-terminal sugar of the oligonucleotide chain). In some instances, O for C(O) (connecting Mod and L001) is omitted (e.g., for Mod013L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC *SfU (SEQ ID NO: 3148), Linkage/Stereochemistry OSSSSSSOSOSSOOSSSSSS);
Various Mods:

(1016) Mod001 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1017) ##STR00400##
Lauric (in Mod013), Myristic (in Mod014), Palmitic (in Mod005), Stearic (in Mod015), Oleic (in Mod016), Linoleic (in Mod017), alpha-Linoleinc (in Mod018), gamma-Linolenic (in Mod019), DHA (in Mod006), Turbinaric (in Mod020), Dilinoleic (in Mod021), TriGlcNAc (in Mod024), TrialphaMannose (in Mod026), MonoSulfonamide (in Mod 027), TriSulfonamide (in Mod029), Lauric (in Mod030), Myristic (in Mod031), Palmitic (in Mod032), and Stearic (in Mod033): Lauric acid (for Mod013), Myristic acid (for Mod014), Palmitic acid (for Mod005), Stearic acid (for Mod015), Oleic acid (for Mod016), Linoleic acid (for Mod017), alpha-Linolenic acid (for Mod018), gamma-Linolenic acid (for Mod019), docosahexaenoic acid (for Mod006), Turbinaric acid (for Mod020), alcohol for Dilinoleyl (for Mod021), acid for TriGlcNAc (for Mod024), acid for TrialphaMannose (for Mod026), acid for MonoSulfonamide (for Mod 027), acid for TriSulfonamide (for Mod029), Lauryl alcohol (for Mod030), Myristyl alcohol (for Mod031), Palmityl alcohol (for Mod032), and Stearyl alcohol (for Mod033), respectively, conjugated to oligonucleotide chains, e.g., through an amide group, a linker (e.g., C6 amino linker, (L001)), and/or a linkage group (e.g., phosphodiester linkage (PO), phosphorothioate linkage (PS), etc.): e.g., Mod013 (Lauric acid with C6 amino linker and PO or PS), Mod014 (Myristic acid with C6 amino linker and PO or PS), Mod005 (Palmitic acid with C6 amino linker and PO or PS), Mod015 (Stearic acid with C6 amino linker and PO or PS), Mod016 (Oleic acid with C6 amino linker and PO or PS), Mod017 (Linoleic acid with C6 amino linker and PO or PS), Mod018 (alpha-Linolenic acid with C6 amino linker and PO or PS), Mod019 (gamma-Linolenic acid with C6 amino linker and PO or PS), Mod006 (DHA with C6 amino linker and PO or PS), Mod020 (Turbinaric acid with C6 amino linker and PO or PS), Mod021 (alcohol (see below) with PO or PS), Mod024 (acid (see below) with C6 amino linker and PO or PS), Mod026 (acid (see below) with C6 amino linker and PO or PS), Mod027 (acid (see below) with C6 amino linker and PO or PS), Mod029 (acid (see below) with C6 amino linker and PO or PS), Mod030 (Lauryl alcohol with PO or PS), Mod031 (Myristyl alcohol with PO or PS), Mod032 (Palmityl alcohol with PO or PS), and Mod033 (Stearyl alcohol with PO or PS), with PO or PS for each oligonucleotide indicated in Table A1. For example, WV-3557 Steary alcohol conjugated to oligonucleotide chain of WV-3473 via PS: Mod033*fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*SfC*Sf U (SEQ ID NO: 3149) (Description), XSSSSSSOSOSSOOSSSSSS (Stereochemistry); and WV-4106 Stearic acid conjugated to oligonucleotide chain of WV-3473 via amide group, C6, and PS:
Mod015L001*fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*Sf C*SfU (SEQ ID NO: 3150) (Description), XSSSSSSOSOSSOOSSSSSS (Stereochemistry). Certain moieties for conjugation, and example reagents (many of which were previously known and are commercially available or can be readily prepared using known technologies in accordance with the present disclosure, e.g., Lauric acid (for Mod013), Myristic acid (for Mod014), Palmitic acid (for Mod005), Stearic acid (for Mod015), Oleic acid (for Mod016), Linoleic acid (for Mod017), alpha-Linolenic acid (for Mod018), gamma-Linolenic acid (for Mod019), docosahexaenoic acid (for Mod006), Turbinaric acid (for Mod020), alcohol for Dilinoleyl (for Mod021), Lauryl alcohol (for Mod030), Myristyl alcohol (for Mod031), Palmityl alcohol (for Mod032), Stearyl alcohol (for Mod033), etc.) are listed below. Certain example moieties (e.g., lipid moieties, targeting moiety, etc.) and/or example preparation reagents (e.g., acids, alcohols, etc.) for conjugation to oligonucleotide chains include the below with a non-limiting example of a linker:
Mod005 (with C(O) connecting to, e.g., NH of a linker such as L001) and Palmitic acid:

(1018) ##STR00401##
Mod005L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1019) ##STR00402##
Mod006 (with C(O) connecting to, e.g., NH of a linker such as L001) and DHA:

(1020) ##STR00403##
Mod006L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1021) ##STR00404##
Mod009 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1022) ##STR00405##
Mod012 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1023) ##STR00406##
Mod013 (with C(O) connecting to, e.g., NH of a linker such as L001) and Lauric acid:

(1024) ##STR00407##
Mod013L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1025) ##STR00408##
Mod014 (with C(O) connecting to, e.g., NH of a linker such as L001) and Myristic acid:

(1026) ##STR00409##
Mod014L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1027) ##STR00410##
Mod015 (with C(O) connecting to, e.g., NH of a linker such as L001) and Stearic acid:

(1028) ##STR00411##
Mod015L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1029) ##STR00412##
Mod016 (with C(O) connecting to, e.g., NH of a linker such as L001) and Oleic acid:

(1030) ##STR00413##
Mod016L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1031) ##STR00414##
Mod017 (with C(O) connecting to, e.g., NH of a linker such as L001) and Linoleic acid:

(1032) ##STR00415##
Mod 017L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1033) ##STR00416##
Mod018 (with C(O) connecting to, e.g., NH of a linker such as L001) and alpha-Linolenic acid:

(1034) ##STR00417##
Mod018L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1035) ##STR00418##
Mod019 (with C(O) connecting to, e.g., NH of a linker such as L001) and gamma-Linolenic acid:

(1036) ##STR00419##
Mod019L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1037) ##STR00420##
Mod020 (with C(O) connecting to, e.g., NH of a linker such as L001) and Turbinaric acid:

(1038) ##STR00421##
Mod020L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1039) ##STR00422##
Mod021 (with PO or PS connecting to 5-O of an oligonucleotide chain) and alcohol:

(1040) ##STR00423##
Mod024 (with C(O) connecting to, e.g., NH of a linker such as L001) and acid:

(1041) ##STR00424##
Mod024L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1042) ##STR00425##
Mod026 (with C(O) connecting to, e.g., NH of a linker such as L001) and acid:

(1043) ##STR00426##
Mod026L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1044) ##STR00427##
Mod027 (with C(O) connecting to, e.g., NH of a linker such as L001) and acid:

(1045) ##STR00428##
Mod027L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1046) ##STR00429##
Mod028 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1047) ##STR00430##
Mod029 (with C(O) connecting to, e.g., NH of a linker such as L001) and acid:

(1048) ##STR00431##
Mod029L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1049) ##STR00432##
Mod030 (with PO or PS connecting to 5-O of an oligonucleotide chain) and Lauryl alcohol:

(1050) ##STR00433##
Mod031 (with PO or PS connecting to 5-O of an oligonucleotide chain) and Myristyl alcohol:

(1051) ##STR00434##
Mod032 (with PO or PS connecting to 5-O of an oligonucleotide chain) and Palmityl alcohol:

(1052) ##STR00435##
Mod033 (with PO or PS connecting to 5-O of an oligonucleotide chain) and Stearyl alcohol:

(1053) ##STR00436##
Mod053 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1054) ##STR00437##
Mod070 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1055) ##STR00438##
Mod071 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1056) ##STR00439##
Mod086 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1057) ##STR00440##
Mod092 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1058) ##STR00441##
Mod093 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1059) ##STR00442##
Mod007 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1060) ##STR00443##
Mod050 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1061) ##STR00444##
Mod043 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1062) ##STR00445##
Mod057 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1063) ##STR00446##
Mod058 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1064) ##STR00447##
Mod059 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1065) ##STR00448##
Mod066 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1066) ##STR00449##
Mod074 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1067) ##STR00450##
Mod085 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1068) ##STR00451##
Mod091L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1069) ##STR00452##
(e.g., in WV-11114, X=O(PO) and connecting to 5-O of the oligonucleotide chain)
Mod097 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1070) ##STR00453##
Mod098 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1071) ##STR00454##
Mod099 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1072) ##STR00455##
Mod100 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1073) ##STR00456##
Mod102 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1074) ##STR00457##
Mod103 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1075) ##STR00458##
Mod104 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1076) ##STR00459##
Mod105 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1077) ##STR00460##
Mod106 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1078) ##STR00461##
(e.g., in WV-15844, X=O(PO) and connecting to 5-O of the oligonucleotide chain)
Mod107 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1079) ##STR00462##
(e.g., in WV-15845 and WV-16011, X=O(PO) and connecting to 5-O of the oligonucleotide chain)
Mod108 (with C(O) connecting to, e.g., NH of a linker such as L001):

(1080) ##STR00463##
Mod109:

(1081) ##STR00464##
Mod109L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1082) ##STR00465##
(e.g., in WV-19792, X=O)
Mod110:

(1083) ##STR00466##
Mod110L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1084) ##STR00467##
(e.g., in WV-19793, X=O)
Mod111:

(1085) ##STR00468##
Mod111L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1086) ##STR00469##
Mod 112:

(1087) ##STR00470##
Mod112L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1088) ##STR00471##
Mod113:

(1089) ##STR00472##
Mod113L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1090) ##STR00473##
Mod114:

(1091) ##STR00474##
Mod114L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1092) ##STR00475##
Mod115:

(1093) ##STR00476##
Mod115L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1094) ##STR00477##
Mod118:

(1095) ##STR00478##
Mod118L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1096) ##STR00479##
Mod119L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1097) ##STR00480##
Mod120:

(1098) ##STR00481##
Mod120L001 (with PO or PS connecting to 5-O of an oligonucleotide chain):

(1099) ##STR00482##
L009n001L009n001L009n001L009: connected to the 5-position of the 5 terminal sugar of an oligonucleotide chain (e.g., for WV-23576 and WV-23578, sugar of fU) through a phosphodiester:

(1100) ##STR00483##
L009n001L009n001L009n001: connected to the 5-position of the 5 terminal sugar of an oligonucleotide chain (e.g., for WV-23577 and WV-23579, sugar of fU) through n001:

(1101) ##STR00484##
L010n001L010n001L010n001L009: connected to the 5-position of the 5 terminal sugar of an oligonucleotide chain (e.g., for WV-23936 and WV-23938, sugar of fU) through a phosphodiester:

(1102) ##STR00485##
L010n001L010n001L010n001: connected to the 5-position of the 5 terminal sugar of an oligonucleotide chain (e.g., for WV-23937 and WV-23939, sugar of fU) through n001:

(1103) ##STR00486##

(1104) In some embodiments, some functional groups are optionally protected, e.g., for Mod024 and/or Mod 026, the hydroxyl groups are optionally protected as AcO, before and/or during conjugation to oligonucleotide chains, and the functional groups, e.g., hydroxyl groups, can be deprotected, for example, during oligonucleotide cleavage and/or deprotection:

(1105) ##STR00487##

(1106) Applicant notes that presented in Table A1 are example ways of presenting structures of provided oligonucleotides, for example, WV-3546 (Mod020L001fU*SfC*SfA*SfA*SfG*SfG*SmAfA*SmGmA*SfU*SmGmGfC*SfA*SfU*SfU*SfU*Sf C*SfU) (SEQ ID NO: 3151) can be presented as a lipid moiety (Mod020,

(1107) ##STR00488##
connected via C(O)(OOSSSSSSOSOSSOOSSSSSS, which O may be omitted as in Table A1) to the NH of NH(CH.sub.2).sub.6, wherein the (CH.sub.2).sub.6 is connected to the 5-end of the oligonucleotide chain via a phosphodiester linkage (OOSSSSSSOSOSSOOSSSSSS). One having ordinary skill in the art understands that a provided oligonucleotide can be presented as combinations of lipid, linker and oligonucleotide chain units in many different ways, wherein in each way the combination of the units provides the same oligonucleotide. For example, WV-3546, can be considered to have a structure of A.sup.c-[-L.sup.LD-(R.sup.LD).sub.a].sub.b, wherein a is 1, b is 1, and have a lipid moiety R.sup.LD of

(1108) ##STR00489##
connected to its oligonucleotide chain (A.sup.c) unit through a linker L.sup.LD having the structure of C(O)NH(CH.sub.2).sub.6OP(O)(OH)O, wherein C(O) is connected to R.sup.LD, and O is connected to A.sup.c (as 5-O of the oligonucleotide chain); one of the many alternative ways is that R.sup.LD is

(1109) ##STR00490##
and L.sup.LD is NH(CH.sub.2).sub.6OP(O)(OH)O, wherein NH is connected to R.sup.LD, and O is connected to A.sup.c (as 5-O of the oligonucleotide chain).

(1110) In some embodiments, each phosphorothioate internucleotidic linkage of an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, a provided oligonucleotide composition is a chirally controlled oligonucleotide composition of an oligonucleotide type listed in Table A1, wherein each phosphorothioate internucleotidic linkage of the oligonucleotide is independently a chirally controlled internucleotidic linkage.

(1111) In some embodiments, the present disclosure provides compositions comprising or consisting of a plurality of provided oligonucleotides (e.g., chirally controlled oligonucleotide compositions). In some embodiments, all oligonucleotides of the plurality are of the same type, i.e., all have the same base sequence, pattern of backbone linkages, pattern of backbone chiral centers, and pattern of backbone phosphorus modifications. In some embodiments, all oligonucleotides of the same type are structural identical. In some embodiments, provided compositions comprise oligonucleotides of a plurality of oligonucleotides types, typically in controlled amounts. In some embodiments, a provided chirally controlled oligonucleotide composition comprises a combination of two or more provided oligonucleotide types.

(1112) In some embodiments, an oligonucleotide composition of the present disclosure is a chirally controlled oligonucleotide composition, wherein the sequence of the oligonucleotides of its plurality comprises or consists of a base sequence listed in Table A1.

(1113) In some experiments, provided oligonucleotides can provide surprisingly high activities, e.g., when compared to those of Drisapersen and/or Eteplirsen. For example, chirally controlled oligonucleotide compositions of WV-887, WV-892, WV-896, WV-1714, WV-2444, WV-2445, WV-2526, WV-2527, WV-2528, and WV-2530, and many others, each showed a superior capability, in some embodiments many fold higher, to mediate skipping of an exon in dystrophin, compared to Drisapersen and/or Eteplirsen. Certain data are provided in the present disclosure as examples.

(1114) In some embodiments, the present disclosure pertains to a composition comprising a chirally controlled oligonucleotide selected from any DMD oligonucleotide listed herein, or any DMD oligonucleotide having a base sequence comprising at least 15 consecutive bases of any DMD oligonucleotide listed herein.

(1115) In some embodiments, a provided oligonucleotide is no more than 25 bases long. In some embodiments, a provided oligonucleotide is no more than 25 to 60 bases long. In some embodiments, a U can be replaced with T, or vice versa.

(1116) In some embodiments, when assaying example oligonucleotides in mice, oligonucleotides (e.g., WV-3473, WV-3545, WV-3546, WV-942, etc.) are intravenous injected via tail vein in male C57BL/10ScSndmdmdx mice (4-5 weeks old), at tested amounts, e.g., 10 mg/kg, 30 mg/kg, etc. In some embodiments, tissues are harvested at tested times, e.g., on Day, e.g., 2, 7 and/or 14, etc., after injection, in some embodiments, fresh-frozen in liquid nitrogen and stored in 80 C. until analysis.

(1117) Various assays can be used to assess oligonucleotide levels in accordance with the present disclosure. In some embodiments, hybrid-ELISA is used to quantify oligonucleotide levels in tissues using test article serial dilution as standard curve: for example, in an example procedure, maleic anhydride activated 96-well plate (Pierce 15110) was coated with 50 l of capture probe at 500 nM in 2.5% NaHCO.sub.3(Gibco, 25080-094) for 2 hours at 37 C. The plate was then washed 3 times with PBST (PBS+0.1% Tween-20), and blocked with 5% fat free milk-PBST at 37 C. for 1 hour. Test article oligonucleotide was serial diluted into matrix. This standard together with original samples were diluted with lysis buffer (4 M Guanidine; 0.33% N-Lauryl Sarcosine; 25 mM Sodium Citrate; 10 mM DTT) so that oligonucleotide amount in all samples is less than 100 ng/ml. 20 l of diluted samples were mixed with 180 l of 333 nM detection probe diluted in PBST, then denatured in PCR machine (65 C., 10 min, 95 C., 15 min, 4 C ). 50 l of denatured samples were distributed in blocked ELISA plate in triplicates, and incubated overnight at 4 C. After 3 washes of PBST, 1:2000 streptavidin-AP in PBST was added, 50 l per well and incubated at room temperature for 1 hour. After extensive wash with PBST, 100 l of AttoPhos (Promega S1000) was added, incubated at room temperature in dark for 10 min and read on plate reader (Molecular Device, M5) fluorescence channel: Ex435 nm, Em555 nm. Oligonucleotides in samples were calculated according to standard curve by 4-parameter regression.

(1118) In some embodiments, provided oligonucleotides are stable in both plasma and tissue homogenates.

(1119) Additional Embodiments and Examples of Oligonucleotides and Compositions, Including Dystrophin (DMD) Oligonucleotides and Compositions

(1120) Among other things, the present disclosure provides oligonucleotides, compositions, and methods for, modulating splicing, reducing target levels, treating various conditions, disorders, diseases, etc. For example, in some embodiments, the present disclosure provides dystrophin (DMD) oligonucleotides and/or DMD oligonucleotide compositions that are useful for various purposes. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 23 in the mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 44 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 46 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 47 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 51 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 52 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 53 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 54 in the human or mouse DMD gene. In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of exon 55 in the human or mouse DMD gene.

(1121) In some embodiments, a DMD oligonucleotide and/or composition is capable of mediating skipping of multiple exons in the human or mouse DMD gene.

(1122) In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises a modification. In some embodiments, a DMD oligonucleotide comprises a sugar modification. In some embodiments, a DMD oligonucleotide comprises a sugar modification at the 2 position. In some embodiments, a DMD oligonucleotide comprises a sugar modification at the 2 position selected from 2-F, 2-OMe and 2-MOE.

(1123) In some embodiments, a DMD oligonucleotide comprises a 2-F, 2-OMe and/or 2-MOE. In some embodiments, a DMD oligonucleotide comprises a 2-F. In some embodiments, in a DMD oligonucleotide, each sugar comprises a 2-F.

(1124) In some embodiments, a DMD oligonucleotide comprises a 2-OMe. In some embodiments, in a DMD oligonucleotide, each sugar comprises a 2-OMe. In some embodiments, a DMD oligonucleotide comprises a 2-MOE. In some embodiments, in a DMD oligonucleotide, each sugar comprises a 2-MOE.

(1125) In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide comprises a 2-OMe and a 2-F. In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises a pattern of 2 sugar modifications, wherein the pattern comprises a sequence selected from: fm, mf, ffm, fffm, ffffm, fffffm, ffffffm, fffffffm, ffffffffm, fffffffffm, mf, mff, mfff, mffff, mfffff, mffffff, mfffffff, mfffffff, fmf, fmmf, fmmmf, fmmmmf, fmmmmmf, fmmmmmmf, fmmmmmmmf, fmmmmmmmmf, fmmmmmmmmmf, ffffffmmmmmmmmffffff, fffffmmmmmmmmmmfffff, ffffmmmmmmmmmmmmffff, fffmmmmmmmmmmmmmmfff, ffmmmmmmmmmmmmmmmmff, fmmmmmmmmmmmmmmmmmmf, ffffffffffmmmmmmmmmm, fffffmmmmmmmmffffff, ffffmmmmmmmmmmfffff, fffmmmmmmmmmmmmffff, ffmmmmmmmmmmmmmmfff, fmmmmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmmmmf, fffffffffmmmmmmmmmm, ffffmmmmmmmmffffff, fffmmmmmmmmmmfffff, ffmmmmmmmmmmmmffff, fmmmmmmmmmmmmmmfff, mmmmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmmmf, ffffffffmmmmmmmmmm, fffmmmmmmmmffffff, ffmmmmmmmmmmfffff, fmmmmmmmmmmmmffff, mmmmmmmmmmmmmmfff, mmmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmmf, fffffffmmmmmmmmmm, ffmmmmmmmmffffff, fmmmmmmmmmmfffff, mmmmmmmmmmmmffff, mmmmmmmmmmmmmfff, mmmmmmmmmmmmmmff, mmmmmmmmmmmmmmmf, ffffffmmmmmmmmmm, fmmmmmmmmffffff, mmmmmmmmmmfffff, mmmmmmmmmmmffff, mmmmmmmmmmmmfff, mmmmmmmmmmmmmff, mmmmmmmmmmmmmmf, fffffmmmmmmmmmm, mmmmmmmmffffff, mmmmmmmmmfffff, mmmmmmmmmmffff, mmmmmmmmmmmfff, mmmmmmmmmmmmff, mmmmmmmmmmmmmf, ffffmmmmmmmmmm, ffffffmmmmmmmmfffff, fffffmmmmmmmmmmffff, ffffmmmmmmmmmmmmfff, fffmmmmmmmmmmmmmmff, ffmmmmmmmmmmmmmmmmf, fmmmmmmmmmmmmmmmmmm, ffffffffffmmmmmmmmm, ffffffmmmmmmmmffff, fffffmmmmmmmmmmfff, ffffmmmmmmmmmmmmff, fffmmmmmmmmmmmmmmf, ffmmmmmmmmmmmmmmmm, fmmmmmmmmmmmmmmmmm, ffffffffffmmmmmmmm, ffffffmmmmmmmmfff, fffffmmmmmmmmmmff, ffffmmmmmmmmmmmmf, fffmmmmmmmmmmmmmm, ffmmmmmmmmmmmmmmm, fmmmmmmmmmmmmmmmm, ffffffffffmmmmmmm, ffffffmmmmmmmmff, fffffmmmmmmmmmmf, ffffmmmmmmmmmmmm, fffmmmmmmmmmmmmm, ffmmmmmmmmmmmmmm, fmmmmmmmmmmmmmmm, ffffffffffmmmmmm, ffffffmmmmmmmmf, fffffmmmmmmmmmm, ffffmmmmmmmmmmm, fffmmmmmmmmmmmm, ffmmmmmmmmmmmmm, fmmmmmmmmmmmmmm, ffffffffffmmmmm, ffffffmmmmmmmm, fffffmmmmmmmmm, ffffmmmmmmmmmm, fffmmmmmmmmmmm, ffmmmmmmmmmmmm, fmmmmmmmmmmmmm, ffffffffffmmmm, ffffffmmmmmmm, fffffmmmmmmmm, ffffmmmmmmmmm, fffmmmmmmmmmm, ffmmmmmmmmmmm, fffffmmmmmmm, ffffmmmmmmmm, ffffffffffmm, ffffffmmmmm, fffmmmmmmmmm, ffmmmmmmmmmm, fmmmmmmmmmmm, fmmmmmmmmmmmm, ffffffffffmmm, ffffffmmmmmm, fffffmmmmmm, ffffmmmmmmm, fffmmmmmmmm, ffmmmmmmmmm, fmmmmmmmmmm, ffffffffffm, mmmmmmmmmmffffffffff, ffffffmmmmmmmmmmmmmm, ffmmmmmmmmfmmfmfffff, mmffffffffmffmfmmmmm, mmmmmmmmmmmmmmffffff, mfmfmfmfmfmfmfmfmfmf, mmmmmmffffffffmmmmmm, ffffffmmmmmmmmffffff, mfmmffmmfmmfffmmmmfm, fmffmmffmffmmmffffmf, fmff, mffm, fmffm, mfmmf, fmmf, fmffmm, mfmmff, mmff, fmmff, mmffm, fmffmmf, mfmmffm, mfmm, mfmmf, mfmmff, fmffmmf, mfmmffm, mmffm, ffmmf, fmfff, mfffm, fmfffm, fmfffmm, mfmmfff, mmfff, fmmfff, mmfffm, fmfffmmf, mfmmfffm, mfmm, mfmmf, mfmmfff, fmfffmmf, mfmmfffm, mmfffm, fffmmf, mfmmmf, fmmmf, fmffmmm, mfmmmff, mmmff, fmmmff, mmmffm, fmffmmmf, mfmmmffm, mfmmm, mfmmmf, mfmmmff, fmffmmmf, mfmmmffm, mmmffm, ffmmmf, or any portion thereof comprising at least five consecutive modifications, wherein f is 2-F and m is 2-OMe.

(1126) In some embodiments, a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises a pattern which comprises any of: O, OO, OOO, OOOO, OOOOO, OOOOOO, OOOOOOO, OOOOOOOO, OOOOOOOOO, OOOOOOOOOO, OOOOOOOOOOO, S, SS, SSS, SSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS, SSSSSSSSSS, SSSSSSSSSSS, X, XX, XXX, XXXX, XXXXX, XXXXXX, XXXXXXX, XXXXXXXX, XXXXXXXXX, XXXXXXXXXX, XXXXXXX, R, RR, RRR, RRRR, RRRRR, RRRRRR, RRRRRRR, RRRRRRRR, RRRRRRRRR, RRRRRRRRRR, RRRRRRRRRRR, OSOOO, OSOO, OSO, SOOO, OXOOO, OXOO, OXO, XOO, ROOOR, ROROR, ROROR, ROORR, RROOR, ROOR, OOR, RRROR, RRRO, RROR, ROR, SOOOR, ROOOS, ROOO, ROO, RO, OOOS, SOOOS, SOOO, SOOSS, SOSOS, SOSO, OSOS, SOS, SSOOS, SSOO, SSO, SOO, SSSOS, SSSO, SOS, XOOOX, XOOO, XOO, XO, OOOX, OOX, OX, SOOOS, SOOO, SOO, SO, OOOS, OOS, XXXXXXXXXXXXX, XXXXXXXXXXX, XXXXXXXXXXX, XXXXXXXXXX, XXXXXX, XXXXXXXXX, XXXXXXX, XXXXXX, XXXXX, XXXX, SSSSRSSRSS, SSSSRSSRS, SSSSRSSR, SSSSRSS, SSSSRS, SSSS, SSS, SSSRSSRSS, SSRSSRSS, SRSSRSS, RSSRSS, SSRSS, SSRS, SSSRSSRSSS, SSRSSRSSS, SSSRSSRSS, SSRSSRSSSS, SRSSRSSSS, SSRSSRSSS, SSRSSSSSSS, SRSSSSSSS, SSRSSSSSS, SSSSSSRSSS, SSSSSRSSS, SSSSSSRSS, SSO, SOS, OSO, OSSO, SOS, SSOSS, SSOSSO, ssOSSOS, SSOSSOSS, XO, XXO, XOX, XXOX, XXOXX, XXXOXX, XXXOX, XXOXX, XXXOXXX, XXOXXO, XXOXX, XXOXXOX, or XXOXXOXX, or any portion thereof comprising at least 5 consecutive internucleotidic linkages, wherein X is a stereorandom phosphorothioate linkage, S is a phosphorothioate linkage of the Sp configuration, and R is a phosphorothioate linkage of the Rp configuration.

(1127) Various oligonucleotides, including DMD oligonucleotides, having these modifications and patterns thereof, or portions thereof, are described in the present disclosure, including those listed in Table A1.

(1128) In some embodiments, a DMD oligonucleotide comprises a non-negatively charged internucleotidic linkage. Non-limiting examples of such an oligonucleotide include, inter alia: WV-11237, WV-11238, WV-11239, WV-11340, WV-11341, WV-11342, WV-11343, WV-11344, WV-11345, WV-11346, WV-11347, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, WV-12136, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, WV-12876, WV-12877, WV-12878, WV-12879, WV-12880, WV-12881, WV-12882, WV-12883, WV-12884, WV-12885, WV-12887, WV-12888, WV-13408, WV-13409, WV-13594, WV-13595, WV-13596, WV-13597, WV-13812, WV-13813, WV-13814, WV-13815, WV-13816, WV-13817, WV-13820, WV-13821, WV-13822, WV-13823, WV-13824, WV-13825, WV-13857, WV-13858, WV-13859, WV-13860, WV-13861, WV-13862, WV-13863, WV-13864, WV-13865, WV-14342, WV-14343, WV-14344, WV-14345, WV-14522, WV-14523, WV-14525, WV-14526, WV-14528, WV-14529, WV-14530, WV-14532, WV-14533, WV-14565, WV-14566, WV-14773, WV-14774, WV-14776, WV-14777, WV-14778, WV-14779, WV-14790, WV-14791, WV-15052, WV-15053, WV-15143, WV-15322, WV-15323, WV-15324, WV-15325, WV-15326, WV-15327, WV-15328, WV-15329, WV-15330, WV-15331, WV-15332, WV-15333, WV-15334, WV-15335, WV-15336, WV-15337, WV-15338, WV-15366, WV-15369, WV-15589, WV-15647, WV-15844, WV-15845, WV-15846, WV-15850, WV-15851, WV-15852, WV-15853, WV-15854, WV-15855, WV-15856, WV-15857, WV-15858, WV-15859, WV-15860, WV-15861, WV-15862, WV-15912, WV-15913, WV-15928, WV-15929, WV-15930, WV-15931, WV-15932, WV-15933, WV-15934, WV-15935, WV-15937, WV-15939, WV-15940, WV-15941, WV-15942, WV-15943, WV-15944, WV-15945, WV-15946, WV-15947, WV-15948, WV-15949, WV-15962, WV-15963, WV-15964, WV-15965, WV-15966, WV-15967, WV-15968, WV-15969, WV-15970, WV-15971, WV-15972, WV-15973, WV-16004, WV-16005, WV-16010, WV-16011, WV-16366, WV-16368, WV-16369, WV-16371, WV-16372, WV-16499, WV-16505, WV-16506, WV-16507, WV-17765, WV-17774, WV-17775, WV-17801, WV-17802, WV-17803, WV-17831, WV-17832, WV-17833, WV-17834, WV-17838, WV-17839, WV-17840, WV-17841, WV-17842, WV-17843, WV-17854, WV-17855, WV-17856, WV-17857, WV-17858, WV-17859, WV-17860, WV-17861, WV-17862, WV-17863, WV-17864, WV-17865, WV-17866, WV-17881, WV-17882, WV-17883, WV-18853, WV-18854, WV-18855, WV-18856, WV-18857, WV-18858, WV-18859, WV-18860, WV-18861, WV-18862, WV-18863, WV-18864, WV-18865, WV-18866, WV-18867, WV-18868, WV-18869, WV-18870, WV-18871, WV-18872, WV-18873, WV-18874, WV-18875, WV-18876, WV-18877, WV-18878, WV-18879, WV-18880, WV-18881, WV-18882, WV-18883, WV-18884, WV-18885, WV-18886, WV-18887, WV-18888, WV-18889, WV-18890, WV-18891, WV-18892, WV-18893, WV-18894, WV-18895, WV-18896, WV-18897, WV-18898, WV-18899, WV-18900, WV-18901, WV-18902, WV-18903, WV-18904, WV-18905, WV-18906, WV-18907, WV-18908, WV-18909, WV-18910, WV-18911, WV-18912, WV-18913, WV-18914, WV-18915, WV-18916, WV-18917, WV-18918, WV-18919, WV-18920, WV-18921, WV-18922, WV-18923, WV-18924, WV-18925, WV-18926, WV-18927, WV-18928, WV-18929, WV-18930, WV-18931, WV-18932, WV-18933, WV-18934, WV-18935, WV-18936, WV-18937, WV-18938, WV-18939, WV-18940, WV-18941, WV-18942, WV-18944, WV-18945, WV-19790, WV-19791, WV-19792, WV-19793, WV-19794, WV-19795, WV-19796, WV-19797, WV-19798, WV-19803, WV-19804, WV-19805, WV-19806, WV-19886, WV-19887, WV-19888, WV-19889, WV-19890, WV-19891, WV-19892, WV-19893, WV-19894, WV-19895, WV-19896, WV-19897, WV-19898, WV-19899, WV-19900, WV-19901, WV-19902, WV-19903, WV-19904, WV-19905, WV-19906, WV-19907, WV-19908, WV-19909, WV-19910, WV-19911, WV-19912, WV-19913, WV-19914, WV-19915, WV-19916, WV-19917, WV-19918, WV-19919, WV-19920, WV-19921, WV-19922, WV-19923, WV-19924, WV-19925, WV-19926, WV-19927, WV-19928, WV-19929, WV-19930, WV-19931, WV-19932, WV-19933, WV-19934, WV-19935, WV-19936, WV-19937, WV-19938, WV-19939, WV-19940, WV-19941, WV-19942, WV-19943, WV-19944, WV-19945, WV-19946, WV-19947, WV-19948, WV-19949, WV-19950, WV-19951, WV-19952, WV-19953, WV-19954, WV-19955, WV-19956, WV-19957, WV-19958, WV-19959, WV-19960, WV-19961, WV-19962, WV-19963, WV-19964, WV-19965, WV-19966, WV-19967, WV-19968, WV-19969, WV-19970, WV-19971, WV-19972, WV-19973, WV-19974, WV-19975, WV-19976, WV-19977, WV-19978, WV-19979, WV-19980, WV-19981, WV-19982, WV-19983, WV-19984, WV-19985, WV-19986, WV-19987, WV-19988, WV-19989, WV-19990, WV-19991, WV-19992, WV-19993, WV-19994, WV-19995, WV-19996, WV-19997, WV-19998, WV-19999, WV-20000, WV-20001, WV-20002, WV-20003, WV-20004, WV-20005, WV-20006, WV-20007, WV-20008, WV-20009, WV-20010, WV-20011, WV-20012, WV-20013, WV-20014, WV-20015, WV-20016, WV-20017, WV-20018, WV-20019, WV-20020, WV-20021, WV-20022, WV-20023, WV-20024, WV-20025, WV-20026, WV-20027, WV-20028, WV-20029, WV-20030, WV-20031, WV-20032, WV-20033, WV-20034, WV-20035, WV-20036, WV-20037, WV-20038, WV-20039, WV-20040, WV-20041, WV-20042, WV-20043, WV-20044, WV-20045, WV-20046, WV-20047, WV-20048, WV-20049, WV-20050, WV-20051, WV-20052, WV-20053, WV-20054, WV-20055, WV-20056, WV-20057, WV-20058, WV-20059, WV-20060, WV-20061, WV-20062, WV-20063, WV-20064, WV-20065, WV-20066, WV-20067, WV-20068, WV-20069, WV-20070, WV-20071, WV-20072, WV-20073, WV-20074, WV-20075, WV-20076, WV-20077, WV-20078, WV-20079, WV-20080, WV-20081, WV-20082, WV-20083, WV-20084, WV-20085, WV-20086, WV-20087, WV-20088, WV-20089, WV-20090, WV-20091, WV-20092, WV-20093, WV-20094, WV-20095, WV-20096, WV-20097, WV-20098, WV-20099, WV-20100, WV-20101, WV-20102, WV-20103, WV-20104, WV-20105, WV-20106, WV-20107, WV-20108, WV-20109, WV-20110, WV-20111, WV-20112, WV-20113, WV-20114, WV-20115, WV-20116, WV-20117, WV-20118, WV-20119, WV-20120, WV-20121, WV-20122, WV-20123, WV-20124, WV-20125, WV-20126, WV-20127, WV-20128, WV-20129, WV-20130, WV-20131, WV-20132, WV-20133, WV-20134, WV-20135, WV-20136, WV-20137, WV-20138, WV-20139, WV-20140, WV-20141, WV-20142, WV-20143, WV-20144, WV-20145, WV-20146, WV-20147, WV-20148, WV-20149, WV-20150, WV-20151, WV-20152, WV-20153, WV-20154, WV-20155, WV-20156, WV-20157, WV-20158, WV-20159, WV-20160, WV-21210, WV-21211, WV-21212, WV-21217, WV-21218, WV-21219, WV-21226, WV-21245, WV-21252, WV-21253, WV-21257, WV-21258, WV-21374, WV-21375, WV-21376, WV-21377, WV-21378, WV-21379, WV-21380, WV-21381, WV-21382, WV-21383, WV-21384, WV-21385, WV-21386, WV-21387, WV-21388, WV-21389, WV-21390, WV-21578, WV-21579, WV-21580, WV-21581, WV-21582, WV-21583, WV-21584, WV-21585, WV-21586, WV-21587, WV-21588, WV-21589, WV-21590, WV-21591, WV-21592, WV-21593, WV-21594, WV-21595, WV-21596, WV-21597, WV-21598, WV-21599, WV-21600, WV-21601, WV-21602, WV-21603, WV-21604, WV-21605, WV-21606, WV-21607, WV-21608, WV-21609, WV-21610, WV-21611, WV-21612, WV-21613, WV-21614, WV-21615, WV-21616, WV-21617, WV-21618, WV-21619, WV-21620, WV-21621, WV-21622, WV-21623, WV-21624, WV-21625, WV-21626, WV-21627, WV-21628, WV-21629, WV-21630, WV-21631, WV-21632, WV-21633, WV-21634, WV-21635, WV-21636, WV-21637, WV-21638, WV-21639, WV-21640, WV-21641, WV-21642, WV-21643, WV-21644, WV-21645, WV-21646, WV-21647, WV-21648, WV-21649, WV-21650, WV-21651, WV-21652, WV-21653, WV-21654, WV-21655, WV-21656, WV-21657, WV-21658, WV-21659, WV-21660, WV-21661, WV-21662, WV-21663, WV-21664, WV-21665, WV-21666, WV-21667, WV-21668, WV-21669, WV-21670, WV-21671, WV-21672, WV-21673, WV-21723, WV-21724, WV-21725, WV-21726, WV-21727, WV-21728, WV-21729, WV-21730, WV-21731, WV-21732, WV-21733, WV-21734, WV-21735, WV-21736, WV-21737, WV-21738, WV-21739, WV-21740, WV-21741, WV-21742, WV-21743, WV-21744, WV-21745, WV-21746, WV-21747, WV-21748, WV-21749, WV-21750, WV-21751, WV-21752, WV-21753, WV-21754, WV-21755, WV-21756, WV-21757, WV-21758, WV-21759, WV-21760, WV-21761, WV-21762, WV-21763, WV-21764, WV-21765, WV-21766, WV-21767, WV-21768, WV-21769, WV-21770, WV-21771, WV-21772, WV-21773, WV-21774, WV-21775, WV-21776, WV-21777, WV-21778, WV-21779, WV-21780, WV-21781, WV-21782, WV-21783, WV-21784, WV-21785, WV-21786, WV-21787, WV-21788, WV-21789, WV-21790, WV-21791, WV-21792, WV-21793, WV-21794, WV-21795, WV-21796, WV-21797, WV-21798, WV-21799, WV-21800, WV-21801, WV-21802, WV-21803, WV-21804, WV-21805, WV-21806, WV-21807, WV-21808, WV-21809, WV-21810, WV-21811, WV-21812, WV-21813, WV-21814, WV-21815, WV-21816, WV-21817, WV-21818, WV-22753, WV-23576, WV-23577, WV-23578, WV-23579, WV-23936, WV-23937, WV-23938, and WV-23939.

(1129) Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 23

(1130) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 23 in mouse DMD. Non-limiting examples include oligonucleotides and compositions of WV-10256, WV-10257, WV-10258, WV-10259, WV-10260, WV-1093, WV-1094, WV-1095, WV-1096, WV-1097, WV-1098, WV-1099, WV-1100, WV-1101, WV-1102, WV-1103, WV-1104, WV-1105, WV-1106, WV-1121, WV-1122, WV-1123, WV-11231, WV-11232, WV-11233, WV-11234, WV-11235, WV-11236, WV-1124, WV-1125, WV-1126, WV-1127, WV-1128, WV-1129, WV-1130, WV-11343, WV-11344, WV-11345, WV-11346, WV-11347, WV-1141, WV-1142, WV-1143, WV-1144, WV-1145, WV-1146, WV-1147, WV-1148, WV-1149, WV-1150, WV-1678, WV-1679, WV-1680, WV-1681, WV-1682, WV-1683, WV-1684, WV-1685, WV-2733, WV-2734, WV-4610, WV-4611, WV-4614, WV-4615, WV-4616, WV-4617, WV-4618, WV-4619, WV-4620, WV-4621, WV-4622, WV-4623, WV-4624, WV-4625, WV-4626, WV-4627, WV-4628, WV-4629, WV-4630, WV-4631, WV-4632, WV-4633, WV-4634, WV-4635, WV-4636, WV-4637, WV-4638, WV-4639, WV-4640, WV-4641, WV-4642, WV-4643, WV-4644, WV-4645, WV-4646, WV-4647, WV-4648, WV-4649, WV-4650, WV-4651, WV-4652, WV-4653, WV-4654, WV-4655, WV-4656, WV-4657, WV-4658, WV-4659, WV-4660, WV-4661, WV-4662, WV-4663, WV-4664, WV-4665, WV-4666, WV-4667, WV-4668, WV-4669, WV-4670, WV-4671, WV-4672, WV-4673, WV-4674, WV-4675, WV-4676, WV-4677, WV-4678, WV-4679, WV-4680, WV-4681, WV-4682, WV-4683, WV-4684, WV-4685, WV-4686, WV-4687, WV-4688, WV-4689, WV-4690, WV-4691, WV-4692, WV-4693, WV-4694, WV-4695, WV-4696, WV-4697, WV-6010, WV-7677, WV-7678, WV-7679, WV-7680, WV-7681, WV-7682, WV-7683, WV-7684, WV-7685, WV-7686, WV-7687, WV-7688, WV-7689, WV-7690, WV-7691, WV-7692, WV-7693, WV-7694, WV-7695, WV-7696, WV-7697, WV-7698, WV-7699, WV-7700, WV-7701, WV-7702, WV-7703, WV-7704, WV-7705, WV-7706, WV-7707, WV-7708, WV-7709, WV-7710, WV-7711, WV-7712, WV-7713, WV-7714, WV-7715, WV-7716, WV-7717, WV-7718, WV-7719, WV-7720, WV-7721, WV-7722, WV-7723, WV-7724, WV-7725, WV-7726, WV-7727, WV-7728, WV-7729, WV-7730, WV-7731, WV-7732, WV-7733, WV-7734, WV-7735, WV-7736, WV-7737, WV-7738, WV-7739, WV-7740, WV-7741, WV-7742, WV-7743, WV-7744, WV-7745, WV-7746, WV-7747, WV-7748, WV-7749, WV-7750, WV-7751, WV-7752, WV-7753, WV-7754, WV-7755, WV-7756, WV-7757, WV-7758, WV-7759, WV-7760, WV-7761, WV-7762, WV-7763, WV-7764, WV-7765, WV-7766, WV-7767, WV-7768, WV-7769, WV-7770, WV-7771, WV-9163, WV-9164, WV-9165, WV-9166, WV-9167, WV-9168, WV-9169, WV-9170, WV-9171, WV-9172, WV-9173, WV-9174, WV-9175, WV-9176, WV-9177, WV-9178, WV-9179, WV-9180, WV-9181, WV-9182, WV-9183, WV-9184, WV-9185, WV-9186, WV-9187, WV-9188, WV-9189, WV-9190, WV-9191, WV-9192, WV-9193, WV-9194, WV-9195, WV-9196, WV-9197, WV-9198, WV-9199, WV-9200, WV-9201, WV-9202, WV-9203, WV-9204, WV-9205, WV-9206, WV-9207, WV-9208, WV-9209, WV-9210, WV-9408, WV-9409, WV-9410, WV-9411, WV-9412, WV-9413, WV-9414, WV-9415, WV-9416, WV-9417, WV-9418, WV-9419, WV-9420, WV-943, WV-9875, WV-9876, WV-9877, WV-9878, and WV-9879, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1131) In some embodiments, a DMD oligonucleotide is capable of mediating skipping of exon 23. Non-limiting examples of such DMD oligonucleotides include: WV-12566, WV-12567, WV-12568, WV-12884, WV-12885, WV-12886, WV-12887, WV-12888, WV-12571, and WV-12572, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1132) Exon skipping of DMD exon 23 and other exons may be assayed in patient-derived cell lines and in cells from the mdx mouse model (which carries a nonsense point mutation in the in-frame exon 23 (Sicinski et al. 1989 Science 244: 1578-1580). By skipping exon 23 the nonsense mutation is bypassed while the reading frame is maintained). Additional strains of mdx mice, including the mdx2cv mdx4cv and mdx5cv alleles were reported by Wha Bin Im et al. 1996 Hum. Mol. Gen. 5: 1149-1153.

(1133) Data showing the capability of various DMD oligonucleotides to mediate skipping of exon 23 is shown herein, inter alia, in Table 1A.1, Table 1A.2, Table 1A.3, and Table 25C.1 to Table 25C.5.

(1134) Example Dystrophin Oligonucleotides and Compositions Targeting Exon 44 and Adjoining Intronic Region 3 to Exon 44

(1135) In some embodiments, a DMD oligonucleotide targets DMD exon 44 or the adjoining intronic region 3 to DMD exon 44.

(1136) In some embodiments, a DMD oligonucleotide targets DMD exon 44 or the adjoining intronic region 3 to DMD exon 44, and the oligonucleotide is capable of mediating multiple exon skipping (e.g., of exons 45 to 55, or 45 to 57).

(1137) Reportedly, a phenomenon known as back-splicing can occur, in which, for example, a portion of the 3 end of exon 55 interacts with a portion of the 5 end of exon 45, forming a circular RNA (circRNA), which can thus skip multiple exons, e.g., all exons from exon 45 to 55, inclusive. The phenomenon can also reportedly occur between exon 57 and exon 45, skipping multiple exons, e.g., all exons from exon 45 to 57, inclusive. Back-splicing is described in the literature, e.g., in Suzuki et al. 2016 Int. J. Mol. Sci. 17.

(1138) Without wishing to be bound by any particular theory, the present disclosure suggests that it may be possible for a DMD oligonucleotide targeting DMD exon 44 or the adjoining intronic region 3 to exon 44 may be able to mediate splicing of exons 45 to 55, or of exons 45 to 57, which exons are excised as a single piece of circular RNA (circRNA) designated 45-55 (or 55-45) or 45-57 (or 57-45), respectively.

(1139) Several oligonucleotides were designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44. In some embodiments, oligonucleotides designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44 are tested to determine if they can increase the amount of backslicing and/or multiple-exon skipping.

(1140) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating exon skipping in human DMD, wherein the base sequence of the oligonucleotide is a sequence of exon 44 or intron 44, or a portion of both exon 44 and intron 44. Non-limiting examples include oligonucleotides and compositions of WV-13963, WV-13964, WV-13965, WV-13966, WV-13967, WV-13968, WV-13969, WV-13970, WV-13971, WV-13972, WV-13973, WV-13974, WV-13975, WV-13976, WV-13977, WV-13978, WV-13979, WV-13980, WV-13981, WV-13982, WV-13983, WV-13984, WV-13985, WV-13986, WV-13987, WV-13988, WV-13989, WV-13990, WV-13991, WV-13992, WV-13993, WV-13994, WV-13995, WV-13996, WV-13997, WV-13998, WV-13999, WV-14000, WV-14001, WV-14002, WV-14003, WV-14004, WV-14005, WV-14006, WV-14007, WV-14008, WV-14009, WV-14010, WV-14011, WV-14012, WV-14013, WV-14014, WV-14015, WV-14016, WV-14017, WV-14018, WV-14019, WV-14020, WV-14021, WV-14022, WV-14023, WV-14024, WV-14025, WV-14026, WV-14027, WV-14028, WV-14029, WV-14030, WV-14031, WV-14032, WV-14033, WV-14034, WV-14035, WV-14036, WV-14037, WV-14038, WV-14039, WV-14040, WV-14041, WV-14042, WV-14043, WV-14044, WV-14045, WV-14046, WV-14047, WV-14048, WV-14049, WV-14050, WV-14051, WV-14052, WV-14053, WV-14054, WV-14055, WV-14056, WV-14057, and WV-14058, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1141) Data showing the capability of various DMD oligonucleotides targeting exon 44 or the adjacent intron 3 to exon 44 are shown in Table 22A.2 and Table 22A.3.

(1142) TABLE-US-00002 TABLE 1A.1 Example data of certain oligonucleotides Oligo- nucleotide 10 3.33 1.11 0.37 0.12 WV-7684 4.2 2.1 1 0.2 0.1 4.1 2.1 0.9 0.2 0.1 5.2 3.2 1.5 0 0 5.1 3.3 1.1 0 0 WV-12886 27.7 17.5 10 5 2.4 28 17.6 9.8 5 2.3 29.8 22.8 13.1 3.7 32.7 21.5 11.9 3.5 WV-11231 3.8 2.1 1.4 0.4 0.3 3.8 2.1 1.3 0.5 0.3 5.3 2.7 1.4 0.7 0.2 5.1 2.4 1.6 0.8 0.2 WV-10258 24.5 19.9 9.5 4.8 2.8 25.3 20.1 9.1 4.8 2.7 24.4 19.4 13.2 6.2 3.4 24.2 19.7 13.6 6.3 3.5 WV-11345 29.2 24.9 15.9 12.1 5 30.2 24.9 15.5 11.9 5.1 30.8 25.8 17.8 32.3 25.3 17.6 WV-12885 26.8 23.3 16.5 8 2.8 27.5 23 17.2 8.2 3.8 32.3 25.8 16.3 6.1 30.7 27.1 16.3 6.3 WV-15589 22.2 14.8 11.2 4.6 2.2 21.7 15 12.3 4.4 2.3 24.1 11.3 11.4 23.5 8.6 10.8

(1143) Oligonucleotides to DMD exon 23 were tested in vitro for their ability to induce skipping of exon 23.

(1144) H2K cells were dosed with oligonucleotide in differentiation media for 4 days. RNA was extracted with Trizol, pre-amp then treated with TaqMan with multiplexed reading of skipped and total DMD transcript; absolute quantification was via standard curve g-Blocks. In these and various other studies, numbers indicate amount of skipping (i.e., skipping efficiency; or the percentage of skipping as a percentage of total mRNA transcript).

(1145) Oligonucleotides were tested at 10, 3.33, 1.11, 0.37 or 0.12 uM.

(1146) TABLE-US-00003 TABLE 1A.2 Activity of certain oligonucleotides PBS WV-11345 WV-17774 WV-18945 Quadriceps 0.01 0.01 28.61 30.25 3.93 3.92 2.1 1.53 0.01 0.12 26.34 24.53 10.82 10.73 1.16 0.91 0.15 0.06 40.29 36.57 14.79 13.47 2.04 0.92 30 30.05 10.13 6.19 5.05 3.97 23.24 25.18 13.92 14.36 2.4 1.77 Gastrocnemius 0.02 0.02 22.27 13.18 36.41 33.55 2.46 1.95 0.02 0.01 14.74 8.03 18.02 19.55 0.6 0.27 0.09 0.11 11.12 3.68 16.17 15.44 0.36 0.41 22.82 28.29 11.22 10.94 0.72 0.75 18.09 15.66 28.85 27.9 0.61 3.14 Diaphram 0.04 0.03 27.05 24 7.11 4.07 0.72 0.82 0.01 1.13 16.22 16.2 18.1 18.6 0.81 0.68 0.04 0.09 15.16 13.23 9.66 10.02 0.33 0.32 33.66 36.52 4.55 4.86 0.63 0.21 20.03 20.55 8.38 9.46 0.56 0.91 Tibialis 0.01 0.01 34.34 35.04 16.2 15.77 0 0 0 0 28.7 23.07 42.94 42.97 0.04 0.02 7.87 9.87 12.1 14.51 17.01 14.68 15.16 13.91 45.6 41.54

(1147) In this study, in vivo skipping activity was measured in MDX mouse model after single IV dose.

(1148) MDX mice received single IV dose of 150 mg/kg. Necropsied flash frozen tissues (Quadriceps, Diaphragm, etc.) were pulverized and RNA extracted with Trizol. Skipping efficiency was determined by multiplex TaqMan assay for total and exon-23 skipped DMD transcripts, normalized to gBlock standard curves.

(1149) Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).

(1150) TABLE-US-00004 TABLE 1A.3 Activity of certain oligonucleotides 10 uM 3.3 uM 1.1 uM 0.3 uM 0.1 uM WV- 32.1 17.7 11.1 3.9 1.9 10258 33.2 19.4 13 4.6 2.1 29 18.5 11.5 11.1 6.4 29 18.6 12.4 11.3 6 WV- 6.8 7.6 0.7 1.6 0.1 11233 6.9 7.8 0.5 1.3 0 11.1 1.3 1.6 0.6 0.7 11 1.3 1.6 0.4 0.7 WV- 11345 42 29.3 16.6 8.1 5 40 27.4 17.4 8.2 4.7 WV- 18944 7.7 4 1.4 1 0.7 8 4 1.7 1 0.8 WV- 44.5 38.2 26.7 11.9 6.6 17774 45.2 37.5 26.3 12.5 6.6 44 37.2 26.7 14.7 4.8 44.7 35.6 27.2 13.2 4.5 WV- 14.1 11.6 5 1.9 1.5 18945 14.3 11.2 4.8 2 1.5 21.4 11.4 4.7 2.4 2.6 21.3 11.1 4.7 2.3 3 Mock 0.2 0.6 0 0.3 0.8 0 2.5 0 0.3 2.5 1.2 2 0 0.4 2.5 1.1

(1151) Oligonucleotides were tested in vitro for ability to skip DMD exon 23.

(1152) Oligonucleotides were tested at 10, 3.3., 1.1, 0.3, and 0.1 uM.

(1153) Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).

(1154) Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 45

(1155) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 45 in DMD (e.g., of mouse, human, etc.).

(1156) In some embodiments, a provided DMD oligonucleotide and/or composition is capable of mediating skipping of exon 45. Non-limiting examples of such DMD oligonucleotides and compositions include those of: WV-11047, WV-11048, WV-11049, WV-11050, WV-11051, WV-11052, WV-11053, WV-11054, WV-11055, WV-11056, WV-11057, WV-11058, WV-11059, WV-11060, WV-11061, WV-11062, WV-11063, WV-11064, WV-11065, WV-11066, WV-11067, WV-11068, WV-11069, WV-11070, WV-11071, WV-11072, WV-11073, WV-11074, WV-11075, WV-11076, WV-11077, WV-11078, WV-11079, WV-11080, WV-11081, WV-11082, WV-11083, WV-11084, WV-11085, WV-11086, WV-11087, WV-11088, WV-11089, WV-11090, WV-11091, WV-11092, WV-11093, WV-11094, WV-11095, WV-11096, WV-11097, WV-11098, WV-11099, WV-11100, WV-11101, WV-11102, WV-11103, WV-11104, WV-11105, WV-9594, WV-9595, WV-9596, WV-9597, WV-9598, WV-9599, WV-9600, WV-9601, WV-9602, WV-9603, WV-9604, WV-9605, WV-9606, WV-9607, WV-9608, WV-9609, WV-9610, WV-9611, WV-9612, WV-9613, WV-9614, WV-9615, WV-9616, WV-9617, WV-9618, WV-9619, WV-9620, WV-9621, WV-9622, WV-9623, WV-9624, WV-9625, WV-9626, WV-9627, WV-9628, WV-9629, WV-9630, WV-9631, WV-9632, WV-9633, WV-9634, WV-9635, WV-9636, WV-9637, WV-9638, WV-9639, WV-9640, WV-9641, WV-9642, WV-9643, WV-9644, WV-9645, WV-9646, WV-9647, WV-9648, WV-9649, WV-9650, WV-9651, WV-9652, WV-9653, WV-9654, WV-9655, WV-9656, WV-9657, WV-9658, WV-9659, WV-9762, WV-9763, WV-9764, WV-9765, WV-9766, WV-9767, WV-9768, WV-9769, WV-9770, WV-9771, WV-9772, WV-9773, WV-9774, WV-9775, WV-9776, WV-9777, WV-9778, WV-9779, WV-9780, WV-9781, WV-9782, WV-9783, WV-9784, WV-9785, WV-9786, WV-9787, WV-9788, WV-9789, WV-9790, WV-9791, WV-9792, WV-9793, WV-9794, WV-9795, WV-9796, WV-9797, WV-9798, WV-9799, WV-9800, WV-9801, WV-9802, WV-9803, WV-9804, WV-9805, WV-9806, WV-9807, WV-9808, WV-9809, WV-9810, WV-9811, WV-9812, WV-9813, WV-9814, WV-9815, WV-9816, WV-9817, WV-9818, WV-9819, WV-9820, WV-9821, WV-9822, WV-9823, WV-9824, WV-9825, and WV-9826, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1157) As shown in various tables from Table 1 to Table 22 (and parts thereof), various DMD oligonucleotides comprising various patterns of modifications were testing for skipping of various exons. The Tables show test results of certain DMD oligonucleotides. To assay exon skipping of DMD, certain DMD oligonucleotides were tested in vitro in 52 human patient-derived myoblast cells (also designated DEL52) and/or 45-52 human patient-derived myoblast cells (human cells wherein the exon 52 or exons 45-52 were already deleted, also designated DEL45-52). Unless noted otherwise, in various experiments, oligonucleotides were delivered gymnotically. In the tables, generally, 100.00 would represent 100% skipping and 0.0 would represent 0% skipping. Various DMD oligonucleotides are described in detail in Table A1.

(1158) Table 1A.4, below, shows example data of some DMD oligonucleotides in skipping exon 45. Procedure: 48-50 (Del48-50 or D48-50) myoblasts were treated with 10 uM oligonucleotides for 4 days in differentiation media.

(1159) TABLE-US-00005 TABLE 1A.4 Example data of certain oligonucleotides. WV-11047 0.024 0.009 0.012 0.016 WV-11051 0.022 0.024 0.046 0.014 WV-11052 0.024 0.032 0.014 0.026 WV-11053 0.027 0.009 0.017 0.023 WV-11054 0.029 0.038 0.035 0.028 WV-11055 0.030 0.025 0.016 0.033 WV-11056 0.029 0.043 0.018 0.031 WV-11057 0.000 0.015 0.000 0.032 WV-11058 0.044 0.029 0.049 0.024 WV-11059 0.025 0.041 0.049 0.024 WV-11062 0.218 0.175 0.151 0.231 WV-11063 0.472 0.730 0.456 0.594 WV-11064 0.297 0.307 0.334 0.345 WV-11065 0.651 0.630 0.675 0.544 WV-11066 0.124 0.087 0.137 0.153 WV-11067 0.183 0.210 0.238 0.224 WV-11068 0.212 0.266 0.244 0.406 WV-11069 0.389 0.715 0.407 0.744 WV-11070 1.677 1.473 1.483 1.677 WV-11071 0.385 0.362 0.413 0.310 WV-11072 0.146 0.250 0.142 0.268 WV-11073 0.709 0.876 0.721 0.835 WV-11074 2.015 2.207 1.992 2.527 WV-11075 0.254 0.238 0.157 0.220 WV-11076 0.000 2.715 0.000 2.315 WV-11077 1.568 1.414 1.388 1.308 WV-11078 3.915 3.122 4.175 3.076 WV-11079 7.178 8.083 8.257 6.955 WV-11080 1.467 1.202 1.726 1.155 WV-11081 9.279 4.780 10.244 4.512 WV-11082 3.377 2.646 3.242 2.256 WV-11083 3.964 2.631 4.001 2.419 WV-11084 11.336 7.481 13.752 8.270 WV-11085 1.818 0.679 1.787 2.003 WV-11086 16.017 15.215 17.207 15.191 WV-11087 1.104 0.766 1.728 1.030 WV-11088 14.320 12.940 14.287 10.746 WV-11089 16.126 13.507 15.515 15.389 WV-11090 1.148 0.596 1.405 0.647 WV-11091 0.105 0.069 0.311 0.049 WV-11092 0.094 0.066 0.111 0.066 WV-11093 0.123 0.060 0.087 0.037 WV-11094 0.054 0.062 0.060 0.038 WV-11095 0.317 0.064 0.241 0.109 WV-11096 0.062 0.061 0.096 0.059 WV-11098 0.026 0.033 0.032 0.024 WV-11100 0.015 0.012 0.014 0.011 WV-11101 0.000 0.021 0.000 0.011 WV-11102 0.019 0.030 0.025 0.017 WV-11103 0.017 0.023 0.014 0.029 WV-11104 0.053 0.050 0.067 0.035 WV-11105 0.017 0.033 0.034 0.051 Mock 0.050 0.018 0.010 0.037 Mock 0.019 0.023 0.009 0.023
Numbers represent level of skipping, wherein 100 would represent 100% skipping and 0 would represent 0% skipping. For various data described herein, Mock is a negative control, in which water was used instead of an oligonucleotide.
Table 1B.1, and 1B.2. Example data of certain oligonucleotides.
The Tables below show example data of some DMD oligonucleotides in skipping exon 45. Procedure: 48-50 (Del48-50 or DEL48-50 or D48-50) myoblasts were treated with 10 or 3 uM oligonucleotides for 4 days in differentiation media.
Oligonucleotides were dosed at 10 M and 3 M for 4 days in DEL48-50 Myoblasts. Certain oligonucleotides comprise a non-negatively charged internucleotidic linkage, as detailed in Table A1.

(1160) TABLE-US-00006 TABLE 1B.1 Example data of certain oligonucleotides. 10 um 3 um WV-13810 7.0 6.5 7.1 6.5 2.7 2.8 2.5 2.3 WV-13811 8.4 8.0 9.1 9.5 3.3 3.2 2.4 2.8 WV-13812 22.8 21.1 22.9 23.7 9.2 9.2 10.0 9.7 WV-13813 19.4 19.9 20.1 20.2 7.6 8.1 7.5 7.4 WV-13814 13.6 13.6 13.5 13.3 5.1 4.3 4.9 4.9 WV-13815 26.9 25.6 23.9 24.3 9.0 8.9 8.2 8.6 WV-13816 37.0 35.0 31.8 33.8 14.0 14.5 14.6 12.0 WV-13817 52.7 55.4 54.3 54.2 24.9 26.1 21.9 21.7 WV-14531 2.9 2.7 2.8 2.9 0.7 0.9 1.0 1.2 WV-14532 4.3 4.3 3.8 4.1 1.4 1.3 1.1 1.0 WV-14533 7.9 7.6 7.3 7.9 1.9 2.1 2.4 2.1 WV-11086 18.3 20.1 18.4 18.4 7.9 7.7 7.6 8.1

(1161) TABLE-US-00007 TABLE 1B.2 Example data of certain oligonucleotides. 10 uM 3 uM WV-13818 3.2 2.8 3.2 2.9 0.9 0.8 1.1 1.2 WV-13819 3.8 3.8 3.0 2.9 1.0 0.9 0.9 1.0 WV-13820 6.6 6.7 6.4 6.3 3.2 3.0 2.9 3.0 WV-13821 7.4 6.5 7.4 6.9 2.2 1.9 2.5 1.9 WV-13822 9.5 9.5 8.1 8.6 3.4 3.5 3.4 3.9 WV-13823 10.4 10.9 11.2 10.5 4.2 5.0 4.1 4.4 WV-13824 17.1 16.3 16.1 15.6 8.1 7.6 7.1 7.0 WV-13825 20.1 19.3 22.5 20.6 9.9 9.8 9.0 9.6 WV-14527 2.2 1.9 1.4 2.0 0.7 0.7 0.9 0.7 WV-14528 2.3 2.2 2.5 2.4 1.0 0.9 1.0 1.0 WV-14529 5.2 1.8 2.0 2.0 0.7 0.7 0.8 0.8 WV-11089 2.6 2.7 2.9 2.5 0.9 0.9 1.4 1.3

(1162) Additional data related to multiple exon skipping mediated by DMD oligonucleotides which target DMD exon 45 are shown in Table 22A.1.

(1163) Example Dystrophin Oligonucleotides and Compositions Which Target Exon 46

(1164) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 46 and/or mediating skipping of exon 46 in human DMD. Non-limiting examples include oligonucleotides and compositions of WV-13701, WV-13702, WV-13703, WV-13704, WV-13705, WV-13706, WV-13707, WV-13708, WV-13709, WV-13710, WV-13711, WV-13712, WV-13713, WV-13714, WV-13715, WV-13716, WV-13780, and WV-13781, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1165) In some embodiments, DMD oligonucleotides are first tested for single exon skipping to select suitable oligonucleotides, then tested combinatorially (in combination with another DMD oligonucleotide) for multi-exon skipping.

(1166) In some embodiments, DMD oligonucleotides targeting DMD exon 46, 47, 52, 54 or 55 are first tested for single exon skipping to select suitable oligonucleotides, then tested combinatorially (in combination with another DMD oligonucleotide) for multi-exon skipping.

(1167) TABLE-US-00008 TABLE 2A Example data of certain oligonucleotides. Numbers indicate percentage of exon 46 skipping. WV-13701 0.3 0.3 0.5 0.4 WV-13702 0.3 0.4 0.5 0.3 WV-13703 0.9 0.9 1.1 0.8 WV-13704 9.7 5.4 WV-13705 4.9 5.1 5.9 3.4 WV-13706 4.6 4.8 WV-13707 8.5 7.4 5.2 5.1 WV-13708 9.4 10.8 6.0 5.6 WV-13709 8.8 12.1 8.1 4.9 WV-13710 0.1 0.1 0.1 0.1 WV-13711 0.1 0.1 0.0 0.1 WV-13712 3.4 4.7 2.4 2.4 WV-13713 0.5 0.7 0.5 WV-13714 0.6 0.5 0.4 WV-13715 0.9 0.6 0.7 WV-13716 1.5 3.9 1.1 2.8 WV-13780 10.1 5.2 6.1 WV-13781 7.7 6.4 5.0 Mock 0.0 0.0 0.0 0.0 Mock 0.0 0.0
Example Dystrophin Oligonucleotides and Compositions Which Target Exon 47

(1168) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 47 and/or mediating skipping of exon 47 in human DMD. Non-limiting examples include oligonucleotides and compositions of exon 47 oligos include: WV-13717, WV-13718, WV-13719, WV-13720, WV-13721, WV-13722, WV-13723, WV-13724, WV-13725, WV-13726, WV-13727, WV-13728, WV-13729, WV-13730, WV-13731, WV-13732, WV-13788, and WV-13789, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1169) TABLE-US-00009 TABLE 3A Example data of certain oligonucleotides. Numbers represent percentage of exon 47 skipping. WV-13717 0.0 0.0 WV-13718 0.0 0.0 WV-13719 0.0 0.0 WV-13720 0.0 0.0 WV-13721 0.0 0.0 WV-13722 0.0 0.0 WV-13723 0.5 0.5 WV-13724 1.4 1.8 WV-13725 0.6 0.4 WV-13726 0.0 0.0 WV-13727 1.1 1.1 WV-13728 1.1 1.1 WV-13729 0.2 0.2 WV-13730 0.5 0.6 WV-13731 1.6 1.8 WV-13732 0.1 0.6
Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 51

(1170) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 51 in DMD (e.g., of mouse, human, etc.).

(1171) In some embodiments, a provided DMD oligonucleotide and/or composition is capable of mediating skipping of exon 51. Non-limiting examples of such DMD oligonucleotides and compositions include those of: ONT-395, WV-10255, WV-10261, WV-10262, WV-10634, WV-10635, WV-10636, WV-10637, WV-10868, WV-10869, WV-10870, WV-10871, WV-10872, WV-10873, WV-10874, WV-10875, WV-10876, WV-10877, WV-10878, WV-10879, WV-10880, WV-10881, WV-10882, WV-10883, WV-10884, WV-10885, WV-10886, WV-10887, WV-10888, WV-1107, WV-1108, WV-1109, WV-1110, WV-1111, WV-1112, WV-1113, WV-1114, WV-1115, WV-1116, WV-1117, WV-1118, WV-1119, WV-1120, WV-11237, WV-11238, WV-11239, WV-1131, WV-1132, WV-1133, WV-1134, WV-1135, WV-1136, WV-1137, WV-1138, WV-1139, WV-1140, WV-1151, WV-1152, WV-1153, WV-1154, WV-1155, WV-1156, WV-1157, WV-1158, WV-1159, WV-1160, WV-1709, WV-1710, WV-1711, WV-1712, WV-1713, WV-1714, WV-1715, WV-1716, WV-2095, WV-2096, WV-2097, WV-2098, WV-2099, WV-2100, WV-2101, WV-2102, WV-2103, WV-2104, WV-2105, WV-2106, WV-2107, WV-2108, WV-2109, WV-2165, WV-2179, WV-2180, WV-2181, WV-2182, WV-2183, WV-2184, WV-2185, WV-2186, WV-2187, WV-2188, WV-2189, WV-2190, WV-2191, WV-2192, WV-2193, WV-2194, WV-2195, WV-2196, WV-2197, WV-2198, WV-2199, WV-2200, WV-2201, WV-2202, WV-2203, WV-2204, WV-2205, WV-2206, WV-2207, WV-2208, WV-2209, WV-2210, WV-2211, WV-2212, WV-2213, WV-2214, WV-2215, WV-2216, WV-2217, WV-2218, WV-2219, WV-2220, WV-2221, WV-2222, WV-2223, WV-2224, WV-2225, WV-2226, WV-2227, WV-2228, WV-2229, WV-2230, WV-2231, WV-2232, WV-2233, WV-2234, WV-2235, WV-2236, WV-2237, WV-2238, WV-2239, WV-2240, WV-2241, WV-2242, WV-2243, WV-2244, WV-2245, WV-2246, WV-2247, WV-2248, WV-2249, WV-2250, WV-2251, WV-2252, WV-2253, WV-2254, WV-2255, WV-2256, WV-2257, WV-2258, WV-2259, WV-2260, WV-2261, WV-2262, WV-2263, WV-2264, WV-2265, WV-2266, WV-2267, WV-2268, WV-2273, WV-2274, WV-2275, WV-2276, WV-2277, WV-2278, WV-2279, WV-2280, WV-2281, WV-2282, WV-2283, WV-2284, WV-2285, WV-2286, WV-2287, WV-2288, WV-2289, WV-2290, WV-2291, WV-2292, WV-2293, WV-2294, WV-2295, WV-2296, WV-2297, WV-2298, WV-2299, WV-2300, WV-2301, WV-2302, WV-2303, WV-2304, WV-2305, WV-2306, WV-2307, WV-2308, WV-2309, WV-2310, WV-2311, WV-2312, WV-2313, WV-2314, WV-2315, WV-2316, WV-2317, WV-2318, WV-2319, WV-2320, WV-2321, WV-2322, WV-2323, WV-2324, WV-2325, WV-2326, WV-2327, WV-2328, WV-2329, WV-2330, WV-2331, WV-2332, WV-2333, WV-2334, WV-2335, WV-2336, WV-2337, WV-2338, WV-2339, WV-2340, WV-2341, WV-2342, WV-2343, WV-2344, WV-2345, WV-2346, WV-2347, WV-2348, WV-2349, WV-2350, WV-2351, WV-2352, WV-2353, WV-2354, WV-2361, WV-2362, WV-2363, WV-2364, WV-2365, WV-2366, WV-2367, WV-2368, WV-2369, WV-2370, WV-2381, WV-2382, WV-2383, WV-2384, WV-2385, WV-2432, WV-2433, WV-2434, WV-2435, WV-2436, WV-2437, WV-2438, WV-2439, WV-2440, WV-2441, WV-2442, WV-2443, WV-2444, WV-2445, WV-2446, WV-2447, WV-2448, WV-2449, WV-2526, WV-2527, WV-2528, WV-2529, WV-2530, WV-2531, WV-2532, WV-2533, WV-2534, WV-2535, WV-2536, WV-2537, WV-2538, WV-2578, WV-2579, WV-2580, WV-2581, WV-2582, WV-2583, WV-2584, WV-2585, WV-2586, WV-2587, WV-2588, WV-2625, WV-2627, WV-2628, WV-2660, WV-2661, WV-2662, WV-2663, WV-2664, WV-2665, WV-2666, WV-2667, WV-2668, WV-2669, WV-2670, WV-2737, WV-2738, WV-2739, WV-2740, WV-2741, WV-2742, WV-2743, WV-2744, WV-2745, WV-2746, WV-2747, WV-2748, WV-2749, WV-2750, WV-2752, WV-2783, WV-2784, WV-2785, WV-2786, WV-2787, WV-2788, WV-2789, WV-2790, WV-2791, WV-2792, WV-2793, WV-2794, WV-2795, WV-2796, WV-2797, WV-2798, WV-2799, WV-2800, WV-2801, WV-2802, WV-2803, WV-2804, WV-2805, WV-2806, WV-2807, WV-2808, WV-2812, WV-2813, WV-2814, WV-3017, WV-3018, WV-3019, WV-3020, WV-3022, WV-3023, WV-3024, WV-3025, WV-3026, WV-3027, WV-3028, WV-3029, WV-3030, WV-3031, WV-3032, WV-3033, WV-3034, WV-3035, WV-3036, WV-3037, WV-3038, WV-3039, WV-3040, WV-3041, WV-3042, WV-3043, WV-3044, WV-3045, WV-3046, WV-3047, WV-3048, WV-3049, WV-3050, WV-3051, WV-3052, WV-3053, WV-3054, WV-3055, WV-3056, WV-3057, WV-3058, WV-3059, WV-3060, WV-3061, WV-3070, WV-3071, WV-3072, WV-3073, WV-3074, WV-3075, WV-3076, WV-3077, WV-3078, WV-3079, WV-3080, WV-3081, WV-3082, WV-3083, WV-3084, WV-3085, WV-3086, WV-3087, WV-3088, WV-3089, WV-3113, WV-3114, WV-3115, WV-3116, WV-3117, WV-3118, WV-3120, WV-3121, WV-3152, WV-3153, WV-3357, WV-3358, WV-3359, WV-3360, WV-3361, WV-3362, WV-3363, WV-3364, WV-3365, WV-3366, WV-3463, WV-3464, WV-3465, WV-3466, WV-3467, WV-3468, WV-3469, WV-3470, WV-3471, WV-3472, WV-3473, WV-3506, WV-3507, WV-3508, WV-3509, WV-3510, WV-3511, WV-3512, WV-3513, WV-3514, WV-3515, WV-3516, WV-3517, WV-3518, WV-3519, WV-3520, WV-3543, WV-3544, WV-3545, WV-3546, WV-3547, WV-3548, WV-3549, WV-3550, WV-3551, WV-3552, WV-3553, WV-3554, WV-3555, WV-3556, WV-3557, WV-3558, WV-3559, WV-3560, WV-3753, WV-3754, WV-3820, WV-3821, WV-3855, WV-3856, WV-3971, WV-4106, WV-4107, WV-4191, WV-4231, WV-4232, WV-4233, WV-4890, WV-6137, WV-6409, WV-6410, WV-6560, WV-6826, WV-6827, WV-6828, WV-7109, WV-7110, WV-7333, WV-7334, WV-7335, WV-7336, WV-7337, WV-7338, WV-7339, WV-7340, WV-7341, WV-7342, WV-7343, WV-7344, WV-7345, WV-7346, WV-7347, WV-7348, WV-7349, WV-7350, WV-7351, WV-7352, WV-7353, WV-7354, WV-7355, WV-7356, WV-7357, WV-7358, WV-7359, WV-7360, WV-7361, WV-7362, WV-7363, WV-7364, WV-7365, WV-7366, WV-7367, WV-7368, WV-7369, WV-7370, WV-7371, WV-7372, WV-7373, WV-7374, WV-7375, WV-7376, WV-7377, WV-7378, WV-7379, WV-7380, WV-7381, WV-7382, WV-7383, WV-7384, WV-7385, WV-7386, WV-7387, WV-7388, WV-7389, WV-7390, WV-7391, WV-7392, WV-7393, WV-7394, WV-7395, WV-7396, WV-7397, WV-7398, WV-7399, WV-7400, WV-7401, WV-7402, WV-7410, WV-7411, WV-7412, WV-7413, WV-7414, WV-7415, WV-7457, WV-7458, WV-7459, WV-7460, WV-7461, WV-7506, WV-7596, WV-8130, WV-8131, WV-8230, WV-8231, WV-8232, WV-8449, WV-8478, WV-8479, WV-8480, WV-8481, WV-8482, WV-8483, WV-8484, WV-8485, WV-8486, WV-8487, WV-8488, WV-8489, WV-8490, WV-8491, WV-8492, WV-8493, WV-8494, WV-8495, WV-8496, WV-8497, WV-8498, WV-8499, WV-8500, WV-8501, WV-8502, WV-8503, WV-8504, WV-8505, WV-8506, WV-8806, WV-884, WV-885, WV-886, WV-887, WV-888, WV-889, WV-890, WV-891, WV-892, WV-893, WV-894, WV-895, WV-896, WV-897, WV-9222, WV-9223, WV-9224, WV-9225, WV-9226, WV-9227, WV-942, WV-9540, WV-9541, WV-9737, WV-9738, WV-9739, WV-9740, WV-9741, WV-9742, WV-9827, WV-9828, WV-9829, WV-9830, WV-9831, WV-9832, WV-9833, WV-9834, WV-9835, WV-9836, WV-9837, WV-9838, WV-9839, WV-9840, WV-9841, WV-9842, WV-9843, WV-9844, WV-9845, WV-9846, WV-9847, WV-9848, WV-9849, WV-9850, WV-9851, WV-9852, WV-9858, and WV-8937, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1172) Additional non-limiting examples of such DMD oligonucleotides and compositions include those of: WV-2444, WV-2528, WV-2531, WV-2578, WV-2579, WV-2580, WV-2581, WV-2669, WV-2745, WV-3032, WV-3152, WV-3153, WV-3360, WV-3363, WV-3364, WV-3465, WV-3466, WV-3470, WV-3472, WV-3473, WV-3507, WV-3545, WV-3546, WV-3552, WV-4106, WV-4231, WV-4232, WV-4233, WV-887, WV-896, WV-942, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1173) Additional non-limiting examples of such DMD oligonucleotides and compositions include those of: WV-12494, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, WV-12136, WV-12496, WV-12495, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, WV-12876, WV-12877, WV-12878, WV-12879, WV-12880, WV-12881, WV-12882, and WV-12883, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1174) In some embodiments, the sequence of the region of interest for exon 51 skipping differs between the mouse and human.

(1175) Various assays can be utilized to assess oligonucleotides for exon skipping in accordance with the present disclosure. In some embodiments, in order to test the efficacy of a particular combination of chemistry and stereochemistry of an oligonucleotide intended for exon 51 skipping in human, a corresponding oligonucleotide can be prepared which has the mouse sequence, and then tested in mouse. The present disclosure recognizes that in the human and mouse homologs of exon 51, a few differences exist (underlined below):

(1176) TABLE-US-00010 (SEQIDNO:3152) MGTGGTTACTAAGGAAACTGTCATCTCCAAACTAGAAATGCCATCTTCTTTGCTGTTGGAG (SEQIDNO:3153) HGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAG
where M is Mouse, nt 7571-7630; and His Human, nt 7665-7724.

(1177) Because of these differences, slightly different DMD oligonucleotides for skipping exon 51 can be prepared for testing in mouse and human. As a non-limiting example, the following DMD oligonucleotide sequences can be used for testing in human and mouse:

(1178) TABLE-US-00011 HUMANDMDoligonucleotidesequence: (SEQIDNO:3154) UCAAGGAAGAUGGCAUUCU MOUSEDMDoligonucleotidesequence: (SEQIDNO:3155) GCAAAGAAGAUGGCAUUUCU
Mismatches between human and mouse are underlined.

(1179) A DMD oligonucleotide intended for treating a human subject can be constructed with a particular combination of base sequence (e.g., UCAAGGAAGAUGGCAUUUCU (SEQ ID NO: 3156), and a particular pattern of chemistry, internucleotidic linkages, stereochemistry, and additional chemical moieties (if any). Such a DMD oligonucleotide can be tested in vitro in human cells or in vivo in human subjects, but may have limited suitability for testing in mouse, for example, because base sequences of the two have mismatches.

(1180) A corresponding DMD oligonucleotide can be constructed with the corresponding mouse base sequence (GCAAAGAAGAUGGCAUUUCU (SEQ ID NO: 3157)) and the same pattern of chemistry, internucleotidic linkages, stereochemistry, and additional chemical moieties (if any). Such an oligonucleotide can be tested in vivo in mouse. Several DMD oligonucleotides comprising the mouse base sequence were constructed and tested.

(1181) In some embodiments, a human DMD exon skipping oligonucleotide can be tested in a mouse which has been modified to comprise a DMD gene comprising the human sequence.

(1182) Various DMD oligonucleotides comprising various patterns of modifications are described herein. The Tables below show test results of certain DMD oligonucleotides. To assay exon skipping of DMD, DMD oligonucleotides were tested in vitro in 52 human patient-derived myoblast cells and/or 45-52 human patient-derived myoblast cells (human cells wherein the exon 52 or exons 45-52 were already deleted). Unless noted otherwise, in various experiments, oligonucleotides were delivered gymnotically.

(1183) TABLE-US-00012 TABLE 4A Example data of certain oligonucleotides. 10 uM 3 uM WV-942 1.0 2.2 1.5 0.2 0.5 0.2 WV-1709 8.5 12.9 7.7 3.3 5.8 3.7 WV-1710 4.1 6.1 4.7 1.1 2.5 1.3 WV-1711 4.4 5.8 3.7 1.1 2.4 1.4 WV-1712 2.6 4.4 3.1 0.9 2.0 1.7 WV-1713 2.1 3.5 2.3 0.6 1.6 0.3 WV-1714 7.8 10.5 10.2 2.3 4.1 2.3 WV-1715 2.2 3.8 3.3 0.8 1.8 1.1 WV-1716 2.1 3.5 2.4 0.9 1.8 0.9
DMD oligonucleotides were tested in vitro at 10 uM and 3 uM, in triplicates. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. Full descriptions of the oligonucleotides tested in this Table (and other Tables) are provided in Table A1.

(1184) In Table 4B, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

(1185) TABLE-US-00013 TABLE 4B Example data of certain oligonucleotides. 10 uM 3 uM WV-942 1.0 2.2 1.5 0.2 0.5 0.2 WV-1714 7.8 10.5 10.2 2.3 4.1 2.3 WV-2444 22.2 26.7 28.6 9.1 12.6 11.9 WV-2445 17.1 20.7 18.7 7.0 9.7 9.1 WV-2528 32.4 34.6 39.3 16.9 19.9 22.3 WV-2529 3.2 5.8 6.1 2.2 4.5 3.0 WV-2530 18.6 21.1 25.4 7.6 11.5 11.4
DMD oligonucleotides were tested at 10 uM and 3 uM, in triplicates. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1186) In Table 4C, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

(1187) TABLE-US-00014 TABLE 4C Example data of certain oligonucleotides. WV-942 WV-887 WV-1714 WV-2438 10 uM 1.1 0.7 5.1 3.9 3.6 3.7 9.3 9.3 3 uM 0.5 0.3 1.0 2.2 1.6 1.5 3.9 3.1 1 uM 0.2 0.2 0.6 0.7 0.6 0.3 1.4 1.1 WV-2439 WV-2444 WV-2445 Mock 10 uM 3.2 2.1 12.9 14.3 9.7 8.9 0.4 0.1 3 uM 0.8 0.7 4.7 4.1 3.3 3.5 0.1 0.1 1 uM 0.4 0.3 1.4 1.0 1.1 1.0 0.1
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1188) In Table 4D, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

(1189) TABLE-US-00015 TABLE 4D Example data of certain oligonucleotides. 10 uM WV-942 0.6 0.6 0.6 0.6 WV-2660 0.2 0.3 0.1 0.1 WV-2661 0.4 0.4 WV-2662 0.2 0.2 0.1 0.1 WV-2663 0.5 0.5 0.4 0.5 WV-2670 5.1 5.2 6.2 7.3
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1190) In Table 5, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

(1191) TABLE-US-00016 TABLE 5 Example data of certain oligonucleotides. 10 uM 3 uM 1 uM Mock 0.0 0.1 0.0 WV-2531 21.7 8.7 3.2 WV-3152 26.1 15.3 5.7 WV-2745 24.0 10.7 4.8 WV-3463 6.6 3.0 0.8 WV-3464 16.1 6.2 2.4 WV-3465 16.4 6.0 1.8 WV-3466 13.0 5.7 2.0 WV-3467 12.6 5.8 2.6 WV-3469 14.2 6.0 1.5 WV-3470 24.9 11.9 6.4 WV-3471 4.9 1.6 1.0 WV-3472 20.1 12.4 7.2 WV-3473 24.9 11.4 7.6 WV-942 3.3 2.1 0.7
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1192) TABLE-US-00017 TABLE 6 Example data of certain oligonucleotides. 5 uM 1 uM WV-942 .2 PMO .1 WV-6137 1 .9 WV-7333 .3 .2 WV-7334 .7 .4 WV-7335 1.7 .4 WV-7336 2.2 .6 WV-7337 1.7 .4 WV-7343 1.4 .5 WV-7344 2.8 .7 WV-7345 2.9 1 WV-7346 1.9 .7 WV-7347 1.2 .5 WV-7348 2.5 1 WV-7349 3 .6 WV-7350 3.1 1 WV-7351 1.7 .6 WV-7352 2.7 .8 WV-7353 2.8 .2 WV-7354 2.2 .3 WV-7355 2.7 1.6 WV-7356 3.3 1.2 WV-7357 2.7 1.1 WV-7358 2.2 .6 WV-7359 .7 .3 WV-7360 .6 .5 WV-7361 2.8 .8 WV-7362 4.1 .8 WV-7363 2 .7
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. Numbers are approximate. Oligonucleotides were delivered gymnotically to A48-50 patient-derived myoblasts (4 days post-differentiation). The oligonucleotide designated as PMO in this table and other tables related to skipping of DMD exon 51 is WV-8806 CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 3158), which is fully PMO (Morpholino).

(1193) In Table 7, below, additional data of DMD oligonucleotides for skipping exon 51 were presented.

(1194) TABLE-US-00018 TABLE 7 Example data of certain oligonucleotides. Mock .1 WV-942 .2 PMO .1 WV-7364 2 .5 WV-7365 1.8 .5 WV-7366 1.1 5.7 WV-7367 .2 .3 WV-7368 .4 .4 WV-7369 .4 .2 WV-7370 .2 .3 WV-7371 .3 .2 WV-7372 .3 WV-7373 .5 1.3 WV-7374 .3 .4 WV-7375 .2 .8 WV-7376 .2 .5 WV-7377 .3 .5 WV-7378 .4 WV-7379 7.8 1 WV-7380 2.8 .3 WV-7381 4.1 .2 WV-7382 1.3 .1 WV-7383 1.7 .3 WV-7384 2.8 .4 WV-7385 1.8 WV-7386 4 1.6 WV-7387 3 1.8 WV-7388 1.2 .7 WV-7389 .5 .4 WV-7390 1 .5
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. Numbers are approximate.

(1195) In some embodiments, the present disclosure pertains to metabolites of any oligonucleotide, e.g., DMD oligonucleotide, disclosed herein, or any combination thereof. In some embodiments, a metabolite of an oligonucleotide, e.g., a DMD oligonucleotide is the result of an oligonucleotide, e.g., a DMD oligonucleotide being acted upon by a nuclease (e.g., an exonuclease or endonuclease or other enzymes, including those may chemically process one or more modifications of an oligonucleotide). In some embodiments, a metabolite of an oligonucleotide, e.g., a DMD oligonucleotide is not the physical product of such an oligonucleotide being metabolized or physically treated with a nuclease, but rather a compound which corresponds chemically to a product of an oligonucleotide being metabolized or treated with an enzyme, e.g., a nuclease. In some embodiments, metabolite of an oligonucleotide, e.g., a DMD oligonucleotide, is chemically synthesized, without any metabolic process, and optionally administered to a subject.

(1196) In some embodiments, a metabolite is a truncation of an oligonucleotide on the 5 end and/or 3 end by one or two nucleotides or nucleosides. In some embodiments, the present disclosure provides an oligonucleotide, e.g., DMD oligonucleotide which corresponds to an oligonucleotide, e.g., DMD oligonucleotide listed herein, but is truncated at the 5 end by one or two nucleotides. In some embodiments, the present disclosure provides an oligonucleotide, e.g., a DMD oligonucleotide which corresponds to an oligonucleotide, e.g., a DMD oligonucleotide listed herein, but is truncated at the 3 end by one or two nucleotides. In some embodiments, the present disclosure provides an oligonucleotide, e.g., a DMD oligonucleotide which corresponds to an oligonucleotide, e.g., a DMD oligonucleotide listed herein, but is truncated at the 3 end and 5 end by one or two nucleotides. Among other things, such oligonucleotides may perform various of biological functions, e.g., such DMD oligonucleotides can mediate skipping of exon 23, 45, 51, 53, or any other DMD exon.

(1197) In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has the base sequence of a DMD oligonucleotide listed herein, except that the base sequence is shorter on the 5 end by one or two bases. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has the base sequence of a DMD oligonucleotide listed herein, except that the base sequence is shorter on the 3 end by one or two bases. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has the base sequence of a DMD oligonucleotide disclosed herein, except that the base sequence is shorter on the 3 end and the 5 end by one or two bases. Such DMD oligonucleotides, among other things, can mediate skipping of exon 23, 45, 51, 53, or any other DMD exon.

(1198) In some embodiments, a metabolite of a DMD oligonucleotide has removed from the oligonucleotide an additional moiety (e.g., a lipid or other conjugated moiety).

(1199) In some embodiments, an oligonucleotide of the present disclosure may be a metabolite of another oligonucleotide. For example, several oligonucleotides may be metabolite of WV-3473, for example, WV-4231 (3 n-1, truncated at the 3 end by one nucleotide), WV-4232 (3 n-2), WV-4233 (5 n-1), etc. Example data of such metabolite oligonucleotides were presented in Table 9 below (at 1, 3 and 10 uM, in replicates). Generally, an oligonucleotide can be used independently whether or not it can be a metabolite of another oligonucleotide.

(1200) TABLE-US-00019 TABLE 9 Example data of certain oligonucleotides. Oligonucleotide 10 uM 3 uM 1 uM PMO 2.4 1.6 0.4 1.1 0.4 0.6 WV-3473 78.8 73.5 62.5 59.8 38.8 38.8 WV-4231 (3 n-1) 83.8 71.4 65.0 67.2 44.4 43.0 WV-4232 (3 n-2) 48.5 66.5 42.2 57.5 30.0 WV-4233 (5 n-1) 54.2 45.9 37.1 31.6 18.6 14.5
Results of replicate experiments are shown. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown. In this and other tables, PMO is a Morpholino oligonucleotide control.

(1201) In some embodiments, the present disclosure pertains to DMD oligonucleotides corresponding to any DMD oligonucleotide to exon 51 or any other exon listed herein (e.g., in Table A1), but which are truncated by one, two or more nucleotides on the 5 end and/or 3 end.

(1202) In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 15 to 45 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 20 to 45 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 20 to 40 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 35 bases. In some embodiments, the length of a provided oligonucleotide, e.g., a DMD oligonucleotide, is 20 to 25 bases.

(1203) In some experiments, lengths of DMD oligonucleotides for skipping exon 51 are 20 or 25 bases.

(1204) Tables 10A and 10B. Example data of certain oligonucleotides.

(1205) Table 10A shows data of 20-mers for skipping DMD exon 51; Table 10B shows data of 25-mers for skipping DMD exon 51. Sequences are provided in Table A1. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1206) TABLE-US-00020 TABLE 10A 20-mers untreated WV-2313 WV-2314 WV-2315 WV-2316 0.1 0.1 1.0 1.4 1.7 1.6 2.0 2.0 4.6 2.5 WV-2317 WV-2318 WV-2319 WV-2320 WV-942 1.7 1.1 4.3 4.3 5.0 6.5 2.9 3.7 3.9 3.4

(1207) TABLE-US-00021 TABLE 10B 25-mers WV-2223 WV-2224 WV-2225 WV-2226 15.7 14.8 6.6 7.3 13.4 16.1 7.7 7.7 WV-2227 WV-2228 WV-2229 WV-2230 9.8 9.7 15.7 15.6 8.5 8.9 12.9 13.4

(1208) Additional data are provided.

(1209) TABLE-US-00022 TABLE 10C Example data of certain oligonucleotides. 10 uM 3 uM 1 uM WV-2531 21.7 25.1 8.7 10.6 3.2 4.6 WV-3152 26.1 21.7 15.3 10.7 5.7 4.1 WV-3472 20.1 16.3 12.4 8.5 7.2 3.8 WV-3473 24.9 38.4 11.4 11.2 7.6 6.5 WV-942 3.3 0.2 2.1 0.7 0.1
Oligonucleotides were tested in vitro at 10, 3 and 1 M. Results of replicate experiments are shown. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1210) TABLE-US-00023 TABLE 10D Example data of certain oligonucleotides. 10 uM 3 uM 1 uM WV-1714 5.8 6.2 8.1 2.4 3.0 2.7 0.7 0.7 2.0 WV-3030 29.9 27.2 35.2 6.2 5.6 5.6 0.6 0.6 1.6 WV-3032 31.7 29.3 37.9 7.8 6.4 7.7 1.2 1.1 1.1 WV-2669 3.1 3.1 4.1 1.4 1.7 1.7 0.6 0.7 0.8 WV-3035 13.2 16.4 17.6 1.9 2.5 2.8 1.0 1.1 0.8
Oligonucleotides were tested in vitro at 10, 3 and 1 M. Results of replicate experiments are shown. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1211) TABLE-US-00024 TABLE 10E Example data of certain oligonucleotides. 10 uM 3 uM 1 uM WV-2531 24.7 21.7 11.0 8.7 4.8 3.2 WV-3360 25.1 12.9 10.1 3.3 WV-3363 24.0 7.7 3.4 WV-3364 72.8 45.5 17.2 9.8 4.0
Oligonucleotides were tested in vitro at 10, 3 and 1 M. Results of replicate experiments are shown. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency: results from replicate experiments are shown.

(1212) TABLE-US-00025 TABLE 10F Example data of certain oligonucleotides. 10 uM 3 uM 1 uM Mock 0.0 0.1 0.0 WV-2531 21.7 8.7 3.2 WV-3360 25.1 10.1 3.3 WV-3363 24.0 7.7 3.4 WV-3364 45.5 9.8 4.0
Oligonucleotides were tested in vitro at 10, 3 and 1 M. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1213) TABLE-US-00026 TABLE 10G Example data of certain oligonucleotides. 10 uM 3 uM 1 uM WV-1714 5.8 6.2 8.1 2.4 3.0 2.7 0.7 0.7 2.0 WV-3030 29.9 27.2 35.2 6.2 5.6 5.6 0.6 0.6 1.6 WV-3032 31.7 29.3 37.9 7.8 6.4 7.7 1.2 1.1 1.1 WV-2669 3.1 3.1 4.1 1.4 1.7 1.7 0.6 0.7 0.8 WV-3035 13.2 16.4 17.6 1.9 2.5 2.8 1.0 1.1 0.8
Oligonucleotides were tested in vitro at 10, 3 and 1 M. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1214) TABLE-US-00027 TABLE 10H Example data of certain oligonucleotides. 10 uM, 15% serum 10 uM 5% serum Mock 0.0 0.1 0.0 0.1 WV-942 1.0 1.0 0.2 0.2 0.7 0.5 0.4 0.4 WV-2578 3.2 2.2 2.4 2.3 2.2 0.9 WV-2579 3.1 2.9 2.5 2.5 WV-2580 2.5 2.9 2.4 3.1 6.8 6.4 2.8 3.2 WV-2581 3.3 3.6 3.9 3.7 4.4 5.8 5.8 5.4 10 uM 5% serum 10 uM 5% serum 20 mg/ml BSA 4 mg/ml BSA Mock 0.1 0.1 0.1 0.1 WV-942 0.7 0.6 1.4 1.3 0.2 0.3 0.6 0.5 WV-2578 0.9 0.5 0.5 0.6 0.6 0.6 0.5 0.7 WV-2579 0.1 0.1 0.5 0.3 0.1 0.1 0.5 0.4 WV-2580 0.4 0.3 0.2 0.2 0.2 0.1 WV-2581 0.2 0.2 0.4 0.4 0.2 0.2 0.1 0.1 3 uM 15% serum 3 uM 5% serum Mock 0.0 0.0 0.0 0.0 WV-942 0.1 0.0 0.3 0.3 0.1 0.1 0.2 0.2 WV-2578 0.5 0.3 0.3 0.4 0.3 0.5 0.6 0.2 WV-2579 0.6 0.5 1.8 1.5 0.5 0.4 0.3 0.3 WV-2580 1.0 1.0 0.5 0.6 1.2 1.0 0.5 0.7 WV-2581 0.0 0.0 0.6 0.6 0.4 0.5 0.8 0.7 3 uM 5% serum 3 uM 5% serum 20 mg/ml BSA 4 mg/ml BSA Mock 0.0 0.0 0.0 0.0 WV-942 0.1 0.1 0.1 0.1 0.1 0.1 0.4 0.3 WV-2578 0.2 0.2 0.2 0.3 0.2 0.1 0.1 WV-2579 0.4 0.4 0.2 0.2 0.1 0.1 0.2 0.2 WV-2580 0.2 0.2 0.2 0.3 0.0 0.0 0.3 0.3 WV-2581 0.0 0.0 0.3 0.3 0.1 0.1 0.1 0.1 10 uM, 15% serum 10 uM 5% serum Mock 0.0 0.1 0.0 0.1 WV-942 1.0 1.0 0.2 0.2 0.7 0.5 0.4 0.4 WV-2578 3.2 2.2 2.4 2.3 2.2 0.9 WV-2579 3.1 2.9 2.5 2.5 WV-2580 2.5 2.9 2.4 3.1 6.8 6.4 2.8 3.2 WV-2581 3.3 3.6 3.9 3.7 4.4 5.8 5.8 5.4 10 uM 5% serum 10 uM 5% serum 20 mg/ml BSA 4 mg/ml BSA Mock 0.1 0.1 0.1 0.1 WV-942 0.7 0.6 1.4 1.3 0.2 0.3 0.6 0.5 WV-2578 0.9 0.5 0.5 0.6 0.6 0.6 0.5 0.7 WV-2579 0.1 0.1 0.5 0.3 0.1 0.1 0.5 0.4 WV-2580 0.4 0.3 0.2 0.2 0.2 0.1 WV-2581 0.2 0.2 0.4 0.4 0.2 0.2 0.1 0.1 3 uM 15% serum 3 uM 5% serum Mock 0.0 0.0 0.0 0.0 WV-942 0.1 0.0 0.3 0.3 0.1 0.1 0.2 0.2 WV-2578 0.5 0.3 0.3 0.4 0.3 0.5 0.6 0.2 WV-2579 0.6 0.5 1.8 1.5 0.5 0.4 0.3 0.3 WV-2580 1.0 1.0 0.5 0.6 1.2 1.0 0.5 0.7 WV-2581 0.0 0.0 0.6 0.6 0.4 0.5 0.8 0.7 3 uM 5% serum 3 uM 5% serum 20 mg/ml BSA 4 mg/ml BSA Mock 0.0 0.0 0.0 0.0 WV-942 0.1 0.1 0.1 0.1 0.1 0.1 0.4 0.3 WV-2578 0.2 0.2 0.2 0.3 0.2 0.1 0.1 WV-2579 0.4 0.4 0.2 0.2 0.1 0.1 0.2 0.2 WV-2580 0.2 0.2 0.2 0.3 0.0 0.0 0.3 0.3 WV-2581 0.0 0.0 0.3 0.3 0.1 0.1 0.1 0.1
Oligonucleotides were tested in vitro at 10 and 3 M. In this table, in some cases, serum and/or BSA were added to test the effect on exon skipping. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1215) TABLE-US-00028 TABLE 10I Example data of certain oligonucleotides. 10 uM 3 uM 1 uM Mock 0.0 0.1 0.0 WV-2531 21.7 8.7 3.2 WV-3152 26.1 15.3 5.7 WV-2745 24.0 10.7 4.8 WV-3463 6.6 3.0 0.8 WV-3464 16.1 6.2 2.4 WV-3465 16.4 6.0 1.8 WV-3466 13.0 5.7 2.0 WV-3467 12.6 5.8 2.6 WV-3469 14.2 6.0 1.5 WV-3470 24.9 11.9 6.4 WV-3471 4.9 1.6 1.0 WV-3472 20.1 12.4 7.2 WV-3473 24.9 11.4 7.6 WV-942 3.3 2.1 0.7
Oligonucleotides were tested in vitro at 10, 3 and 1 M. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1216) TABLE-US-00029 TABLE 10J Example data of certain oligonucleotides. 10 uM 3 uM 1 uM WV-2531 32.9 32.0 16.9 16.7 6.2 6.2 WV-3360 27.2 26.5 13.4 14.2 6.0 5.9 WV-3361 28.9 28.0 16.7 16.1 6.3 6.0 WV-3362 34.3 32.9 16.2 15.5 6.1 5.8 WV-3363 33.2 33.6 16.4 16.0 6.7 6.4 WV-3364 47.9 47.6 14.2 14.0 6.4 6.5 WV-3365 25.6 24.2 14.7 14.2 6.9 6.4 WV-3366 34.6 34.0 21.1 19.8 8.0 7.4 WV-942 0.6 0.6 0.3 0.3 0.1 0.1 Mock 0.0 0.0 0.1 0.1 0.1 0.0
Oligonucleotides were tested in vitro at 10, 3 and 1 M. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1217) TABLE-US-00030 TABLE 10K Example data of certain oligonucleotides. Activity relative to WV-942 WV-942 1.1 0.9 Mock 0.1 0.0 WV-2526 18.4 15.3 WV-2527 17.0 16.3 WV-2528 34.6 27.2 WV-2529 3.7 2.8 WV-2530 17.0 16.9 WV-2533 4.1 3.6 WV-2534 2.0 1.2 WV-2535 0.4 0.2 WV-2536 0.2 0.1 WV-2537 1.1 1.0
Oligonucleotides were tested in vitro at 10 M. In this table, numbers represent skipping efficiency relative to WV-942 (ave): results from replicate experiments are shown.

(1218) TABLE-US-00031 TABLE 10L Example data of certain oligonucleotides. Activity relative to WV-942 at 10 uM WV-942 0.8 1.8 1.2 WV-1709 7.1 10.7 6.5 WV-1710 3.4 5.1 3.9 WV-1711 3.6 4.9 3.1 WV-1712 2.1 3.7 2.6 WV-1713 1.8 2.9 1.9 WV-1714 6.5 8.8 8.5 WV-1715 1.8 3.1 2.7 WV-1716 1.7 2.9 2.0 WV-2444 18.5 22.2 23.8 WV-2445 14.2 17.2 15.6 WV-2528 27.0 28.8 32.7 WV-2529 2.7 4.8 5.1 WV-2530 15.5 17.6 21.2 Activity relative to WV-942 at 3 uM WV-942 0.7 1.7 0.6 WV-1709 10.9 19.5 12.2 WV-1710 3.6 8.3 4.3 WV-1711 3.6 8.1 4.6 WV-1712 3.0 6.7 5.8 WV-1713 2.0 5.3 0.9 WV-1714 7.5 13.8 7.8 WV-1715 2.6 5.8 3.6 WV-1716 3.2 6.1 3.1 WV-2444 30.3 41.9 39.7 WV-2445 23.4 32.3 30.2 WV-2528 56.3 66.3 74.4 WV-2529 7.5 15.0 10.0 WV-2530 25.2 38.4 37.8
Oligonucleotides were tested in vitro at 10 and 3 M. In this table, numbers represent skipping efficiency relative to WV-942 (ave); results from replicate experiments are shown.

(1219) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide, can be tested in vivo for capability to skip an exon in a tissue in a live animal; in some embodiments, a tissue is gastrocnemius, triceps, quadriceps, diaphragm, and/or heart. In some embodiments, a live animal is a mouse, rat, monkey, dog, or non-human primate. In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide, is capable of mediating skipping, e.g., of exon 23, 45, 51, 53, or any other DMD exon. Various DMD oligonucleotides were shown to mediate skipping of DMD exon 51 in a tissue in a non-human primate (NHP), wherein the tissue was gastrocnemius, triceps, quadriceps, diaphragm, or heart.

(1220) In some embodiments, the present disclosure pertains to methods of administering oligonucleotides, e.g., DMD oligonucleotides, wherein the timeline of pre-differentiation (of myoblast cells to myotubules) and treatment with the oligonucleotide are suitably altered. In some embodiments, in a test in vitro, an oligonucleotide, e.g., a DMD oligonucleotide to exon 51, was tested with treatment of 1 day or 4 day.

(1221) TABLE-US-00032 TABLE 11A Example data of certain oligonucleotides. Oligonucleotide Group A Group B Group C PMO 1.3 0.6 3.3 WV-3473 29.3 23.1 81.6
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. PMO is a Morpholino having the sequence of CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 3159).
Conditions for Groups A to C in Table 11A.

(1222) TABLE-US-00033 Group A Group B Group C Pre-differentiation 1 day 2 day 0 day.sup. ASO treatment 1 day 1 day 4 days Wash-out .sup.2 days .sup.2 days
Example 19 describes various timelines for experiments suitable for testing oligonucleotides, e.g., DMD oligonucleotides, e.g., in patient-derived myoblasts in vitro.

(1223) TABLE-US-00034 TABLE 11B Example data of certain oligonucleotides. Conc. (uM) WV-942 PMO 0.3 0.2 0.0 0.1 0.1 0.5 0.4 0.1 0.0 1 0.6 0.1 0.2 0.1 0.1 0.1 0.1 0.3 3 0.1 0.1 0.1 0.2 0.2 0.5 0.3 0.7 0.2 10 0.5 0.3 0.1 0.8 0.7 1.3 0.8 1.6 0.4 30 0.0 1.0 0.5 2.0 3.4 5.5 2.3 0.9 1.7 Conc. (uM) WV-3473 WV-3545 0.3 5.1 4.7 1.9 8.7 1.4 3.9 6.4 3.0 4.2 0.9 1.1 2.9 1 15.6 8.5 13.8 5.7 6.2 12.9 13.9 11.7 2.8 5.6 5.2 12.0 3 24.4 25.1 7.7 14.7 18.5 27.3 22.6 21.3 16.9 16.9 23.5 10 36.8 38.1 17.3 31.9 33.8 46.9 49.0 51.7 42.9 34.1 31.0 42.1 30 67.7 49.0 47.6 51.6 69.4 91.2 88.9 89.9 83.7 79.8 84.7 Conc. (uM) WV-3546 0.3 6.0 0.7 1.1 0.7 1.6 7.1 1 8.2 12.2 14.2 4.7 5.4 11.1 3 31.5 15.9 29.6 10 62.1 59.1 74.0 49.9 43.6 65.1 30 98.9 98.8 97.4 97.4 95.6 98.1
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. PMO is a control oligonucleotide which is a Morpholino corresponding to Eteplirsen. WV-942 is an oligonucleotide corresponding to Drisapersen. Oligonucleotides were delivered gymnotically.

(1224) TABLE-US-00035 TABLE 11C Example data of certain oligonucleotides. Conc. (uM) WV-942 PMO WV-3473 0.3 0.2 0.0 0.1 0.4 0.1 0.0 5.1 4.7 1.9 1 0.6 0.1 0.2 0.1 0.1 0.3 15.6 8.5 13.8 3 0.1 0.1 0.1 0.3 0.7 0.2 24.4 25.1 7.7 10 0.5 0.3 0.1 0.8 1.6 0.4 36.8 38.1 17.3 30 0.0 1.0 0.5 2.3 0.9 1.7 67.7 49.0 Conc. (uM) WV-3545 WV-3546 WV-3543 0.3 6.4 3.0 4.2 6.0 0.7 1.1 5.1 2.1 4.6 1 13.9 11.7 2.8 8.2 12.2 14.2 8.2 2.8 9.2 3 22.6 21.3 16.9 31.5 17.9 21.6 18.8 10 49.0 51.7 42.9 62.1 59.1 74.0 26.7 28.9 31.2 30 91.2 88.9 89.9 98.9 98.8 97.4 83.2 82.5 75.5 Conc. (uM) WV-3544 WV-3554 WV-4107 0.3 5.6 3.0 3.1 2.2 2.0 4.0 1.1 1.0 0.8 1 12.4 9.8 12.0 12.6 4.5 8.4 3.9 2.3 4.0 3 22.7 23.9 15.7 18.6 15.7 18.3 15.7 14.1 13.5 10 37.8 32.0 35.1 42.3 36.8 33.0 70.0 53.6 64.3 30 80.4 81.3 79.1 86.4 91.1 84.3 93.6 92.0 93.0
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. PMO is a control oligonucleotide which is a Morpholino corresponding to Eteplirsen. WV-942 is an oligonucleotide corresponding to Drisapersen. Oligonucleotides were delivered gymnotically.

(1225) In some embodiments, an oligonucleotide comprises a derivative of U. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises a derivative of U. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises a derivative of U and at least one chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises a derivative of U and at least one chirally controlled phosphorothioate internucleotidic linkage. In some embodiments, a derivative of U is BrU or Acet5U

(1226) ##STR00491##

(1227) In some embodiments, an oligonucleotide comprises BrU. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises BrU. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises BrU and at least one chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises BrU and at least one chirally controlled phosphorothioate internucleotidic linkage.

(1228) In some embodiments, an oligonucleotide comprises Acet5U. In some embodiments, Acet5U is also designated AcetU or acetU. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises Acet5U. In some embodiments, in an oligonucleotide, e.g., DMD oligonucleotide, any U or T can be optionally replaced by Acet5U (e.g., in a first wing, a core, a second wing, or anywhere in the oligonucleotide). In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD comprises an Acet5mU nucleoside unit, wherein the base is Acet5U and the sugar is the common natural RNA sugar wherein the 2-OH is replaced with 2-OMe. In some embodiments, an oligonucleotide comprises an Acet5fU nucleoside unit, wherein the base is Acet5U and the sugar is the common natural RNA sugar wherein the 2-OH is replaced with 2-F. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises Acet5U and at least one chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide capable of mediating skipping of an exon of DMD and comprises Acet5U and at least one chirally controlled phosphorothioate internucleotidic linkage.

(1229) As shown in Table 11D, Table 11E, and Table A1, certain oligonucleotides, e.g., DMD oligonucleotides, were designed and constructed comprising BrU or acet5U. In some oligonucleotides, the nucleoside at the 5 end comprises BrU or acet5U. In some embodiments, oligonucleotides comprise a BrfU nucleoside unit, wherein the base is BrU and the sugar is the common natural RNA sugar wherein the 2-OH is replaced with 2-F. In some oligonucleotides, the oligonucleotide comprises a BrdU nucleoside unit, wherein the base is BrU and the sugar is 2-deoxyribose (common natural DNA sugar). In some embodiments, any U or T can be replaced by BrU (e.g., in a first wing, a core, a second wing, or anywhere within an oligonucleotide). In some embodiments, in an oligonucleotide, e.g., a DMD oligonucleotide, any number of U or T can be replaced by BrU and/or Acet5U.

(1230) In some embodiments, an oligonucleotide comprises an acet5fU nucleoside unit, wherein the base is acet5U and the sugar is the common natural RNA sugar wherein the 2-OH is replaced with 2-F.

(1231) Table 11D shows data of various DMD oligonucleotides which mediate skipping of exon 51, including oligonucleotide WV-7410, which comprises BrfU, and WV-7413, which comprises acet5fU. Percentage was measured using RT-qPCR. Gymnotic delivery of 10 M and 3 M oligonucleotides in 48-50 patient derived myoblasts (4 days post-differentiation). The experiment was done in technical replicates.

(1232) TABLE-US-00036 TABLE 11D Example data of certain oligonucleotides. WV-3152 WV-3516 WV-7410 WV-7413 10 M 39 10 49 11 3 M 20 6 34 6
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. Approximate numbers are provided.
In some embodiments, the present disclosure provides oligonucleotides, e.g., various DMD oligonucleotides, that comprise BrdU at or near the center of the oligonucleotides (e.g., in a core region, middle region, etc.). In some embodiments, example such oligonucleotides include WV-2812, WV-2813, and WV-2814. Certain exon skipping data of these oligonucleotides were presented below.

(1233) TABLE-US-00037 TABLE 11E Example data of certain oligonucleotides. 10 uM 3 uM WV-1714 0.035 0.034 0.012 0.013 WV-2812 0.094 0.095 0.023 0.024 WV-942 0.004 0.004 0.001 0.001 WV-2814 0.004 0.005 0.002 0.002 WV-2813 0.041 0.042 0.017 0.017
Numbers represent skipping efficiency, wherein 1.000 would represent 100% skipping and 0.0 represents 0% efficiency. Approximate numbers are provided.

(1234) TABLE-US-00038 TABLE 11F Example data of certain oligonucleotides. 10 uM 3 uM WV-9738 44.7 44.0 46.1 45.4 26.6 25.9 25.6 24.4 WV-9739 51.8 49.9 53.2 50.9 32.3 35.4 31.0 33.2 WV-9740 49.9 48.8 47.8 46.1 32.5 30.3 29.0 29.6 WV-9741 36.1 37.8 35.0 35.6 23.5 22.3 21.4 24.6 WV-9742 53.4 54.8 59.1 56.8 41.7 40.4 37.6 40.3 WV-7410 64.8 63.9 65.4 67.0 45.1 43.5 43.9 40.6 WV-7410 66.0 67.2 64.7 64.5 44.9 40.3 33.7 31.7 WV-3152 47.0 45.7 47.1 45.0 28.3 30.2 25.3 22.6 WV-3516 12.5 12.5 9.7 10.4 5.0 4.9 5.2 4.6 MOCK 0.5 0.3 0.5 0.3 0.5 0.6 0.8 0.4 MOCK 0.6 0.4 0.5 0.5 0.6 0.6 0.3 0.4 MOCK 0.3 0.3 0.6 0.2 0.4 0.4 0.2 0.6
Additional DMD oligonucleotides for skipping Exon 51 were constructed. Various DMD oligonucleotides comprise BrU. In some cases, a BrU is attached to a sugar which is 2-F modified (BrfU). D48-50 myoblasts were dosed at 10 uM and 3 uM in differentiation media for 4 days. Percentage of skipping is shown, wherein 100 would represent 100% skipping and 0 would represent 0% skipping.

(1235) TABLE-US-00039 TABLE 11G Activity of certain oligonucleotides 10 3.3 1.1 10 3.3 1.1 WV- 20.8 9 4.1 WV- 36.9 10.4 4.7 3152 22 10 4.9 14522 27.4 10.4 4.2 17.3 9.3 3.2 21 12.6 5.6 21.3 7.2 4.4 26.5 10.4 5.7 WV- 27.4 13.2 12.7 WV- 27.2 8.1 6.2 15860 30.4 15.4 9 14523 28.3 8.5 4.9 33 14.2 6 18.4 9.1 3.6 33.4 16.9 5.9 18.7 9.6 4.4 WV- 26.6 9.2 5.6 Mock 0.21 15861 28.5 6.1 5.4 0.35 34.1 8.2 5.2 0.48 29.9 11.1 4 0.24 WV- 30.7 7.8 15862 33.3 7.2 21.9 15.1 6.8 26.4 13.2 7.2
Activity of various DMD exon 51 oligonucleotides was tested in vitro.
Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).
Amounts tested were: 10, 3.3 and 1.1 M.

(1236) TABLE-US-00040 TABLE 11H Activity of certain oligonucleotides 10 3.3 1.1 10 3.3 1.1 uM uM uM uM uM uM Mock 0.2 0.3 0.2 WV- 37.6 22.6 9 0.3 0.2 0.3 17861 38.8 22.5 8.9 0.2 0 0.2 40.7 24.4 13.2 0.2 0.6 0.2 41.7 25.4 11.6 WV- 3.1 1.6 0.7 WV- 38.4 18.9 8.1 7336 8.9 1.8 0.1 17862 34.1 19.6 9 5.4 1.4 0.9 34.8 26 10 4.9 1.5 0.7 36.1 21.4 9.5 WV- 32.4 26.5 7.5 WV- 32.7 18.2 9.2 3152 27.2 22.2 8.4 17863 35.1 18.9 9.3 28 14.5 7.6 34.8 18.2 8.6 26.8 14.8 7.3 30.7 17 9 WV- 43.3 25.7 10.2 WV- 37.3 23.6 11.7 15860 37.9 23.8 9.6 17864 41.4 23.3 10.6 38.4 24.5 11.2 39.9 20.6 17.5 42.4 21.9 11 38.8 21.7 10.2 WV- 42.3 26.7 16.3 WV- 35.9 16.5 9.3 17859 41.3 26 16.8 17865 34 16.7 7.5 39.9 22.9 15.5 34.4 17.5 11.9 48.6 23.6 14.9 34.1 17.8 9.8 WV- 38.1 19.3 11.7 WV- 48.7 28.4 17.7 17860 35.3 19.2 12 17866 43.3 28.6 13.1 41 28.2 16.4 44.5 24.8 15.4 40.4 21.9 11.1 45.1 30.5 16.3
Oligonucleotides for skipping DMD exon 51 were tested in vitro.
Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).
Concentrations of oligonucleotides used: 10, 3.3 and 1.1 uM.

(1237) TABLE-US-00041 TABLE 11I Activity of certain oligonucleotides 10 uM 3.3 uM Mock 0 0 0 0 0 0 0 0 WV- 15.9 7 20034 17.1 8.4 16.1 7.3 15.3 7.2 WV- 29.7 18.3 20037 27.2 17.5 26.6 19.4 29.2 18.4 WV- 9.6 4.9 20040 9.1 5.2 11.4 3.5 10.9 2.9 WV- 20.2 9.6 20043 20.4 9.8 18.9 9.8 21 10.4 WV- 28.5 14.7 20046 29.8 14.2 29.2 15.8 26.6 14.5 WV- 20.9 11.6 20049 18.6 12.2 18.4 11.7 WV- 28.8 18.8 20052 30.1 18.6 29.6 20.1 WV- 26.8 17 20055 25.3 16.6 24.1 17 WV- 14.6 4.8 20058 12 3.7 12.6 3.5 WV- 35.8 26.5 20061 39.3 24.2 39.9 22.8 WV- 26.5 17.6 20064 24.5 16.4 27.5 17.1 WV- 15.7 8.3 20067 16.8 9.3 17.3 8.6 16.3 8.7 WV- 41.3 26.4 20070 31.7 22.3 39.7 27.2 38.4 26.9 WV- 30.9 21.1 20073 26.9 17.9 31.1 20.2 30.7 22.2 WV- 23.2 16.8 20076 18.9 11.4 21.8 16.9 22.8 15.8 WV- 35.7 24.8 3152 33.5 24.9 32.1 25.3 WV- 41.9 27.5 15860 43.6 30.7 42.4 30
Oligonucleotides for skipping DMD exon 51 were tested in vitro.
Numbers indicate amount of skipping DMD exon 23 (as a percentage of total mRNA, where 100 would represent 100% skipped).
Concentrations of oligonucleotides used: 10 and 3.3 uM.

(1238) TABLE-US-00042 TABLE 11J Activity of certain oligonucleotides WV-3152 19 20 12 14 WV-15860 29 31 26 23 WV-20140 1 1 1 1 WV-20139 3 3 2 2 WV-20138 2 3 WV-20137 4 5 WV-20136 WV-20135 5 5 5 5 WV-20134 5 6 5 4 WV-20133 17 17 13 13 WV-20132 8 8 6 6 WV-20131 14 16 12 12 WV-20130 10 9 8 8 WV-20129 12 14 11 11 WV-20128 9 9 8 8 WV-20127 8 8 WV-20126 7 8 8 7 WV-20125 8 8 8 8 WV-20124 22 21 21 21 WV-20123 13 13 14 12 WV-20122 11 12 12 11 WV-20121 21 22 22 21 WV-20120 28 30 32 33 WV-20119 52 50 WV-20118 39 37 27 26 WV-20117 18 17 15 18 WV-20116 20 20 17 17 WV-20115 8 8 8 6 WV-20114 19 20 15 14 WV-20113 20 18 17 15 WV-20112 16 15 12 12 WV-20111 31 30 33 31 WV-20110 14 14 14 12 WV-20109 20 21 25 24 WV-20108 27 25 22 22 WV-20107 20 19 16 14 WV-20106 44 42 34 37 WV-20105 23 22 18 18 WV-20104 41 40 33 28 WV-20103 48 52 53 53 WV-20102 54 52 55 59 WV-20101 38 39 38 43 WV-20100 52 51 48 50 WV-20099 53 51 47 48 WV-20098 46 44 45 46 WV-20097 47 46 51 48 WV-20096 45 41 42 43 WV-20095 43 41 50 47 WV-20094 55 50 57 55 WV-20093 35 34 35 38 WV-20092 25 26 25 25 WV-20091 28 27 30 32 WV-20090 21 19 22 22 WV-20089 8 7 8 9 WV-20088 22 21 26 25 WV-20087 28 28 33 32 WV-20086 25 25 27 26 WV-20085 33 31 30 31 WV-20084 21 22 21 21 WV-20083 21 21 19 17 WV-20082 42 37 32 30 WV-20081 41 41 30 30 WV-20080 49 44 26 25 WV-20079 42 38 53 51 WV-20078 27 28 36 35 WV-20077 10 10 10 10 WV-20076 45 45 45 41 WV-20075 40 31 37 42 WV-20074 55 57 53 56 WV-20073 51 55 51 50 WV-20072 41 36 37 36 WV-20071 42 40 44 46 WV-20070 18 18 25 25 WV-20069 11 11 10 9 WV-20068 20 17 20 18 WV-20067 12 9 11 11 WV-20066 12 11 13 12 WV-20065 16 15 16 14 WV-20064 37 35 37 36 WV-20063 19 24 22 WV-20062 6 6 7 7 WV-20061 24 23 26 24 WV-20060 16 17 16 17 WV-20059 55 42 62 67 WV-20058 28 30 33 33 WV-20057 37 38 37 34 WV-20056 35 34 33 35 WV-20055 40 40 WV-20054 25 25 35 36 WV-20053 43 45 46 46 WV-20052 47 47 53 46 WV-20051 30 33 30 30 WV-20050 29 28 28 26 WV-20049 41 41 38 38 WV-20049 24 23 22 21
Oligonucleotides for skipping DMD exon 51 were tested in vitro.
Oligonucleotides were dosed 4d at 10 uM.
Numbers indicate amount of skipping DMD exon 51 (as a percentage of total mRNA, where 100 would represent 100% skipped).
Example Dystrophin Oligonucleotides and Compositions Which Target Exon 52

(1239) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 52 and/or mediating skipping of exon 52 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 52 oligos include: WV-13733, WV-13734, WV-13735, WV-13736, WV-13737, WV-13738, WV-13739, WV-13740, WV-13741, WV-13742, WV-13743, and WV-13744, WV-13782, and WV-13783, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1240) TABLE-US-00043 TABLE 12A Example data of certain oligonucleotides. WV-13733 0.3 0.2 WV-13734 0.0 0.0 WV-13735 1.6 0.3 WV-13736 3.9 1.3 WV-13737 0.7 0.4 WV-13738 0.0 0.0 WV-13739 28.3 29.3 WV-13740 29.9 33.3 WV-13741 1.6 1.6 WV-13742 12.9 14.1 WV-13743 0.9 1.0 WV-13744 0.6 0.7 WV-13782 0.1 0.1 WV-13783 0.8 0.0 Mock 0.0 0.0 Mock 0.1 0.1
Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 52.
Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Exon 53

(1241) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating skipping of exon 53 in DMD (e.g., of mouse, human, etc.).

(1242) In some embodiments, an oligonucleotide, e.g., a human DMD exon 53 skipping oligonucleotide can be tested in a mouse which has been modified to comprise a DMD gene comprising the human exon 53 sequence.

(1243) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide, is capable of mediating skipping of exon 53. Non-limiting examples of such oligonucleotides include: WV-10439, WV-10440, WV-10441, WV-10442, WV-10443, WV-10444, WV-10445, WV-10446, WV-10447, WV-10448, WV-10449, WV-10450, WV-10451, WV-10452, WV-10453, WV-10454, WV-10455, WV-10456, WV-10457, WV-10458, WV-10459, WV-10460, WV-10461, WV-10462, WV-10463, WV-10464, WV-10465, WV-10466, WV-10467, WV-10468, WV-10469, WV-10470, WV-10487, WV-10488, WV-10489, WV-10490, WV-10491, WV-10492, WV-10493, WV-10494, WV-10495, WV-10496, WV-10497, WV-10498, WV-10499, WV-10500, WV-10501, WV-10502, WV-10503, WV-10504, WV-10505, WV-10506, WV-10507, WV-10508, WV-10509, WV-10510, WV-10511, WV-10512, WV-10513, WV-10514, WV-10515, WV-10516, WV-10517, WV-10518, WV-10519, WV-10520, WV-10521, WV-10522, WV-10523, WV-10524, WV-10525, WV-10526, WV-10527, WV-10528, WV-10529, WV-10530, WV-10531, WV-10532, WV-10533, WV-10534, WV-10535, WV-10536, WV-10537, WV-10538, WV-10539, WV-10540, WV-10541, WV-10542, WV-10543, WV-10544, WV-10545, WV-10546, WV-10547, WV-10548, WV-10549, WV-10550, WV-10551, WV-10552, WV-10553, WV-10554, WV-10555, WV-10556, WV-10557, WV-10558, WV-10559, WV-10560, WV-10561, WV-10562, WV-10563, WV-10564, WV-10565, WV-10566, WV-10567, WV-10568, WV-10569, WV-10570, WV-10571, WV-10572, WV-10573, WV-10574, WV-10575, WV-10576, WV-10577, WV-10578, WV-10579, WV-10580, WV-10581, WV-10582, WV-10583, WV-10584, WV-10585, WV-10586, WV-10587, WV-10588, WV-10589, WV-10590, WV-10591, WV-10592, WV-10593, WV-10594, WV-10595, WV-10596, WV-10597, WV-10598, WV-10599, WV-10600, WV-10601, WV-10602, WV-10603, WV-10604, WV-10605, WV-10606, WV-10607, WV-10608, WV-10609, WV-10610, WV-10611, WV-10612, WV-10613, WV-10614, WV-10615, WV-10616, WV-10617, WV-10618, WV-10619, WV-10620, WV-10621, WV-10622, WV-10623, WV-10624, WV-10625, WV-10626, WV-10627, WV-10628, WV-10629, WV-10630, WV-10670, WV-10671, WV-10672, WV-11340, WV-11341, WV-11342, WV-11544, WV-11545, WV-11546, WV-11547, WV-13835, WV-13864, WV-14344, WV-4698, WV-4699, WV-4700, WV-4701, WV-4702, WV-4703, WV-4704, WV-4705, WV-4706, WV-4707, WV-4708, WV-4709, WV-4710, WV-4711, WV-4712, WV-4713, WV-4714, WV-4715, WV-4716, WV-4717, WV-4718, WV-4719, WV-4720, WV-4721, WV-4722, WV-4723, WV-4724, WV-4725, WV-4726, WV-4727, WV-4728, WV-4729, WV-4730, WV-4731, WV-4732, WV-4733, WV-4734, WV-4735, WV-4736, WV-4737, WV-4738, WV-4739, WV-4740, WV-4741, WV-4742, WV-4743, WV-4744, WV-4745, WV-4746, WV-4747, WV-4748, WV-4749, WV-4750, WV-4751, WV-4752, WV-4753, WV-4754, WV-4755, WV-4756, WV-4757, WV-4758, WV-4759, WV-4760, WV-4761, WV-4762, WV-4763, WV-4764, WV-4765, WV-4766, WV-4767, WV-4768, WV-4769, WV-4770, WV-4771, WV-4772, WV-4773, WV-4774, WV-4775, WV-4776, WV-4777, WV-4778, WV-4779, WV-4780, WV-4781, WV-4782, WV-4783, WV-4784, WV-4785, WV-4786, WV-4787, WV-4788, WV-4789, WV-4790, WV-4791, WV-4792, WV-4793, WV-9067, WV-9068, WV-9069, WV-9070, WV-9071, WV-9072, WV-9073, WV-9074, WV-9075, WV-9076, WV-9077, WV-9078, WV-9079, WV-9080, WV-9081, WV-9082, WV-9083, WV-9084, WV-9085, WV-9086, WV-9087, WV-9088, WV-9089, WV-9090, WV-9091, WV-9092, WV-9093, WV-9094, WV-9095, WV-9096, WV-9097, WV-9098, WV-9099, WV-9100, WV-9101, WV-9102, WV-9103, WV-9104, WV-9105, WV-9106, WV-9107, WV-9108, WV-9109, WV-9110, WV-9111, WV-9112, WV-9113, WV-9114, WV-9115, WV-9116, WV-9117, WV-9118, WV-9119, WV-9120, WV-9121, WV-9122, WV-9123, WV-9124, WV-9125, WV-9126, WV-9127, WV-9128, WV-9129, WV-9130, WV-9131, WV-9132, WV-9133, WV-9134, WV-9135, WV-9136, WV-9137, WV-9138, WV-9139, WV-9140, WV-9141, WV-9142, WV-9143, WV-9144, WV-9145, WV-9146, WV-9147, WV-9148, WV-9149, WV-9150, WV-9151, WV-9152, WV-9153, WV-9154, WV-9155, WV-9156, WV-9157, WV-9158, WV-9159, WV-9160, WV-9161, WV-9162, WV-9422, WV-9423, WV-9424, WV-9425, WV-9426, WV-9427, WV-9428, WV-9429, WV-9511, WV-9512, WV-9513, WV-9514, WV-9515, WV-9516, WV-9517, WV-9518, WV-9519, WV-9520, WV-9521, WV-9522, WV-9523, WV-9524, WV-9525, WV-9534, WV-9535, WV-9536, WV-9537, WV-9538, WV-9539, WV-9680, WV-9681, WV-9682, WV-9683, WV-9684, WV-9685, WV-9686, WV-9687, WV-9688, WV-9689, WV-9690, WV-9691, WV-9699, WV-9700, WV-9701, WV-9702, WV-9703, WV-9704, WV-9709, WV-9710, WV-9711, WV-9712, WV-9713, WV-9714, WV-9715, WV-9743, WV-9744, WV-9745, WV-9746, WV-9747, WV-9748, WV-9749, WV-9750, WV-9751, WV-9752, WV-9753, WV-9754, WV-9755, WV-9756, WV-9757, WV-9758, WV-9759, WV-9760, WV-9761, WV-9897, WV-9898, WV-9899, WV-9900, WV-9901, WV-9902, WV-9903, WV-9904, WV-9905, WV-9906, WV-9907, WV-9908, WV-9909, WV-9910, WV-9911, WV-9912, WV-9913, WV-9914, WV-7436, WV-7437, WV-7438, WV-7439, WV-7440, WV-7441, WV-7442, WV-7443, WV-7444, WV-7445, WV-7446, WV-7447, WV-7448, WV-7449, WV-7450, WV-7451, WV-7452, WV-7453, WV-7454, WV-7455, and WV-7456, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1244) Additional examples of such DMD oligonucleotides include: WV-9422, WV-9425, WV-9426, WV-9517, WV-9519, WV-9521, WV-9522, WV-9524, WV-9710, WV-9714, WV-9715, WV-9743, WV-9744, WV-9745, WV-9746, WV-9747, WV-9748, WV-9749, WV-9750, WV-9751, WV-9756, WV-9757, WV-9758, WV-9759, WV-9760, WV-9761, WV-9897, WV-9898, WV-9899, WV-9900, WV-9906, and WV-9912, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1245) Non-limiting examples of such DMD oligonucleotides also include: WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, WV-12876, WV-12877, WV-12878, WV-12879, WV-12880, WV-12881, WV-12882, and WV-12883, and other DMD oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1246) Results of various experiments for skipping Dystrophin exon 53 are described in the present disclosure. For example, data from a sequence identification screen are shown below, in Table 13A.

(1247) TABLE-US-00044 TABLE 13A Example data of certain oligonucleotides. Oligonucleotide Replicate 1 Replicate 2 WV-4698 1.9 2.1 WV-4699 2.0 2.2 WV-4700 2.8 3.0 WV-4701 3.7 2.9 WV-4702 2.9 2.7 WV-4703 1.8 2.4 WV-4704 3.2 3.4 WV-4705 3.7 4.3 WV-4706 2.6 2.6 WV-4707 3.2 3.6 WV-4708 4.8 6.0 WV-4709 6.6 5.2 WV-4710 3.9 4.6 WV-4711 5.4 6.7 WV-4712 5.3 6.4 WV-4713 5.8 8.0 WV-4714 2.9 3.6 WV-4715 3.3 4.3 WV-4716 3.8 4.3 WV-4717 6.8 7.0 WV-4718 4.3 5.0 WV-4719 5.5 6.0 WV-4720 7.7 8.6 WV-4721 2.7 3.8 WV-4722 3.8 4.6 WV-4723 3.4 5.6 WV-4724 3.5 4.7 WV-4725 4.9 6.3 WV-4726 4.2 4.4 WV-4727 2.7 4.9 WV-4728 2.6 5.6 WV-4729 3.9 4.1 WV-4730 2.4 3.3 WV-4731 1.8 2.5 WV-4732 1.8 2.3 WV-4733 2.3 2.1 WV-4734 2.0 2.0 WV-4735 2.5 2.7 WV-4736 2.7 3.0 WV-4737 3.2 3.1 WV-4738 3.1 3.5 WV-4739 2.6 2.4 WV-4740 4.4 3.6 WV-4741 3.7 4.1 WV-4742 4.5 4.9 WV-4743 5.0 5.2 WV-4744 3.6 4.7 WV-4745 4.1 0.0 WV-4746 2.9 2.0 WV-4747 2.5 3.5 WV-4748 2.1 1.7 WV-4749 2.4 2.4 WV-4750 2.3 2.9 WV-4751 1.9 2.5 WV-4752 2.2 1.6 WV-4753 1.6 2.0 WV-4754 1.7 2.0 WV-4755 1.7 1.9 WV-4756 1.7 1.5 WV-4757 1.6 1.9 WV-4758 1.6 2.0 WV-4759 1.6 1.6 WV-4760 1.8 1.8 WV-4761 1.9 1.6 WV-4762 1.2 1.3 WV-4763 0.9 2.0 WV-4764 3.0 2.7 WV-4765 3.4 3.2 WV-4766 2.5 2.3 WV-4767 2.5 2.7 WV-4768 2.3 2.7 WV-4769 2.4 2.4 WV-4770 2.8 2.8 WV-4771 2.3 2.9 WV-4772 4.0 2.5 WV-4773 3.2 1.8 WV-4774 3.0 2.3 WV-4775 4.4 3.3 WV-4776 3.1 3.8 WV-4777 4.5 2.1 WV-4778 0.0 2.0 WV-4779 2.8 3.4 WV-4780 3.2 3.5 WV-4781 2.9 3.2 WV-4782 1.8 2.9 WV-4783 2.1 2.6 WV-4784 2.4 2.4 WV-4785 3.4 3.6 WV-4786 1.8 1.6 WV-4787 2.9 2.7 WV-4788 2.8 3.1 WV-4789 4.3 4.0 WV-4790 3.9 2.6 WV-4791 2.2 2.2 WV-4792 2.5 3.2 WV-4793 2.4 2.6 Mock 1.3 1.6
Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 53 in vitro in Delta 52 human myoblast cells. Oligonucleotides tested were 6-8-6 gapmers (2-F-2-OMe-2-F), wherein each internucleotidic linkage is a stereorandom phosphorothioate. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1248) A number of oligonucleotides were generated and tested for efficacy in skipping DMD Exon 53 in vitro in human patient-derived myoblast cells; certain results are shown below in Tables 13B to 21 (A and B). Oligonucleotides were used at concentrations of 3 and 10 uM, in two replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, wherein 0.0 would indicate no skipping, and 100.0 would indicate 100% skipping. Several base sequences were tested in combination with a variety of chemical formats. For example, in some embodiments, a base sequence is GUACUUCAUCCCACUGAUUC (SEQ ID NO: 3160), GUGUUCTTGTACTTCAUCCC (SEQ ID NO: 3161), UUCUGAAGGTGTTCUUGUAC (SEQ ID NO: 3162), or CUCCGGTTCTGAAGGUGUUC (SEQ ID NO: 3163), wherein U is optionally substituted with T and vice versa. Various chemical formats were utilized, including, e.g., gapmers (for example, 6-8-6 wing-core-wing gapmers). In some embodiments, both wings are 2-F, while the core was all 2-MOE, alternating 2-MOE/2-OMe, alternating 2-OMe/2-MOE, alternating 2-MOE/2-F, alternating 2-F/2-MOE, alternating 2-OMe/2-F, and alternating 2-F/2-OMe, etc. In some embodiments, the first wing was 2-MOE or 2-OMe and the second wing was 2-F (a type of asymmetrical gapmers). In some embodiments, each internucleotidic linkage is a stereorandom phosphorothioate. In some embodiments, some alternating phosphorothioate linkages are replaced by phosphodiester linkages. In some embodiments, 5-methyl 2-MOE C is used. Descriptions of certain oligonucleotides tested are provided in Table A1.

(1249) TABLE-US-00045 TABLE 13B Example data of certain oligonucleotides. Replicate 1 Replicate 2 Oligonucleotide 10 uM 3 uM 10 uM 3 uM WV-9067 6.6 1.9 1.8 WV-9068 6.5 1.5 1.6 WV-9069 6.9 1.8 1.7 1.5 WV-9070 2.9 3.2 2.6 1.9 WV-9071 2.9 1.9 2.0 1.4 WV-9072 9.6 2.4 2.4 1.5 WV-9073 8.6 3.3 2.7 2.1 WV-9074 8.3 2.4 2.5 1.9 WV-9075 7.0 2.1 2.1 2.0 WV-9076 9.6 3.0 3.1 2.0 WV-9077 6.3 1.7 2.0 1.5 WV-9078 6.1 2.3 2.2 1.9 WV-9079 10.0 3.9 3.6 2.3 WV-9080 7.6 3.1 2.8 2.6 WV-9081 5.7 2.2 1.9 1.6 WV-9082 11.2 6.1 6.4 3.2 WV-9083 6.0 1.9 2.1 1.6 WV-9084 6.6 2.4 2.9 2.1 WV-9085 0.0 7.5 7.6 3.4 WV-9086 7.5 3.4 3.1 2.0 WV-9087 7.1 2.4 2.1 1.7 WV-9088 9.0 3.0 2.6 1.6 WV-9089 8.2 2.5 2.3 1.9 WV-9090 0.0 2.3 2.2 1.6 WV-9091 9.9 4.7 3.7 3.2 WV-9092 9.0 3.4 3.4 2.0 WV-9093 8.7 2.9 3.2 2.0 WV-9094 11.9 6.0 5.2 3.1 WV-9095 7.5 3.4 2.6 2.5 WV-9096 10.1 4.0 4.0 2.9 WV-9097 10.7 5.7 4.5 2.8 WV-9098 8.5 3.6 2.9 2.3 WV-9099 8.1 2.9 2.4 2.4 WV-9100 12.7 6.0 4.7 2.9 WV-9101 7.6 2.9 3.1 2.0 WV-9102 9.9 4.0 3.6 2.5 WV-9103 12.6 6.9 6.1 3.0 WV-9104 11.3 3.7 4.3 2.1 WV-9105 6.5 2.9 2.3 2.4 WV-9106 15.1 7.7 5.5 4.3 WV-9107 7.8 2.5 2.2 2.6 WV-9108 11.3 3.3 3.5 2.2 WV-9109 16.1 10.6 8.9 4.1 WV-9110 8.8 3.5 3.4 1.7 WV-9111 7.3 3.4 2.5 1.7 WV-9112 11.5 4.6 3.4 2.2 WV-9113 10.6 4.2 3.1 2.3 WV-9114 10.8 4.9 4.1 2.6 WV-9115 8.4 0.0 2.5 2.1 WV-9116 7.5 0.0 1.6 1.8 WV-9117 6.8 0.0 2.0 1.5 WV-9118 9.3 0.0 2.7 2.1 WV-9119 7.2 0.6 2.0 2.0 WV-9120 8.5 6.1 2.5 2.0 WV-9121 11.8 5.7 3.9 2.5 WV-9122 8.6 4.0 2.4 2.4 WV-9123 10.7 5.2 2.0 2.0 WV-9124 11.0 5.3 3.6 3.2 WV-9125 8.7 3.5 2.3 2.2 WV-9126 10.5 3.4 3.4 2.4 WV-9127 8.5 3.4 2.7 2.5 WV-9128 8.2 2.9 2.0 2.2 WV-9129 7.5 2.6 1.6 1.7 WV-9130 12.6 0.0 5.4 2.7 WV-9131 7.6 2.3 2.2 1.8 WV-9132 8.4 0.7 3.4 2.3 WV-9133 16.2 7.0 6.9 3.2 WV-9134 8.5 3.9 3.0 1.9 WV-9135 12.5 2.8 2.9 1.7 WV-9136 8.7 4.1 3.1 2.2 WV-9137 7.5 2.5 1.7 1.6 WV-9138 7.2 2.7 2.1 1.7 WV-9139 9.3 5.3 5.1 2.8 WV-9140 8.0 3.1 2.5 2.1 WV-9141 7.7 3.3 2.9 1.8 WV-9142 11.9 6.4 6.0 3.2 WV-9143 7.0 3.2 3.9 1.8 WV-9144 9.8 4.0 3.6 2.7 WV-9145 13.0 6.6 5.3 2.6 WV-9146 7.9 3.7 3.4 1.9 WV-9147 8.2 3.9 3.1 2.0 WV-9148 15.0 8.8 6.4 3.3 WV-9149 6.9 2.9 2.3 3.1 WV-9150 10.8 6.9 5.6 1.9 WV-9151 12.9 7.2 5.1 2.7 WV-9152 8.4 3.4 2.6 1.5 WV-9153 7.2 3.9 2.9 1.7 WV-9154 21.5 14.1 12.4 4.3 WV-9155 6.9 3.3 2.5 1.6 WV-9156 11.0 6.4 4.9 2.4 WV-9157 16.7 10.5 9.7 3.9 WV-9158 7.7 3.7 2.3 1.7 WV-9159 7.7 3.1 3.3 1.5 WV-9160 8.0 3.1 2.8 1.8 WV-9161 8.4 4.5 3.2 2.2 WV-9162 8.9 4.5 4.7 2.2 Mock 2.4 Mock 2.1 WV-9746 2.5 2.5 4.6 3.4 WV-9747 3.0 3.1 5.5 4.8 WV-9748 4.9 2.5 4.3 4.0 WV-9749 2.9 2.7 4.5 4.1 WV-9750 3.2 2.5 4.4 3.8 WV-9751 3.5 2.7 4.7 4.8 WV-9758 1.7 1.9 2.1 3.5 WV-9759 2.6 3.6 2.8 6.1 WV-9760 3.1 3.9 3.4 4.8 WV-9761 3.0 4.8 4.6 7.2 WV-9756 3.9 4.4 5.3 8.4 WV-9757 3.7 4.3 6.8 8.1 WV-9517 3.3 2.7 7.1 5.3 WV-9519 2.4 2.1 5.1 4.6 WV-9521 2.4 2.5 6.3 4.9 WV-9522 2.6 2.3 5.8 4.3 WV-9715 4.6 5.7 10.5 4.2 WV-9714 4.5 3.4 9.0 8.5 WV-9422 2.1 2.0 6.2 4.3 WV-9743 4.1 2.4 7.3 6.2 WV-9744 3.4 1.9 4.4 5.1 WV-9745 2.7 2.4 5.6 6.2 Mock 2.4 1.8 1.7 2.5
Efficacy of DMD Exon 53 skipping of various DMD oligonucleotides in vitro. Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency. Results from replicate experiments are shown.

(1250) TABLE-US-00046 TABLE 14 Example data of certain oligonucleotides. 3 uM-R1 3 uM-R2 10 uM-R1 10 uM-R2 WV-9746 2.5 2.5 4.6 3.4 WV-9747 3.0 3.1 5.5 4.8 WV-9748 4.9 2.5 4.3 4.0 WV-9749 2.9 2.7 4.5 4.1 WV-9750 3.2 2.5 4.4 3.8 WV-9751 3.5 2.7 4.7 4.8 WV-9758 1.7 1.9 2.1 3.5 WV-9759 2.6 3.6 2.8 6.1 WV-9760 3.1 3.9 3.4 4.8 WV-9761 3.0 4.8 4.6 7.2 WV-9756 3.9 4.4 5.3 8.4 WV-9757 3.7 4.3 6.8 8.1 WV-9517 3.3 2.7 7.1 5.3 WV-9519 2.4 2.1 5.1 4.6 WV-9521 2.4 2.5 6.3 4.9 WV-9522 2.6 2.3 5.8 4.3 WV-9715 4.6 5.7 10.5 4.2 WV-9714 4.5 3.4 9.0 8.5 WV-9422 2.1 2.0 6.2 4.3 WV-9743 4.1 2.4 7.3 6.2 WV-9744 3.4 1.9 4.4 5.1 WV-9745 2.7 2.4 5.6 6.2 Mock 2.4 1.8 1.7 2.5
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments (R1 and R2) are shown.

(1251) TABLE-US-00047 TABLE 15 Example data of certain oligonucleotides. 10 uM 3 uM WV-9897 7.4 4.8 WV-9898 11.8 4.6 WV-9899 10.1 4.1 WV-9900 10.3 4.7 WV-9901 5.7 2.5 WV-9902 8.8 3.5 WV-9903 7.3 3.4 WV-9904 6.9 3.0 WV-9905 6.7 3.1 WV-9906 12.1 5.0 WV-9907 11.1 3.8 WV-9908 12.6 5.1 WV-9909 11.3 3.9 WV-9910 9.8 4.3 WV-9911 3.5 4.0 WV-9912 11.3 4.7 WV-9913 10.3 3.9 WV-9914 9.4 2.8 WV-9747 7.6 3.4 WV-9749 6.4 3.6 WV-9750 6.0 3.5 WV-9758 3.5 2.5 WV-9517 9.6 4.1 Mock 2.5 2.6
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency.

(1252) Additional oligonucleotides were generated and tested for skipping DMD exon 53 in vitro in cells. Certain data are shown below in Table 16. Oligonucleotides were used at concentrations of 3 and 10 uM, in two replicates. Numbers indicate the percentage of skipping of DMD exon 53. As shown, oligonucleotides can have different base sequences in combination with a variety of chemical formats. In some embodiments, oligonucleotides tested were 20-mers, each having a gapmer format of wing-core-wing, wherein each wing was 2-F, and the core was 2-OMe or a mixture of 2-OMe and 2-F. In some embodiments, each internucleotidic linkage was a chirally controlled phosphorothioate internucleotidic linkage in Sp configuration. In some embodiments, oligonucleotides comprise one or more natural phosphate linkages. In some embodiments, oligonucleotides of the present disclosure comprise one or more 5-methyl 2-F C (5MSfC,

(1253) ##STR00492##
nucleoside is

(1254) ##STR00493##
wherein BA is nucleobase C, R.sup.2s is F).

(1255) TABLE-US-00048 TABLE 16 Example data of certain oligonucleotides. Group A (3 uM) Group B (10 uM) WV-9746 8.0 7.5 13.7 7.5 WV-9747 10.2 9.3 17.4 9.3 WV-9748 8.8 8.2 14.1 8.2 WV-9749 9.9 8.7 15.8 8.7 WV-9750 10.0 9.3 17.3 9.3 WV-9751 9.3 8.4 14.5 8.4 WV-9758 6.9 6.1 8.8 6.1 WV-9759 7.5 7.7 11.3 7.7 WV-9760 8.1 7.3 10.2 7.3 WV-9761 7.3 8.2 12.7 8.2 WV-9756 10.9 10.3 20.2 10.3 WV-9757 22.7 10.1 32.1 10.1 WV-9517 10.3 9.2 20.1 9.2 WV-9519 8.8 8.1 16.2 8.1 WV-9521 9.2 8.0 16.0 8.0 WV-9522 9.5 8.8 17.7 8.8 WV-9715 14.3 12.3 26.9 12.3 WV-9714 13.2 11.3 23.7 11.3 WV-9422 8.3 7.3 16.6 7.3 WV-9743 9.8 7.8 20.1 7.8 WV-9744 7.6 6.7 12.9 6.7 WV-9745 9.6 7.4 17.0 7.4 Mock 4.7 4.9 5.2
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1256) A number of DMD oligonucleotides were also designed, constructed and tested for efficacy in skipping DMD Exon 53 in vitro in differentiated myoblast cells. Certain data are shown below in Table 17. Oligonucleotides were delivered gymnotically at concentrations of 3 and 10 M, in two biological replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, as determined by RT-qPCR.

(1257) TABLE-US-00049 TABLE 17 Example data of certain oligonucleotides. 3 uM-R1 3 uM-R2 10 uM-R1 10 uM-R2 WV-9422 2.1 2.0 6.2 4.3 WV-9743 4.1 2.4 7.3 6.2 WV-9744 3.4 1.9 4.4 5.1 WV-9745 2.7 2.4 5.6 6.2 Mock 2.4 1.8 1.7 2.5
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments (R1 and R2) are shown.

(1258) A number of oligonucleotides were designed, constructed and tested for efficacy in skipping DMD Exon 53 in vitro in 52 differentiated myoblast cells. Certain data were shown below in Table 18. In an example procedure, cells were pre-differentiated for 4 days and oligonucleotides were delivered gymnotically for 4 days. Differentiation medium was DMEM, 2% horse serum and 10 g/ml insulin. In some embodiments, with certain oligonucleotides, without pre-differentiating these cells, skipping efficiency was relatively low. Oligonucleotides were delivered gymnotically at concentrations of 1, 3 and 10 M, in biological replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, as determined by RT-qPCR. PMO53 is an oligonucleotide also designated as WV-13405, HumDMDEx53, or PMO (in DMD exon 53 experiments), or PMO SR, which has a base sequence of GTTGCCTCCGGTTCTGAAGGTGTTC (SEQ ID NO: 3164) and is fully PMO (Morpholino).

(1259) - indicates that no data were available for that particular sample.

(1260) TABLE-US-00050 TABLE 18 Example data of certain oligonucleotides. 30 uM- 30 uM- 10 uM- 10 uM- 3 uM- 3 uM- 1 uM- 1 uM- R1 R2 R1 R2 R1 R2 R1 R2 WV-9714 52.1 31.0 25.0 21.7 7.9 9.2 WV-9715 12.6 7.3 11.1 8.7 WV-9517 20.5 20.4 7.3 6.9 WV-9519 39.0 30.5 15.1 13.3 5.3 6.6 WV-9521 43.2 10.2 16.9 15.1 5.1 5.2 WV-9747 83.0 87.5 50.7 46.6 17.0 19.5 6.4 6.2 WV-9748 66.4 68.2 42.9 33.2 14.5 10.2 4.8 3.9 WV-9749 76.8 80.2 39.2 35.4 18.5 13.0 5.7 23.5 WV-9897 26.0 25.3 8.3 8.4 WV-9898 22.8 23.6 8.5 7.9 WV-9900 46.7 45.7 25.5 21.8 7.4 7.9 WV-9899 28.7 27.2 26.1 8.8 8.8 WV-9906 37.9 9.7 9.8 WV-9912 22.5 8.8 9.7 WV-9524 14.6 32.9 15.2 14.5 5.4 6.9 PMO53 112.8 105.4 53.7 49.3 20.4 19.9 6.9 10.4 Mock 2.2 1.7 2.2 1.5 1.6 1.8 2.0 2.0
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping relative to control and 0.0 would represent 0% efficiency; results from replicate experiments (R1 and R2) are shown.

(1261) A number of DMD oligonucleotides were designed, constructed and tested for efficacy in skipping DMD Exon 53 in vitro in 45-52 differentiated myoblast cell. Certain results, normalized to SFSR9, are shown below in Table 19. Oligonucleotides were delivered gymnotically at concentrations of 1, 3 and 10 M, in biological replicates (R1 and R2). Numbers indicate the percentage of skipping of DMD exon 53, as determined by RT-qPCR.

(1262) TABLE-US-00051 TABLE 19 Example data of certain oligonucleotides. 10 uM- 10 uM- 3 uM- 3 uM- 1 uM- 1 uM- R1 R2 R1 R2 R1 R2 MOCK 0.8 0.8 0.8 0.8 0.9 0.9 MOCK 0.7 0.7 0.8 0.8 0.8 0.8 PMO 18.0 18.0 5.6 5.7 3.8 4.0 PMO 19.3 17.9 9.6 9.4 3.1 3.1 WV-9517 39.4 42.3 16.0 16.1 5.3 5.2 WV-9517 43.8 42.9 18.5 17.5 5.5 5.7 WV-9519 33.7 28.5 14.3 13.3 4.5 4.5 WV-9519 27.6 27.9 12.4 11.3 4.1 4.1 WV-9897 30.8 31.1 11.7 12.5 3.9 3.8 WV-9897 32.3 30.7 12.0 11.9 4.6 4.7 WV-9714 46.8 42.8 21.5 20.6 4.5 4.1 WV-9714 46.5 48.1 25.4 25.6 4.2 2.9 WV-9747 31.1 31.8 12.0 12.5 4.7 4.7 WV-9747 27.6 28.0 10.5 11.1 3.5 3.7 WV-9748 21.7 21.7 7.9 8.0 3.3 3.2 WV-9748 21.1 20.9 8.5 8.1 3.1 3.1 WV-9749 23.2 24.2 10.1 9.4 3.7 3.7 WV-9749 25.3 24.6 10.7 10.5 3.7 3.9 WV-9897 53.2 53.1 24.5 24.4 5.4 5.5 WV-9897 48.3 48.7 22.8 22.8 4.8 4.8 WV-9898 46.5 46.8 21.1 21.1 5.2 5.4 WV-9898 46.3 46.4 23.4 23.8 5.0 4.6 WV-9899 45.4 44.1 19.5 19.5 4.8 5.0 WV-9899 44.9 44.0 21.4 21.2 5.5 5.6 WV-9900 34.9 35.0 19.5 19.6 5.0 5.3 WV-9900 30.2 31.5 17.6 17.6 4.4 4.4 WV-9906 42.9 44.6 18.0 19.0 2.9 3.1 WV-9906 37.5 36.3 17.5 18.2 2.8 3.2 WV-9912 39.8 41.6 19.6 17.7 5.0 4.4 WV-9912 41.6 40.8 21.3 19.9 4.2 4.2
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments (R1 and R2) are shown.

(1263) Additional testing of oligonucleotides was performed, and the results were shown below in Tables 20 and 21.

(1264) TABLE-US-00052 TABLE 20 Example data of certain oligonucleotides. 10 uM 10 uM 3 uM 3 uM 1 uM 1 uM WV-9517 34.6 35.6 17.0 19.4 6.7 7.8 WV-9897 43.8 26.8 27.3 9.7 9.8 WV-9898 42.7 30.3 22.8 26.7 8.5 9.3 WV-9899 45.0 16.4 26.8 10.0 8.6 WV-10670 32.4 32.9 15.2 18.2 7.2 8.0 WV-10671 28.7 30.9 14.7 16.1 6.7 8.0 WV-10672 25.6 28.1 11.8 12.2 5.0 5.0 PMO 40.8 36.0 19.1 18.6 10.7 11.7 Mock 1.1 1.9 1.8 1.9 1.7 2.5
Numbers represent skipping efficiency, wherein 100.0 would represent 100% skipping and 0.0 represents 0% efficiency; results from replicate experiments are shown.

(1265) TABLE-US-00053 TABLE 21 Example data of certain oligonucleotides. A. WV- WV- WV- WV- WV- WV- WV- WV- WV- 9422 9425 9426 9517 9519 9521 9522 9524 9536 a) 8, a) 8 a) 3 a) 10, a) 9, a) 8, a) 8, a) 9 a) 7 c) 4 c) 6 c) 4 c) 5 c) 5 WV- WV- WV- WV- WV- WV- WV- WV- WV- 9700 9701 9702 9703 9704 9709 9710 9711 9713 a) 4 a) 4 a) 6 a) 8 a) 7 a) 4 a) 6 a) 6 a) 4 WV- WV- WV- WV- WV- WV- WV- WV- WV- 9714 9715 9746 9747 9748 9749 9750 9751 9756 a) 13, a) 15, c) 4 c) 4 c) 4 c) 4 c) 4 c) 4 c) 7 c) 9 c) 9 WV- WV- WV- WV- WV- WV- WV- WV- 9757 9758 9759 9760 9761 9743 9744 9745 c) 7 c) 2 c) 4 c) 4 c) 6 c) 6 c) 4 c) 6 B. WV- WV- WV- WV- WV- 9422 9425 9426 9429 9517 b) 4 b) 2 b) 2 b) 1 b) 5
Oligonucleotides were tested in vitro in delta 52 cells. A, Exon skipping at 10 uM is shown. B, protein restoration. Different replicates or experiments are designated as a), b), and c).

(1266) Additional DMD oligonucleotides were tested for their ability to mediate skipping of a DMD exon, as shown below. Full PMO (Morpholino) oligonucleotides have the following sequences:

(1267) TABLE-US-00054 (SEQIDNO:3165) PMOSR WV-13405 GTTGCCTCCGGTTCTGAAGGTGTTC (SEQIDNO:3166) PMOWV WV-13406 CTCCGGTTCTGAAGGTGTTC (SEQIDNO:3167) PMO WV-13407 TGCCTCCGGTTCTGAAGGTGTTCTTGTA
WV-13407 is also designated PMO NS.

(1268) TABLE-US-00055 TABLE 21C Example data of certain oligonucleotides. 10 uM 3 uM Mock 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 PMO SR 1.8 1.6 1.1 0.9 0.5 0.5 0.5 0.4 PMO WV 0.8 1.0 1.0 1.1 0.4 0.4 0.5 0.3 PMO 2.3 2.5 1.8 1.8 1.0 0.9 0.6 0.6 WV-10454 5.5 6.1 4.5 3.9 1.3 1.3 0.9 0.7 WV-10455 10.5 13.8 7.3 7.8 2.1 2.8 2.0 2.5 WV-10456 7.2 7.4 5.6 5.0 1.4 1.5 1.7 1.3 WV-10457 9.8 14.2 8.4 9.0 3.8 2.9 3.2 2.9 WV-10458 6.6 5.4 5.6 5.2 1.2 1.1 1.1 1.2 WV-10459 2.4 2.8 2.7 2.5 1.0 1.0 0.5 0.5 WV-10460 7.9 6.0 7.6 7.5 1.9 1.8 1.4 1.4 WV-10461 14.9 11.3 5.7 6.0 2.4 3.7 WV-10462 1.6 2.4 3.4 3.1 0.8 0.8 0.7 0.9 WV-10463 2.6 3.2 2.9 2.7 0.7 0.7 0.7 0.7 WV-10464 1.2 1.1 0.2 0.1 0.4 0.3 0.2 0.3 WV-10465 2.3 1.8 0.6 0.7 0.7 0.7 WV-10466 8.6 9.1 3.9 2.6 1.8 1.6 1.9 1.6 WV-10467 3.2 0.8 1.4 1.1 4.1 4.3 3.3 2.9 WV-10468 2.1 2.0 WV-10469 3.2 3.1 4.8 4.2 0.6 0.6 1.0 0.0 WV-9699 4.6 3.2 2.8 2.4 0.8 0.9 0.7 0.5 WV-9898 19.4 19.0 17.6 18.2 5.4 6.2 5.9 5.4
Numbers represent skipping efficiency, wherein 100 would represent 100% skipping and 0 would represent 0% skipping. Replicate data is shown.
In some embodiments, oligonucleotides, e.g., DMD oligonucleotides, are designed to target Intronic Splice Enhancer elements, e.g., for DMD oligonucleotides for exon 53 skipping, elements within 4 kb of Exon53. In some embodiments, provided oligonucleotides are 30-mers. Example data for certain such oligonucleotides are presented in Table 21D.

(1269) TABLE-US-00056 TABLE 21D Example data of certain oligonucleotides. WV-10490 1.6 1.6 1.8 1.9 WV-10491 1.6 1.7 1.7 1.5 WV-10492 1.4 1.5 1.6 1.4 WV-10493 0.9 0.6 WV-10494 1.4 1.5 1.3 1.6 WV-10495 WV-10496 1.8 1.5 1.8 1.7 WV-10497 1.6 1.6 1.5 1.7 WV-10498 0.7 0.7 2.0 1.8 WV-10499 1.5 1.4 1.7 1.6 WV-10500 0.8 1.3 0.9 0.6 WV-10501 1.2 1.7 1.3 1.4 WV-10502 1.4 1.4 1.5 1.4 WV-10503 1.5 1.0 1.7 WV-10504 1.6 1.8 WV-10505 1.5 1.2 1.9 1.5 WV-10506 0.8 0.8 1.4 1.3 WV-10507 1.4 1.1 0.9 1.4 WV-10508 1.5 1.4 1.8 1.7 WV-10509 1.2 1.5 1.4 1.6 WV-10510 1.3 1.7 1.0 1.6 WV-10511 0.5 0.9 0.8 1.2 WV-10512 1.3 1.5 1.7 1.7 WV-10513 1.5 1.6 1.6 1.7 WV-10514 1.1 1.7 1.8 WV-10515 2.0 1.9 1.9 1.9 WV-10516 8.3 8.7 9.1 8.0 WV-10517 0.5 0.5 1.7 1.5 WV-10518 1.7 1.5 1.5 1.7 WV-10519 1.8 1.6 1.8 1.8 WV-10520 2.1 1.8 1.8 1.7 WV-10521 3.3 3.1 2.6 3.4 WV-10522 1.9 2.0 1.7 2.1 WV-10523 2.3 2.1 1.9 1.9 WV-10524 1.8 1.9 2.1 2.0 WV-10525 2.0 2.1 1.1 1.6 WV-10526 1.7 1.9 1.8 1.7 WV-10527 1.1 1.3 1.4 1.5 WV-10528 1.6 1.6 1.7 1.4 WV-10529 1.6 1.1 WV-10530 0.9 1.7 1.7 1.6 WV-10531 1.2 1.5 1.0 1.3 WV-10532 1.4 1.6 1.6 1.5 WV-10533 1.4 0.5 1.5 1.5 WV-10534 1.3 1.4 1.7 1.6 WV-10535 0.9 0.6 1.7 1.6 WV-10536 1.5 1.0 1.4 1.3 WV-10537 1.4 1.6 1.6 1.4 WV-9517 44.5 42.5 41.6 43.2 WV-9699 13.0 12.7 9.8 9.3 Mock 1.6 1.7 1.4 1.3
Results: Gymnotic delivery of 10 M Intron ASO's in 45-52 patient derived myoblasts (4 days post-differentiation). Done in biological replicates. Numbers represent percentage of exon skipping, as determined by RT-qPCR.

(1270) TABLE-US-00057 TABLE 21E Example data of certain oligonucleotides. Conc. 10 3.33 1.11 0.3704 0.1235 0 WV-13405 35.2 23.1 9.0 4.0 2.2 1.0 (PMO) 36.3 23.1 8.7 4.0 2.3 1.2 33.1 20.6 8.3 3.3 2.1 1.0 33.7 20.7 8.3 3.2 2.2 1.2 WV-9898 31.2 22.2 8.6 1.7 1.3 1.1 30.4 22.5 10.3 1.5 1.2 0.9 49.6 23.3 6.2 1.7 1.4 1.2 48.3 22.3 5.5 1.5 1.6 1.5 WV-12880 73.1 53.5 38.4 10.3 4.5 1.0 72.1 54.3 37.6 10.3 4.8 1.1 69.3 51.5 24.4 5.5 3.5 1.2 69.6 52.6 23.7 6.2 3.2 1.0 WV-9517 40.4 28.1 3.5 2.1 1.4 1.0 39.8 28.2 1.2 2.1 1.3 1.0 29.3 18.1 5.5 1.8 1.3 1.6 28.9 17.4 4.9 1.7 1.3 1.4 WV-9897 21.2 20.0 3.9 1.6 2.1 1.3 23.6 18.5 3.7 1.9 2.1 1.2 39.5 18.7 5.1 1.7 2.0 1.5 40.9 18.5 5.2 1.6 1.8 1.0 WV-12887 79.7 59.4 44.2 9.6 5.5 0.9 78.7 58.8 44.1 9.6 5.6 0.9 76.1 61.0 38.1 12.3 6.7 1.1 75.0 61.3 31.9 9.8 5.1 1.1

(1271) 45-52 DMD patient derived myoblasts, with 7d of pre-differentiation, were treated with oligonucleotides in muscle differentiation medium at indicated concentrations under free uptake condition before being collected and analyzed for RNA skipping efficiency (4d dosing) by qPCR. Relative (SRSF9 normalization) quantification. Oligonucleotides were tested at a concentration of 0 to 10 M. Results of replicate experiments are shown. Some of the oligonucleotides tested comprise a non-negatively charged internucleotidic linkage (WV-12887 and WV-12880).

(1272) TABLE-US-00058 TABLE 21F Example data of certain oligonucleotides. 10 uM 3.3 uM Mock 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.3 WV-13405 4.3 4.5 4.2 4.7 1.2 1.1 1.8 1.9 (PMO) WV-9517 15.0 14.2 5.6 5.8 8.7 9.3 WV-11340 32.4 33.7 35.9 36.9 15.4 13.0 15.9 15.0 WV-12873 38.7 37.5 39.6 39.2 13.6 11.7 17.0 14.5 WV-12872 44.9 41.9 44.1 46.5 15.7 17.5 15.7 19.5 WV-13408 49.0 48.7 50.2 50.3 21.6 22.0 23.0 24.5 WV-12553 18.3 20.7 18.7 24.1 7.4 7.6 9.7 8.4 WV-12557 40.0 39.2 33.8 35.9 15.3 15.5 23.6 23.9 WV-12554 38.8 39.0 43.5 44.9 15.1 14.0 20.5 20.3 WV-13409 34.6 38.4 39.1 40.3 14.7 12.9 18.9 16.5 WV-9898 24.1 22.0 7.9 7.7 9.9 8.5 WV-11342 30.4 34.5 31.3 31.9 14.3 14.4 14.1 13.3 WV-12559 44.3 41.8 16.6 16.5 17.4 19.4 WV-12556 42.5 43.0 39.7 43.3 16.1 17.1 18.8 17.1 WV-9897 20.8 17.9 6.0 5.4 6.8 4.8 WV-11341 36.6 39.4 17.8 16.8 18.2 19.3 WV-12558 41.5 39.4 36.0 18.2 15.1 18.5 16.7 WV-12555 44.3 43.6 20.5 19.0 20.2 22.1 WV12880 41.1 43.2 46.1 45.1 27.4 24.6 25.9 29.1 WV-12877 51.5 53.3 26.2 27.1 30.2 30.7 WV-12125 47.3 49.4 37.8 35.1 21.3 20.6 24.0 23.5 WV-12127 40.0 40.6 41.2 39.7 19.9 15.5 18.3 18.0 WV-12129 33.5 35.0 24.4 24.4 13.9 10.7 14.4 13.7
45-52 DMD patient derived myoblasts were treated with oligos in muscle differentiation medium at indicated concentrations for 4d under free uptake conditions and analyzed for RNA skipping efficiency by qPCR.

(1273) TABLE-US-00059 TABLE 21G Example data of certain oligonucleotides. Oligo Conc [uM] 10 uM 3.3 uM Mock 0.6 0.6 0.6 0.8 0.7 0.6 1.0 0.8 WV-13405 6.9 7.4 10.1 10.9 2.2 1.9 4.1 4.4 (PMO) WV-9517 24.2 22.0 11.5 33.7 9.3 9.8 19.8 20.6 WV-11340 50.8 54.1 61.6 63.9 30.1 22.0 33.2 30.6 WV-12872 70.6 66.4 71.0 74.6 24.7 29.2 27.9 38.9 WV-12873 60.8 59.5 62.9 62.8 20.4 15.3 33.5 24.5 WV-13408 73.5 72.3 75.8 75.6 35.6 35.7 42.2 46.3 WV-12553 32.7 39.1 38.0 51.3 13.7 14.6 22.7 18.9 WV-12557 65.2 64.4 76.7 80.4 26.3 27.1 45.3 45.6 WV-12554 61.0 61.5 69.5 71.7 27.0 22.9 38.5 37.6 WV-13409 57.2 63.6 66.2 69.3 23.6 18.9 34.4 28.4 WV-9898 45.1 40.3 16.3 14.4 13.2 12.1 20.8 16.1 WV-11342 49.9 58.1 57.9 60.0 27.4 27.8 30.3 27.4 WV-12559 72.4 68.4 50.8 56.1 33.3 32.8 35.5 42.5 WV-12556 70.5 71.0 68.4 73.5 31.0 33.5 42.0 37.0 WV-9897 42.0 34.9 41.2 10.2 8.0 17.9 9.4 WV-11341 61.6 67.2 74.1 74.4 37.0 33.8 40.8 42.9 WV-12558 71.6 68.0 66.3 35.6 27.1 40.5 35.5 WV-12555 70.2 68.9 56.0 61.7 35.2 32.4 40.1 45.0 WV12880 58.8 63.0 68.5 66.5 44.4 36.6 44.8 52.1 WV-12877 77.9 80.2 69.5 75.6 46.3 48.2 55.8 58.4 WV-12125 71.1 74.1 83.6 80.4 36.5 34.8 45.6 44.3 WV-12127 61.9 64.0 67.8 66.2 35.0 23.3 35.5 34.7 WV-12129 52.7 55.8 63.1 63.6 23.8 14.7 26.5 24.1
45-52 DMD patient derived myoblasts, with 7d of pre-differentiation, were treated with oligos in muscle differentiation medium at indicated concentrations for 4d under free uptake conditions and analyzed for RNA skipping efficiency by qPCR.

(1274) TABLE-US-00060 TABLE 21H Example data of certain oligonucleotides. WV- 27.2 WV- 74.4 WV- 45.0 12553 30.1 12124 67.6 12127 42.3 32.1 67.7 43.2 WV- 63.6 WV- 65.8 WV- 50.2 11341 55.0 12125 74.2 12129 53.3 55.7 92.6 51.2 WV- 51.7 WV- 65.8 WV- 60.6 11342 54.0 12126 57.9 12882 66.9 50.8 55.8 68.6 WV- 81.1 WV- 65.2 WV- 76.0 12555 12880 63.9 12878 75.1 76.2 60.9 78.1 WV- 73.4 WV- 61.9 WV- 67.0 12556 75.1 12881 60.3 12876 62.0 66.9 57.7 66.4 WV- 59.9 WV- 59.5 12558 78.8 12123 55.1 66.0 49.9 WV- 68.3 WV- 78.9 12559 76.3 12877 78.0 73.3 83.1 WV- 59.9 9897 59.6 58.6 WV- 44.7 9898 39.1 46.3
Full length oligonucleotide stability at 5 day timepoint in Human Liver homogenate was tested. Numbers are replicates and represent percentage of full-length oligonucleotide remaining, wherein 100 would represent 100% oligonucleotide remaining (complete stability) and 0 would represent 0% oligonucleotide remaining (complete instability). Some nucleotides tested comprise a non-negatively charged internucleotidic linkage.

(1275) TABLE-US-00061 TABLE 21I Example data of certain oligonucleotides. Oligo Conc WV- WV- WV- WV- [uM] 9517 13826 13827 13835 Mock 10 uM 45.7 46.5 23.1 40.5 1.2 46.3 45.8 22.9 58.8 1.1 49.3 46.8 26.8 54.5 1.3 48.5 50.3 28.1 55.2 1.2 3.3 uM 18.1 20.3 7.9 24.6 1 17 19.5 8.3 25.3 1.1 22.6 19.7 8.8 26.6 1.1 22.8 20.2 8.3 27.2 1.1 1.1 uM 6 7 2.9 7.9 1 6 6.2 2.7 7.4 1.2 6.9 7.3 0.7 9.6 0.9 6.6 6.8 0.9 9.1 0.7 WV- WV- WV- WV- 9517 12880 13864 14344 MOCK 10 uM 36.1 60.2 66.8 47.9 0.9 38.3 62.0 67.0 46.8 1.0 44.5 60.9 68.7 56.8 1.2 43.9 59.2 69.6 56.3 1.0 3.3 uM 15.4 38.3 45.3 25.1 0.9 15.8 37.3 45.6 27.0 0.9 18.8 37.9 50.5 39.2 1.0 18.8 39.6 49.3 38.9 1.0 1.1 uM 4.7 15.8 21.5 12.2 0.6 4.9 14.4 22.6 12.4 0.9 6.4 18.5 24.9 17.2 1.1 6.2 16.2 13.2 17.1 0.9 0.3 uM 2.2 5.0 6.6 5.7 0.8 1.8 5.0 5.9 5.7 0.9 2.7 7.4 8.2 7.2 1.0 2.7 7.5 8.2 6.9 1.0
Numbers indicate amount of skipping relative to control.

(1276) TABLE-US-00062 TABLE 21I.1 Example data of certain oligonucleotides. 10 uM 3.3 uM 1.1 uM 0.3 uM 0.1 uM Mock 1.1 1.2 0.8 1.0 1.0 1.1 2.0 0.9 1.0 1.1 0.7 1.1 1.0 1.1 1.2 0.7 1.1 0.9 1.0 Wv- 44.8 28.6 18.1 9.5 4.0 13405 44.8 23.4 17.4 8.7 4.0 (PMO) 51.2 26.5 11.4 5.1 3.7 50.8 25.6 11.2 5.5 3.6 WV- 35.9 18.3 6.5 2.2 1.9 9517 36.6 17.3 6.4 2.1 1.9 40.2 23.4 5.5 2.7 1.7 38.7 25.6 5.9 2.2 1.8 Wv- 57.3 36.3 16.4 4.8 7.5 12880 55.8 37.0 18.1 2.8 4.7 57.5 35.9 16.6 8.0 7.4 58.9 33.0 16.5 7.2 6.8 WV- 68.1 45.1 22.6 10.5 7.4 13864 68.0 44.5 23.0 12.0 5.6 67.5 43.1 24.3 8.4 6.0 64.8 44.5 19.9 3.3 6.1 WV- 40.2 21.5 6.3 2.8 2.0 13835 39.4 20.3 9.7 2.5 2.0 50.0 21.0 5.5 3.2 2.0 47.7 20.6 6.0 3.3 2.2 WV- 41.4 25.9 7.4 4.7 0.7 14791 40.3 24.8 5.8 4.0 0.5 40.1 24.9 9.1 4.3 3.9 41.3 27.2 8.9 4.6 3.5 WV- 50.1 28.6 13.6 6.4 3.8 14344 47.4 28.6 8.8 5.8 4.7 54.9 46.1 18.0 11.4 6.6 55.7 38.3 18.7 11.8 6.0
Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 53. Numbers represent skipping of exon 53.
45-52 patient myoblasts were differentiated for 7 days, then treated with oligonucleotide for 4d under gymnotic conditions in differentiation media. RNA was harvested by Trizol extraction and skipping analyzed by TaqMan.

(1277) TABLE-US-00063 TABLE 21I.2 Example data of certain oligonucleotides. 10 uM 3.3 uM 1.1 uM 0.3 uM 0.1 uM Mock 0.7 0.6 0.6 0.6 0.7 0.7 0.7 0.6 0.6 0.7 0.6 0.6 0.6 0.7 0.7 0.5 0.5 0.7 0.6 0.7 Wv- 9.4 1.5 3.4 1.1 0.8 13405 9.3 1.4 3.1 1.1 0.8 (PMO) 6.6 2.8 1.5 0.9 0.8 6.3 2.6 1.5 1.0 0.8 WV- 29.3 8.4 2.6 1.0 0.7 9517 28.7 9.2 3.0 1.1 0.8 16.6 6.6 2.3 1.1 0.7 16.9 6.8 2.2 1.1 0.9 WV- 37.9 17.7 9.6 3.4 1.3 12880 38.8 19.9 9.1 3.3 1.4 31.4 16.1 7.9 3.3 1.6 31.6 16.8 8.0 3.0 1.5 WV- 55.9 28.6 11.7 4.3 2.0 13864 54.3 27.8 11.6 4.6 2.0 43.4 22.2 10.7 4.2 2.0 43.0 22.7 9.8 3.8 2.1 WV- 38.7 11.6 2.9 1.3 0.9 13835 37.2 11.0 2.9 1.3 0.8 42.3 13.1 3.5 1.2 0.9 41.5 10.0 3.1 1.3 0.9 WV- 26.3 12.1 5.2 1.9 1.3 14791 24.8 11.2 4.7 2.1 1.1 28.0 13.0 5.2 2.2 1.2 27.6 12.4 4.9 2.1 1.4 WV- 36.2 17.8 8.0 2.7 1.7 14344 37.4 17.0 7.1 2.7 1.8 37.4 22.3 9.8 3.7 1.7 36.6 22.6 9.9 3.7 1.5
Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 53. Numbers represent skipping of exon 53.
45-52 patient myoblasts were treated with oligonucleotide for 4d (4 days) under gymnotic conditions in differentiation media. RNA was harvested by Trizol extraction and skipping analyzed by TaqMan.
Several oligonucleotides (including WV-9517, WV-13864, WV-13835, and WV-14791) were tested at various concentrations up to 30 uM for TLR9 activation in vitro in HEK-blue-TLR9 cells (16 hour gymnotic uptake). WV-13864 and WV-14791 comprise a chirally controlled non-negatively charged internucleotidic linkage in the Rp configuration. WV-9517, WV-13864, WV-13835, and WV-14791 did not exhibit significant TLR9 activation (less than 2-fold TLR9 induction; data not shown). WV-13864 and WV-14791 also exhibited negligible signal up to 30 uM in PBMC cytokine release assay compared to water (data not shown).
Example Dystrophin Oligonucleotides and Compositions Which Target Exon 54

(1278) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 54 and/or mediating skipping of exon 54 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 54 oligos include: WV-13745, WV-13746, WV-13747, WV-13748, WV-13749, WV-13750, WV-13751, WV-13752, WV-13753, WV-13754, WV-13755, WV-13756, WV-13757, WV-13758, WV-13759, WV-13760, WV-13784, and WV-13785, and other oligonucleotides having a base sequence which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1279) TABLE-US-00064 TABLE 21J Example data of certain oligonucleotides. WV-13745 0.2 0.3 0.2 0.0 WV-13746 0.6 0.6 0.4 0.4 WV-13747 0.4 0.5 0.4 0.4 WV-13748 1.1 1.2 0.7 0.9 WV-13749 2.5 2.1 1.7 1.8 WV-13750 1.9 2.1 1.4 1.4 WV-13751 4.3 5.1 4.4 5.7 WV-13752 0.0 0.0 3.1 3.9 WV-13753 0.0 0.0 0.0 0.0 WV-13754 6.0 1.4 1.7 WV-13755 1.1 1.2 0.5 0.5 WV-13756 4.7 5.0 2.3 2.4 WV-13757 1.9 2.1 1.1 1.4 WV-13758 2.0 2.2 0.9 1.2 WV-13759 0.7 0.7 0.4 0.2 WV-13760 0.7 0.6 0.3 0.5 WV-13784 0.0 0.0 0.0 0.0 WV-13785 0.0 0.0 0.0 0.0 Mock 0.0 0.0 Mock 0.0 0.0
Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 54.
Example Dystrophin Oligonucleotides and Compositions Which Target Exon 55

(1280) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 55 and/or mediating skipping of exon 55 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 55 oligos include: WV-13761, WV-13762, WV-13763, WV-13764, WV-13765, WV-13766, WV-13767, WV-13768, WV-13769, WV-13770, WV-13771, WV-13772, WV-13773, WV-13774, WV-13775, WV-13776, WV-13777, WV-13778, WV-13779, WV-13786, and WV-13787, and other oligonucleotides having a base sequence (naked sequence) which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1281) In some embodiments, two or more oligonucleotides capable of skipping or targeting exon 44, 46, 47, 51, 52, 53, 54 and/or 55 can be used in any combination to mediate multiple exon skipping.

(1282) TABLE-US-00065 TABLE 21K Example data of certain oligonucleotides. WV-13761 0.5 0.5 0.3 0.4 WV-13762 0.3 0.2 0.1 0.1 WV-13763 0.2 0.2 0.2 0.2 WV-13764 0.1 0.1 0.1 0.1 WV-13765 1.0 1.0 0.4 0.4 WV-13766 2.6 2.7 1.7 1.8 WV-13767 0.2 0.0 1.4 1.6 WV-13768 1.1 1.1 0.7 0.7 WV-13769 1.6 1.8 1.1 1.1 WV-13770 1.4 1.4 0.8 0.9 WV-13771 0.3 0.4 0.2 0.2 WV-13772 1.8 1.7 0.9 0.9 WV-13773 0.0 0.0 0.1 0.1 WV-13774 0.0 0.0 0.0 0.0 WV-13775 1.0 0.8 0.3 0.4 WV-13776 0.7 0.6 0.3 0.7 WV-13777 2.8 2.2 0.4 1.1 WV-13778 0.3 0.3 0.2 0.3 WV-13779 0.0 0.0 0.4 0.4 WV-13786 0.0 0.0 2.0 2.3 WV-13787 0.0 0.0 0.2 0.1 Mock 0.0 0.0 0.0 0.0 Mock 0.0 0.0 0.0 0.0
Skipping efficiency of various DMD oligonucleotides, tested for skipping of DMD exon 55.
Example Dystrophin Oligonucleotides and Compositions Which Target Exon 57

(1283) In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for targeting exon 57 and/or mediating skipping of exon 57 in human DMD. Non-limiting examples include oligonucleotides and compositions of Exon 57 oligos include: WV-18853, WV-18854, WV-18855, WV-18856, WV-18857, WV-18858, WV-18859, WV-18860, WV-18861, WV-18862, WV-18863, WV-18864, WV-18865, WV-18866, WV-18867, WV-18868, WV-18869, WV-18870, WV-18871, WV-18872, WV-18873, WV-18874, WV-18875, WV-18876, WV-18877, WV-18878, WV-18879, WV-18880, WV-18881, WV-18882, WV-18883, WV-18884, WV-18885, WV-18886, WV-18887, WV-18888, WV-18889, WV-18890, WV-18891, WV-18892, WV-18893, WV-18894, WV-18895, WV-18896, WV-18897, WV-18898, WV-18899, WV-18900, WV-18901, WV-18902, WV-18903, WV-18904, and other oligonucleotides having a base sequence (naked sequence) which comprises at least 15 contiguous bases of any of these DMD oligonucleotides.

(1284) Example Dystrophin Oligonucleotides and Compositions for Exon Skipping of Mutiple Exons (Multi-Exon Skipping)

(1285) In some embodiments, the present disclosure provides oligonucleotides, compositions, and methods for splicing modulation, including skipping of multiple exons. In some embodiments, a DMD oligonucleotide or composition thereof is capable of mediating skipping of multiple exons in the human or mouse Dystrophin gene.

(1286) In some embodiments, in a patient with muscular dystrophy, the symptoms of muscular dystrophy can at least be partially relieved and/or the disorder at least partially treated by administration of a DMD oligonucleotide capable of skipping one exon or multiple exons. Without wishing to be bound by any particular theory, the present disclosure notes that BMD patients with a deletion of exons 45 to 55 of DMD showed a milder or asymptomatic phenotype.

(1287) A non-limiting example of a scheme for multiple exon skipping is shown in FIG. 1. In this Figure, various numbers (43 to 57) indicate exons; and the shapes of the exons (e.g., <, > or |) indicate which reading frame is represented at the 5 and 3 end of each exon. Normally exon 44 is joined to exon 45. In a non-limiting example of multiple exon skipping, exons 45 to 55 are skipped, allowing exon 44 to join to exon 56. The 3 end of exon 44 is represented by the same reading frame (<) as the 5 end of exon 56; thus skipping exons 45 to 55 maintains or restores the correct reading frame. In some embodiments, skipping multiple exons restores the reading frame if one of the skipped exons comprises a mutation which alters the reading frame (in many cases, for example, producing a missense or prematurely truncated protein).

(1288) Among other things, the present disclosure notes that various exons represent at their 5 and/or 3 ends different reading frames; thus, some combinations of skipping adjacent reading frames but not other combinations are capable of maintaining or restoring the reading frame. In some embodiments, provided compositions and methods for multiple exon skipping skip, as non-limiting examples, exons 45-46, 45-47, 45-48, 45-49, 45-51, 45-53, 45-55, 47-48, 47-49, 47-51, 47-53, 47-55, 48-49, 48-51, 48-53, 46-55, 50-51, 50-53, 50-55, 49-51, 49-53, 49-55, 52-53, 52-55, 44-45, 44-54, or 44-56, wherein in each case multiple exon skipping maintains or restores the correct reading frame. In some embodiments, skipping of non-overlapping sets of exons is capable of maintaining or restoring reading frame, e.g., skipping of exons 45-46 and exons 49-55; skipping of exons 45-47 and 49-55; skipping of exons 45-49 and 52-55; etc.

(1289) Without wishing to be bound by any particular theory, the present disclosure notes that some DMD exons may be spliced transcriptionally, while others are spliced post-transcriptionally. For example, each of exons 45 to 55 are reportedly not simultaneously spliced, but rather first as three groups: exons 45 to 49, 50 to 52, and 53 to 55, the individual exons within each group being spliced transcriptionally. Reportedly, the remaining introns (between exons 44/45, 49/50, 52/53, and 55/56) are later spliced post-transcriptionally. Without wishing to be bound by any particular theory, the present disclosure notes that this lag in the timing of splicing may be exploited by oligonucleotides capable of increasing the splicing between exons whose adjacent introns are spliced post-transcriptionally, such as exon 44 and 56. It is reported that in nature, such multi-exon skipping joining exon 44 to exon 56 occurs at a low but detectable frequency (approximately 1/600). Without wishing to be bound by any particular theory, the present disclosure pertains in part to DMD oligonucleotides capable of skipping multiple exons at a therapeutically and clinically significant level.

(1290) In some embodiments, a composition capable of mediating multiple exon skipping comprises a DMD oligonucleotide. In some embodiments, a composition capable of mediating multiple exon skipping comprises a combination of (e.g., two or more different) DMD oligonucleotides. In some embodiments, a composition capable of mediating multiple exon skipping comprises a combination of (e.g., two or more different) DMD oligonucleotides, wherein at least one oligonucleotide recognizes a target associated with skipping the 5 exon to be skipped, and at least one oligonucleotide recognizes a target associated with skipping the 3 exon to be skipped. In some embodiments, a composition capable of mediating multiple exon skipping comprises a oligonucleotide capable of recognizes both (1) a target associated with skipping the 5 exon to be skipped and (2) a target associated with skipping the 3 exon to be skipped.

(1291) In some embodiments, an advantage of a composition capable of multiple exon skipping is that it is useful for treatment of dystrophy associated with a mutation in any individual exon included in the group of exons which is skipped. As a non-limiting example, a DMD oligonucleotide capable of mediating skipping of exon 48 is only capable of treating mutations within that exon (or, in some cases, an adjacent or nearby exon) but not mutations within other exons. However, a composition capable of mediating skipping of exons 45 to 55 is capable of treating mutations in any of exons 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. Thus, both a patient with a mutation in exon 48 and a patient with a mutation in exon 54 can be treated with a composition capable of skipping exons 45 to 55. In some embodiments, a composition capable of mediating skipping of exons 45 to 55 is capable of treating up to about 63% of DMD patients.

(1292) In some embodiments, a composition comprises one or more DMD oligonucleotides, wherein the composition is capable of mediating skipping of multiple (two or more) DMD exons.

(1293) In some embodiments, a MESO(a composition comprising one or more oligonucleotides, which composition is capable of mediating multiple exon skipping) has an advantage over a DMD oligonucleotide capable of skipping only one exon. In some embodiments, a composition which is capable of mediating skipping of a single exon, is only useful for treating patients treatable by skipping that exon (e.g., patients having a genetic lesion in that exon). In some embodiments, a MESO is useful for treating patients treatable by skipping any of the exons which the MESO is able to skip, which is likely a larger percentage of the patient population. In some embodiments, double or multiple exon skipping can potentially be applicable to 90% of patients.

(1294) In addition, in some embodiments, because the 5 and 3 ends of an exon are sometimes not in the same frame, deletion of such an exon would cause a frameshift. Skipping of multiple exons, in various such cases, can restore the reading frame.

(1295) In some embodiments, multiple exon skipping is useful to treat DMD patients with deletion, duplication, and nonsense mutations.

(1296) In addition, in some embodiments, skipping of multiple exons can mimic the genetics of the milder Becker muscular dystrophy. In some embodiments, the more severe Duchenne muscular dystrophy, mediated by a genetic lesion in one exon, can be converted into a milder Becker muscular dystrophy, mediated by an in-frame deletion of multiple exons. It is reported that some BMD patients and an asymptomatic person have in-frame deletions of exons 48 to 51 or 45 to 51. Singh et al. 1997 Hum. Genet. 99:206-208; Melacini et al. 1993 J. Am. Col. Cardiol. 22:1927-1934; Melis et al. 1998 Eur. J. Paediatr. Neurol. 2:255-261; and Aartsma-Rus et al. 2003 Hum. Mol. Genet. 8:907-914.

(1297) In some embodiments, certain exons may be more challenging than others to skip. In some embodiments, the present disclosure provides technologies to skip such exons, e.g., through chemical modifications, linkage phosphorus stereochemistry, and combinations thereof. In some embodiments, the present disclosure encompasses the recognition that multiple exon skipping can be useful for skipping such challenging exons. In some embodiments, the present disclosure provides multiple exon skipping technologies for skipping such challenging exons.

(1298) In some embodiments, exon skipping, e.g., DMD exon skipping, can be used to treat patients, e.g., DMD patients, with circular or circularized RNA transcripts (e.g., those of DMD). Circular DMD transcripts are reported in, as a non-limiting example: Gualandi et al. 2003 J. Med. Gen. 40: e100.

(1299) In some embodiments, a composition capable of mediating multiple exon skipping (MESO) comprises one DMD oligonucleotide capable of mediating skipping of multiple exons. In some embodiments, a composition capable of mediating multiple exon skipping (MESO) comprises two DMD oligonucleotides which are together (e.g., when used in combination) capable of mediating skipping of multiple exons. In some embodiments, a composition capable of mediating multiple exon skipping (MESO) comprises a cocktail of (e.g., a mixture of three or more) DMD oligonucleotides which are together (e.g., when used in combination as a cocktail) capable of mediating skipping of multiple exons. Combinations or cocktails of oligonucleotides capable of mediating skipple of multiple exons have been reported by, for example, Yokota et al. 2009 Arch. Neurol. 66:32; Yokota et al. 2012 Nucl. Acid Ther. 22:306; Adkin et al. 2012 Neur. Dis. 22:297-305; Echigoya et al. 2013 Nucl. Acid. Ther.; and Echigoya et al. 2015 Molecular TherapyNucleic Acids 4: e225. Among other things, the present disclosure provides more effective combinations, through, e.g., selected sequences, chemical modifications, and/or linkage phosphorus chemistry, etc.

(1300) In some embodiments, the present disclosure provides oligonucleotides that, when combined with other oligonucleotides, can provide dramatically increased activities compared to either oligonucleotides individually prior to combination. For example, in some embodiments, the present disclosure provides DMD oligonucleotides which are individually incapable of mediating efficient skipping of a particular exon; when combined with other oligonucleotides, such oligonucleotides are capable of mediating skipping of multiple exons. Among other things, the present disclosure provides combination therapy, wherein two or more oligonucleotides are used together to provide desired and/or enhanced properties and/or activities. When used in combination therapy, the two or more agents, e.g., oligonucleotides, may be administered concurrently, or separately in suitable ways for them to achieve their combination effects. In some embodiments, two or more oligonucleotides in a combination are all (primarily) for skipping of the same exon, and their combination provides enhanced skipping of such exon, in some embodiments, significantly more than the addition of their separate effects. In some embodiments, two or more oligonucleotide in a combination are for skipping of difference exons, and their combination provides effective skipping, sometimes more than the oligonucleotides individually can achieve, of two or more exons. In some embodiments, the present disclosure provide combinations of oligonucleotides with synergies between two or more different oligonucleotides. In some embodiments, the present disclosure provides combinations of different oligonucleotides wherein one or more, or each oligonucleotide by itself is not effective for exon skipping. Certain combinations are described in Adams et al. 2007 BMC Mol. Biol. 8:57. Among other things, the present disclosure provides more effective combinations, through, e.g., designed control of one or more or all structural elements of oligonucleotides. In some embodiments, a provided combination provides exon skipping of DMD exon 45. In some embodiments, a provided combination provides exon skipping of another DMD exon, including those described herein or otherwise desirable for skipping (e.g., for prevention or treatment of one or more conditions, diseases or disorders etc.) as known in the art.

(1301) In some embodiments, cocktails, combinations and mixtures of oligonucleotides, e.g., for multiple exon skipping may have disadvantages compared to single oligonucleotides which can perform the same or comparable functions, such as higher costs of goods, complications in manufacturing and delivery, increased regulatory burden, etc. In accordance with FDA regulations, each component in a combination may need to be separately tested for toxicity, as well as the entire combination. In some embodiments, the present disclosure provides single oligonucleotides that can achieve the same or comparable functions of oligonucleotide combinations, and may be utilized to replace oligonucleotide combinations, through precise and designed control of one or more structural elements of oligonucleotides, e.g., chemical modifications, stereochemistry, and combinations thereof.

(1302) Various technologies are suitable for assessing multiple exon skipping in accordance with the present disclosure. Non-limiting examples are described in Example 20 and FIG. 2.

(1303) In some embodiments, a composition for skipping multiple DMD exons comprises a DMD oligonucleotide capable of skipping DMD exon 45. Various DMD oligonucleotides were tested for their capability to skip exon 45, as shown in Table 1A. Various DMD oligonucleotides for skipping exon 45 were also tested for their ability to skip multiple exons, as shown in Table 22A. Among other things, the present disclosure demonstrates that several oligonucleotides, including WV-11088 and WV-11089, can provide low levels of skipping of exons 45-55 (creating a junction between exon 44 and exon 56 or 44-56).

(1304) In another experiment, oligonucleotides WV-11047, WV-11051 to WV-11059 did not demonstrate significant skipping under the specific tested condition, and oligonucleotides WV-11062 to WV-11069 each exhibited detectable levels of skipping which were <1% under the specific tested condition. Oligonucleotides WV-11091 to WV-11096, WV-11098, and WV-11100 to WV-11105 exhibited <0.5% skipping of exon 45 under the specific tested condition.

(1305) TABLE-US-00066 TABLE 22A Example data of certain oligonucleotides. WV-11070 1.6 WV-11071 .3 WV-11072 .2 WV-11073 .7 WV-11074 2.2 WV-11075 .2 WV-11076 1.2 WV-11077 1.3 WV-11078 3.3 WV-11079 7.5 WV-11080 1.3 WV-11081 7.2 WV-11082 2.8 WV-11083 3.1 WV-11084 10.1 WV-11085 1.5 WV-11086 15.8 WV-11087 1.1 WV-11088 13 WV-11089 15.1 WV-11090 .9
Oligonucleotides were tested for their ability to skip DMD exon 45 in 48-50 cells.
Numbers indicate skipping level, wherein 100 would represent 100% skipping and 0 would represent 0% skipping.
Several oligonucleotides, including WV-11088 and WV-11089, showed detectable levels of multiple exon skipping (specifically exons 45-55) (approximately 0.1% skipping).

(1306) In another experiment, various DMD oligonucleotides targeting exon 45 were tested in 48-50 for an ability to skip multiple exons (specifically 45 to 53, creating a junction between exon 44 and exon 54 or 44-54). Oligonucleotides tested were: WV-11047, WV-11051, WV-11052, WV-11053, WV-11054, WV-11055, WV-11056, WV-11057, WV-11058, WV-11059, WV-11062, WV-11063, WV-11064, WV-11065, WV-11066, WV-11067, WV-11068, WV-11069, WV-11070, WV-11071, WV-11072, WV-11073, WV-11074, WV-11075, WV-11076, WV-11077, WV-11078, WV-11079, WV-11080, WV-11081, WV-11082, WV-11083, WV-11084, WV-11085, WV-11086, WV-11087, WV-11088, WV-11089, WV-11090, WV-11091, WV-11092, WV-11093, WV-11094, WV-11095, WV-11096, WV-11098, WV-11100, WV-11101. All these oligonucleotides, in one experiment, demonstrated on average about 0.05% or less skipping of exons 44-54 (data not shown).

(1307) Oligonucleotides targeting exon 45 were also tested for skipping of exons 45 to 57, as shown in Table 22A.1.

(1308) TABLE-US-00067 TABLE 22A.1 Example data of certain oligonucleotides. WV-11047 0.064 0.118 0.048 0.099 WV-11051 0.044 0.101 0.034 0.079 WV-11052 0.076 0.089 0.078 0.090 WV-11053 0.082 0.076 0.078 0.072 WV-11054 0.126 0.083 0.110 0.100 WV-11055 0.037 0.071 0.048 0.073 WV-11056 0.133 0.102 0.116 0.092 WV-11057 0.000 0.001 0.000 0.097 WV-11058 0.102 0.030 0.071 0.042 WV-11059 0.171 0.100 0.157 0.075 WV-11062 0.070 0.112 0.081 0.088 WV-11063 0.088 0.078 0.051 0.081 WV-11064 0.085 0.071 0.071 0.075 WV-11065 0.073 0.114 0.077 0.143 WV-11066 0.083 0.100 0.004 0.143 WV-11067 0.115 0.069 0.094 0.068 WV-11068 0.112 0.071 0.125 0.053 WV-11069 0.075 0.075 0.083 0.053 WV-11070 0.062 0.107 0.067 0.101 WV-11071 0.085 0.116 0.073 0.118 WV-11072 0.080 0.097 0.052 0.084 WV-11073 0.052 0.148 0.047 0.118 WV-11074 0.155 0.098 0.116 0.101 WV-11075 0.145 0.079 0.126 0.113 WV-11076 0.000 0.105 0.000 0.111 WV-11077 0.050 0.087 0.080 0.058 WV-11078 0.087 0.095 0.077 0.103 WV-11079 0.076 0.063 0.079 0.062 WV-11080 0.059 0.058 0.052 0.070 WV-11081 0.077 0.086 0.058 0.055 WV-11082 0.117 0.071 0.112 0.080 WV-11083 0.077 0.108 0.091 0.091 WV-11084 0.080 0.102 0.053 0.069 WV-11085 0.047 0.143 0.041 0.140 WV-11086 0.085 0.087 0.084 0.074 WV-11087 0.114 0.034 0.000 0.056 WV-11088 0.134 0.112 0.057 0.063 WV-11089 0.074 0.113 0.109 0.082 WV-11090 0.119 0.076 0.074 0.081 WV-11091 0.000 0.055 0.031 0.054 WV-11092 0.039 0.057 0.068 0.058 WV-11093 0.147 0.061 0.138 0.061 WV-11094 0.108 0.078 0.061 0.080 WV-11095 0.062 0.061 0.056 0.072 WV-11096 0.104 0.071 0.072 0.101 WV-11098 0.072 0.095 0.081 0.065 WV-11100 0.068 0.079 0.078 0.068 WV-11101 0.000 0.058 0.000 0.048
Oligonucleotides were tested in 48-50 for their ability to skip DMD exons 45 to 57, creating a junction between exon 44 and exon 58 or 44-58. Numbers indicate skipping level, wherein 100 would represent 100% skipping and 0 would represent 0% skipping. Replicate data in this and other tables are shown.

(1309) In some embodiments, a DMD oligonucleotide targets DMD exon 44 or the adjoining intronic region 3 to DMD exon 44 and is capable of mediating multiple exon skipping.

(1310) In some embodiments, a DMD oligonucleotide targets DMD exon 44 or the adjoining intronic region 3 to DMD exon 44, and the oligonucleotide is capable of mediating multiple exon skipping (e.g., of exons 45 to 55, or 45 to 57).

(1311) Reportedly, a phenomenon known as back-splicing can occur, in which, for example, a portion of the 3 end of exon 55 interacts with a portion of the 5 end of exon 45, forming a circular RNA (circRNA), which can thus skip multiple exons, e.g., all exons from exon 45 to 55, inclusive. The phenomenon can also reportedly occur between exon 57 and exon 45, skipping multiple exons, e.g., all exons from exon 45 to 57, inclusive. Back-splicing is described in the literature, e.g., in Suzuki et al. 2016 Int. J. Mol. Sci. 17.

(1312) Without wishing to be bound by any particular theory, the present disclosure suggests that it may be possible for a DMD oligonucleotide targeting DMD exon 44 or the adjoining intronic region 3 to exon 44 may be able to mediate splicing of exons 45 to 55, or of exons 45 to 57, which exons are excised as a single piece of circular RNA (circRNA) designated 45-55 (or 55-45) or 45-57 (or 57-45), respectively.

(1313) Several oligonucleotides were designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44. In some embodiments, oligonucleotides designed to target exon 44 or intron 44, or which straddle exon 44 and intron 44 are tested to determine if they can increase the amount of backslicing and/or multiple-exon skipping.

(1314) As shown in Table 22A.2 and Table 22A.3, below, DMD oligonucleotides targeting Exon44 were tested for the ability to increase circRNA 55-45 (e.g., mediate multiple exon skipping of exons 45 to 55); or for the ability to increase circRNA 57-45 (e.g., mediate multiple exon skipping of exons 45 to 57). Various DMD oligonucleotides comprise various difference including, inter alia, base sequence and length (18 or 20 bases). Numbers indicate relative amount of circRNA 55-45 (Table 22A.2) or circRNA 57-45 (Table 22A.3). In this and various other tables, Rep indicates Replicate.

(1315) TABLE-US-00068 TABLE 22A.2 Example data of certain oligonucleotides. WV-13964 0.9 1 WV-13965 1.1 1.1 WV-13966 1.1 0.6 WV-13967 1.3 1.2 WV-13969 1 0.8 WV-13971 0.3 0.9 WV-13972 1.1 1.3 WV-13973 1.1 1.3 WV-13976 1.2 1.2 WV-13979 0.5 0.5 WV-13980 1.3 0.4 WV-13981 0.9 0.7 WV-13982 1 1 WV-13983 0.9 0.6 WV-13984 1.1 WV-13985 1.3 0.8 WV-13987 1.2 1 WV-13988 1.4 0.9 WV-13989 1.6 1 WV-13990 1.7 1 WV-13991 1.4 1 WV-13992 1.6 1 WV-13993 1.2 1 WV-13994 1.2 0.6 WV-13995 1.1 0.9 WV-13996 1.4 1 WV-13997 1.2 1.3 WV-13998 1.2 0.8 WV-13999 1.2 1.3 WV-14000 0.9 0.9 WV-14001 1.1 1.5 WV-14002 1 1.1 WV-14003 2 2.1 WV-14004 1.9 1.2 WV-14005 1.1 1 WV-14006 1.2 1.4 WV-14007 1.3 1.7 WV-14008 1.4 1.1 WV-14009 1.3 1.3 WV-14010 1 1.1 WV-14011 3.2 3.7 WV-14012 1.8 2 WV-14013 1.4 1.8 WV-14014 1.1 1.3 WV-14015 1.1 1.3 WV-14016 1.2 1.5 WV-14017 1.5 1.5 WV-14018 0.8 1 WV-14019 1.2 1.4 WV-14020 1 1 WV-14021 1 1.3 WV-14022 1.3 1.5 WV-14023 1.3 1.7 WV-14024 1.2 1.2 WV-14025 1.5 1.6 WV-14026 2.4 0.6 WV-14027 1.2 1.2 WV-14028 1.1 1.2 WV-14029 1.2 1.4 WV-14030 1.3 1.6 WV-14031 1.3 1.6 WV-14032 1.2 1.5 WV-14033 1.3 WV-14034 1.1 1.2 WV-14035 1.2 1.4 WV-14036 1.1 1.1 WV-14037 1.1 1.2 WV-14038 1.4 1.4 WV-14039 1.2 1.2 WV-14040 2.2 3 WV-14041 2.3 2.4 WV-14042 1.3 1.3 WV-14043 1.1 1.4 WV-14044 1.3 1.5 WV-14045 1.8 2.1 WV-14046 1.3 1.6 WV-14047 1.2 1.6 WV-14048 3.8 4.9 WV-14049 2.1 2.6 WV-14050 1.4 1.5 WV-14051 1.5 1.7 WV-14052 1.4 2.2 WV-14053 1.5 1.4 WV-14054 1.4 1.8 WV-14055 1.3 1.6 WV-14056 1.3 1.4 WV-14057 1.7 2.1 WV-14058 1.8 1.4

(1316) TABLE-US-00069 TABLE 22A.3 Example data of certain oligonucleotides. Biological Biological Rep1 Rep2 mock 0.9 mock 0.8 1 mock 1 1.4 mock 1 0.5 mock 1.9 1.2 mock 0.7 0.7 mock 0.9 0.6 mock 0.3 1.6 WV-13964 0.8 1 WV-13965 0.8 0.7 WV-13966 1 0.7 WV-13967 1.2 0.9 WV-13969 1.2 1.3 WV-13971 0.5 WV-13972 0.9 1.3 WV-13973 0.6 1.4 WV-13976 1.3 1.6 WV-13979 0.5 0.3 WV-13980 1.4 0.6 WV-13981 0.8 1.3 WV-13982 1.1 1 WV-13983 1 0.8 WV-13984 0.8 0.4 WV-13985 1.3 1.6 WV-13987 1.4 1.1 WV-13988 1.4 1 WV-13989 1.5 0.7 WV-13990 1.3 0.6 WV-13991 1.3 0.8 WV-13992 1.6 2.4 WV-13993 0.9 0.9 WV-13994 0.6 1 WV-13995 0.9 1.6 WV-13996 1.2 0.8 WV-13997 1.4 0.7 WV-13998 1.2 0.8 WV-13999 0.9 0.9 WV-14000 0.6 0.3 WV-14001 0.8 0.9 WV-14002 0.6 1.3 WV-14003 2.1 2 WV-14004 2.1 0.7 WV-14005 0.9 0.8 WV-14006 1.3 1.1 WV-14007 0.9 1.6 WV-14008 1.3 1.1 WV-14009 0.9 1 WV-14010 1 0.6 WV-14011 3.1 4.7 WV-14010 1 0.6 WV-14011 3.1 4.7 WV-14012 1.3 1.7 WV-14013 0.9 1 WV-14014 0.9 1.1 WV-14015 0.4 1.2 WV-14016 0.4 2.1 WV-14017 1.4 1.3 WV-14018 0.8 0.7 WV-14019 1.3 1.5 WV-14020 0.6 1.2 WV-14021 1.2 1.4 WV-14022 1.6 1.6 WV-14023 1.2 1.3 WV-14024 1.4 1.1 WV-14025 0.5 1.6 WV-14026 1.9 WV-14027 1.1 0.9 WV-14028 0.8 1 WV-14029 1.1 1.3 WV-14030 1.2 1.4 WV-14031 1.2 1.5 WV-14032 0.9 1.7 WV-14033 0.9 WV-14034 0.8 1.1 WV-14035 1.3 1.1 WV-14036 0.7 0.9 WV-14037 1.2 1 WV-14038 1.4 1.6 WV-14039 1.1 0.5 WV-14040 2.5 4.4 WV-14041 2 2.8 WV-14042 1.4 1.2 WV-14043 1.4 1.4 WV-14044 1.7 1.2 WV-14045 1.7 2 WV-14046 1.1 1.9 WV-14047 1.3 0 WV-14048 3.1 7.1 WV-14049 1.9 2.5 WV-14050 1.6 1.4 WV-14051 1.8 1.7 WV-14052 0.9 2.6 WV-14053 1.1 1.8 WV-14054 1.2 2 WV-14055 1.2 2 WV-14056 1.4 0.9 WV-14057 1.5 1.9 WV-14058 1.3 1

(1317) In some embodiments, a composition capable of mediating exon skipping of a particular DMD exon comprises two or more oligonucleotides targeting a particular exon. In some embodiments, a combination of two or more oligonucleotides provides skipping levels significantly higher than the addition of the skipping level of each oligonucleotide individually. In some embodiments, a combination of two or more oligonucleotides provides significant (1%, 5%, 10%, or more) and/or detectable levels of skipping while each oligonucleotide individually does not provide detectable levels of skipping. Combinations of traditional oligonucleotides (e.g., stereorandom oligonucleotide and/or oligonucleotides without non-negatively charged internucleotidic linkages described in the present disclosure) has been reported to provide certain improved effects, e.g., in Wilton et al. 2007 Mol. Ther. 7:1288-1296 (exons 10, 20, 34, 65, etc.). Among other things, provided combinations comprise at least one oligonucleotide comprising one or more chirally controlled internucleotidic linkages and/or one or more non-negatively charged internucleotidic linkages, and can provide significantly increased levels of exon skipping.

(1318) Among other things, the present disclosure recognizes that certain exons are particularly challenging for skipping. For example, in one report, for exons 47 and 57. individual DMD oligonucleotides were not capable of mediating exon skipping, but pairs of oligonucleotides were capable of mediating exon skipping. In one report, effective skipping of exon 45 was mediated by combining two DMD oligonucleotides which were individually not effective in skipping of this exon. Aartsma-Rus et al. 2006 Mol. Ther. 14:401. Aartsma-Rus et al. 2006 Mol. Ther. 14:401. In some embodiments, the present disclosure provides oligonucleotides (e.g., chirally controlled oligonucleotides), and compositions and methods of use thereof, for exon skipping of such challenging exons. With chemistry modifications and/or stereochemistry technologies described herein, the present disclosure provides technologies with greatly improved exon skipping efficiency. In some embodiments, the present disclosure provides single oligonucleotide (e.g., a chirally controlled oligonucleotide) and compositions thereof (e.g., a chirally controlled oligonucleotide composition) for exon skipping of one or more exons that are challenging to skip. In some embodiments, the present disclosure provides combinations of oligonucleotides (e.g., chirally controlled oligonucleotides) and compositions thereof (e.g., chirally controlled oligonucleotide compositions) for exon skipping of one or more exons that are challenging to skip. In some embodiments, combinations of DMD oligonucleotides targeting the same exon mediate increased exon skipping levels relative to individual DMD oligonucleotides.

(1319) In some embodiments, a composition comprises two or more DMD oligonucleotides, wherein each individual DMD oligonucleotide mediates low levels of exon skipping, while the combination mediates a higher level of skipping (higher than the addition of levels achieved by each oligonucleotide individually).

(1320) In some embodiments, a composition comprises two or more DMD oligonucleotides, wherein the oligonucleotides target different exons.

(1321) In some embodiments, a combination of multiple DMD oligonucleotides targeting different exons is capable of mediating skipping of two or more (e.g., multiple) exons.

(1322) In some embodiments, a composition comprises two or more DMD oligonucleotides. In some embodiments, a composition comprises two or more DMD oligonucleotides, at least one of which is described herein or has a base sequence, stereochemistry or other chemical characteristic described herein.

(1323) Oligonucleotides Comprising Non-Negatively Charged Internucleotidic Linkages Can Provide Significantly Improved Activities.

(1324) In some embodiments, the present disclosure provides oligonucleotides comprising one or more non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, the present disclosure provides oligonucleotides comprising one or more neutral internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

(1325) In some embodiments, a non-negatively charged internucleotidic linkage comprises a triazole moiety. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(1326) ##STR00494##
In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(1327) ##STR00495##
In some embodiments, a non-negatively charged internucleotidic linkage comprises a substituted triazolyl group. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(1328) ##STR00496##
wherein W is O or S. In some embodiments, a non-negatively charged internucleotidic linkage comprises an optionally substituted alkynyl group. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of

(1329) ##STR00497##
wherein W is O or S.

(1330) In some embodiments, the present disclosure provides oligonucleotides comprising an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, which comprises a cyclic guanidine moiety. In some embodiments, an internucleotidic linkage comprises a cyclic guanidine and has the structure of:

(1331) ##STR00498##
In some embodiments, an internucleotidic linkage, e.g., a non-negatively charged internucleotidic linkage, comprising a cyclic guanidine is stereochemically controlled.

(1332) In some embodiments, a non-negatively charged internucleotidic linkage, or a neutral internucleotidic linkage, is or comprising a structure selected from

(1333) ##STR00499##
wherein W is O or S. In some embodiments, a non-negatively charged internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, a neutral internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, a nucleic acid or an oligonucleotide comprising a modified internucleotidic linkage comprising a cyclic guanidine moiety is a siRNA, double-straned siRNA, single-stranded siRNA, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir, ribozyme, Ul adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant.

(1334) In some embodiments, an oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide comprises a neutral internucleotidic linkage and a chirally controlled internucleotidic linkage which is a phosphorothioate in the Rp or Sp configuration. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more non-negatively charged internucleotidic linkages and one or more phosphorothioate internucleotidic linkage, wherein each phosphorothioate internucleotidic linkage in the oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, the present disclosure provides an oligonucleotide comprising one or more neutral internucleotidic linkages and one or more phosphorothioate internucleotidic linkage, wherein each phosphorothioate internucleotidic linkage in the oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, a provided oligonucleotide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled phosphorothioate internucleotidic linkages.

(1335) Without wishing to be bound by any particular theory, the present disclosure notes that a neutral internucleotidic linkage is more hydrophobic than a phosphorothioate internucleotidic linkage (PS), which is more hydrophobic than a phosphodiester linkage (natural phosphate linkage, PO). Typically, unlike a PS or PO, a neutral internucleotidic linkage bears less charge. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages into an oligonucleotide may increase oligonucleotides' ability to be taken up by a cell and/or to escape from endosomes. Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more neutral internucleotidic linkages can be utilized to modulate melting temperature between an oligonucleotide and its target nucleic acid.

(1336) Without wishing to be bound by any particular theory, the present disclosure notes that incorporation of one or more non-negatively charged internucleotidic linkages, e.g., neutral internucleotidic linkages, into an oligonucleotide may be able to increase the oligonucleotide's ability to mediate a function such as exon skipping or gene knockdown. In some embodiments, an oligonucleotide capable of altering skipping of one or more exons in a target gene comprises one or more neutral internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating skipping of an exon(s) in a target gene comprises one or more neutral internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating skipping of one or more DMD exon(s) comprises one or more neutral internucleotidic linkages.

(1337) In some embodiments, an oligonucleotide capable of mediating knockdown of level of a nucleic acid or a product encoded thereby comprises one or more non-negatively charged internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating knockdown of expression of a target gene comprises one or more non-negatively charged internucleotidic linkages. In some embodiments, an oligonucleotide capable of mediating knockdown of expression of a target gene comprises one or more neutral internucleotidic linkages.

(1338) In some embodiments, a non-negatively charged internucleotidic linkage is not chirally controlled. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Rp. In some embodiments, a non-negatively charged internucleotidic linkage is chirally controlled and its linkage phosphorus is Sp.

(1339) In some embodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-negatively charged internucleotidic linkages. In some embodiments, a provided oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more neutral internucleotidic linkages. In some embodiments, each of non-negatively charged internucleotidic linkage and/or neutral internucleotidic linkages is optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotidic linkage in an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, each neutral internucleotidic linkage in an oligonucleotide is independently a chirally controlled internucleotidic linkage. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1340) ##STR00500##
wherein W is O or S. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1341) ##STR00501##
In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1342) ##STR00502##
In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1343) ##STR00503##
wherein W is O or S. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1344) ##STR00504##
In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1345) ##STR00505##
In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1346) ##STR00506##
wherein W is O or S. In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1347) ##STR00507##
In some embodiments, at least one non-negatively charged internucleotidic linkage/neutral internucleotidic linkage has the structure of

(1348) ##STR00508##
In some embodiments, a provided oligonucleotide comprises at least one non-negatively charged internucleotidic linkage wherein its linkage phosphorus is in Rp configuration, and at least one non-negatively charged internucleotidic linkage wherein its linkage phosphorus is in Sp configuration.

(1349) In some embodiments, an oligonucleotide capable of increasing the frequency of skipping of an exon of a target gene comprises a non-negatively charged internucleotidic linkage. In some embodiments, an oligonucleotide capable of increasing the frequency of skipping of an exon of a target gene comprises a non-negatively charged internucleotidic linkage and is useful for treatment of a disease wherein the exon comprises a deleterious or disease-associated mutation. A non-limiting example is the DMD gene, wherein the skipping of an exon comprising a mutation contributes to muscular dystrophy.

(1350) Various oligonucleotides, including DMD oligonucleotides, that comprise one or more non-negatively charged internucleotidic linkages/neutral internucleotidic linkages were designed and/or constructed and/or tested, for example, WV-11343, WV-11344, WV-11345, WV-11346, WV-11347, WV-11237, WV-11238, WV-11239, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, WV-12136, WV-11340, WV-11341, WV-11342, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, WV-12873, etc. Example DMD oligonucleotides for skipping exon 23 and comprising a non-negatively charged internucleotidic linkage (e.g., a neutral internucleotidic linkage) include: WV-11343, WV-11344, WV-11345, WV-11346, and WV-11347. Example DMD oligonucleotides for skipping exon 51 and comprising a non-negatively charged internucleotidic linkage (e.g., a neutral internucleotidic linkage) include: WV-11237, WV-11238, WV-11239, WV-12130, WV-12131, WV-12132, WV-12133, WV-12134, WV-12135, and WV-12136. Example DMD oligonucleotides for skipping exon 53 and comprising a non-negatively charged internucleotidic linkage (e.g., a neutral internucleotidic linkage) include: WV-11340, WV-11341, WV-11342, WV-12123, WV-12124, WV-12125, WV-12126, WV-12127, WV-12128, WV-12129, WV-12553, WV-12554, WV-12555, WV-12556, WV-12557, WV-12558, WV-12559, WV-12872, and WV-12873. Certain oligonucleotides are in Table A1.

(1351) Additional DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage were designed and/or constructed. These include DMD oligonucleotides for skipping DMD exon 45, WV-14528, WV-14529, WV-14532, and WV-14533.

(1352) The efficacy of various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in skipping DMD exon 45 is shown in Table 1B.1 and Table 1B.2 herein.

(1353) The efficacy of various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in skipping DMD exon 53 is shown in Table 21E, Table 21F, Table 21G, and Table 21H herein.

(1354) In some embodiments, a non-negatively charged internucleotidic linkage may be designated as nX if stereorandom, or nS chirally controlled and linkage phosphorus in the Sp configuration, or nR if chirally controlled and the linkage phosphorus in the Rp configuration.

(1355) In some embodiments, a non-negatively charged internucleotidic linkage may be designated as n001 if stereorandom, or n001S chirally controlled and linkage phosphorus in the Sp configuration, or n001R if chirally controlled and the linkage phosphorus in the Rp configuration (e.g., in Table A1).

(1356) Various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in the Rp configuration were constructed, including WV-12872, WV-13408, WV-12554, WV-13409, WV-12555, and WV-12556.

(1357) Various DMD oligonucleotides comprising a non-negatively charged internucleotidic linkage in the Sp configuration were constructed, including WV-12557, WV-12558, and WV-12559.

(1358) Data showing activity and stability of various oligonucleotides comprising a non-negatively charged internucleotidic linkage in the Rp or Sp configuration are shown in Table 21H Table 21I, Table 21I.1, and Table 21I.2

(1359) Several oligonucleotides (including WV-9517, WV-13864, WV-13835, and WV-14791) were tested at various concentrations up to 30 uM for TLR9 activation in HEK-blue-TLR9 cells (16 hour gymnotic uptake). WV-13864 and WV-14791 comprise a chirally controlled non-negatively charged internucleotidic linkage in the Rp configuration. WV-9517, WV-13864, WV-13835, and WV-14791 did not exhibit significant TLR9 activation (data not shown).

(1360) Several oligonucleotides which target a gene other than DMD were designed and/or constructed which comprise a non-negatively charged internucleotidic linkage.

(1361) Below are presented oligonucleotides comprising a cyclic guanidine moiety which target DMD or Malat-1 (Malat1). The DMD oligonucleotides are designed to mediate skipping of exon 23 (in mouse) or exon 51 or exon 53 (in human). The Malat-1 oligonucleotides are designed to for Malat1 mRNA knockdown, e.g., mediated through RNase H.

(1362) TABLE-US-00070 TABLE22B ExampleMalat-1oligonucleotidescomprisinganeutralbackbone. Oligonucleotide Description Stereochemistry WV-11533 mU*SGeon001m5Ceon001m5Ceon001mA*SG*SG* SnXnXnXSSRSSR RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* SSRSSSSSS SmC*SmU*SmC(SEQIDNO:3169) WV-12504 Mod001L00mU*SGeon001m5Ceon001m5Ceon001mA* OSnXnXnXSSRSS SG*SG*RC*ST*SG*RG*ST*ST*RA*ST*SmG RSSRSSSSSS *SmA*SmC*SmU*SmC(SEQIDNO:3170) WV-12505 L001mU*SGeon001m5Ceon001m5Ceon001mA*SG*SG OSnXnXnXSSRSS *RC*ST*SG*RG*ST*ST*RA*ST*SmG*SmA* RSSRSSSSSS SmC*SmU*SmC(SEQIDNO:3171)
All of these oligonucleotides have the base sequence of UGCCAGGCTGGTTATGACUC (SEQ ID NO: 3168).

(1363) Oligonucleotides comprising non-negatively charged internucleotidic linkages and targeting other gene targets were also designed, constructed and/or tested for their properties and activities, including activities for reducing levels of target mRNAs and/or proteins, e.g., via RNaseH-mediated knockdown. Such oligonucleotides are active in reducing target levels.

(1364) Various Malat1 oligonucleotides were designed, constructed and tested which comprise a non-negatively charged internucleotidic linkage. Various Malat1 oligonucleotides comprise 1, 2 or 3 non-negatively charged internucleotidic linkages in a wing and/or a core.

(1365) TABLE-US-00071 TABLE22C Malat1oligonucleotides SEQID Oligonucleotide Sequence NO: Stereochemistry WV-8587 mU*SGeom5Ceom5CeomA*SG*SG*RC*ST*SG*RG 3173 SOOOSSRSSR *ST*ST*RA*ST*SmG*SmA*SmC*SmU*SmC SSRSSSSSS WV-14733 mU*SGeom5Ceom5CeomA*SG*SG*SC*ST*SG*SG 3174 SOOOSSSSSS *ST*ST*SA*ST*SmG*SmA*SmC*SmU*SmC SSSSSSSSS WV-15351 mU*SGeom5Ceom5CeomA*SG*SGn001C*ST* 3175 SOOOSSIASS SGn001G*ST*STn001A*ST*SmG*SmA*SmC*SmU nXSSnXSSSSSS *SmC WV-15352 mU*SGeom5Ceom5CeomA*SG*SGn001C*ST*SG* 3176 SOOOSSnXSS RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU*SmC RSSRSSSSSS WV-15353 mU*SGeom5Ceom5CeomA*SG*SG*RC*ST* 3177 SOOOSSRSSnX SGn001G*ST*ST*RA*ST*SmG*SmA*SmC*SmU* SSRSSSSSS SmC WV-15354 mU*SGeom5Ceom5CeomA*SG*SG*RC*ST*SG*RG 3178 SOOOSSRSSRSS *ST*STn001A*ST*SmG*SmA*SmC*SmU*SmC nXSSSSSS WV-15356 mU*SGeom5Ceom5CeomA*SG*SG*RCn001Tn001G* 3179 SOOOSSRnXnX RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU*SmC RSSRSSSSSS WV-15357 mU*SGeom5Ceom5CeomA*SG*SG*RC*ST*SG* 3180 SOOOSSRSSR RGn001Tn001T*RA*ST*SmG*SmA*SmC*SmU*S nXnXRSSSSSS mC WV-15358 mU*SGeom5Ceom5CeomA*SG*SG*RC*ST*SG*RG 3181 SOOOSSRSSRS *ST*ST*RAn001Tn001mG*SmA*SmC*SmU*SmC SRnXnXSSSS WV-8582 mU*SGeom5Ceom5CeomA*SG*SG*SC*ST*SG*SG 3182 SOOOSSSSSSS *ST*ST*RA*ST*SmG*SmA*SmC*SmU*SmC SRSSSSSS WV-15359 mU*SGeom5Ceom5CeomA*SG*SG*SC*ST*SG*SG 3183 SOOOSSSSSSS *ST*STn001An001Tn001mG*SmA*SmC*SmU*SmC SnXnXnXSSSS WV-15360 mU*SGeom5Ceom5CeomA*SG*SG*SC*ST*SG*SG 3184 SOOOSSSSSSS *ST*STn001A*ST*SmG*SmA*SmC*SmU*SmC SnXSSSSSS WV-15361 mU*SGeom5Ceom5CeomA*SG*SG*SC*ST*SG*SG 3185 SOOOSSSSSSS *ST*ST*RA*STn001mGn001mA*SmC*SmU*SmC SRSnXnXSSS WV-15362 mU*SGeom5Ceom5CeomA*SG*SG*SC*ST*SG*SG 3186 SOOOSSSSSSS *ST*ST*RAn001T*SmG*SmA*SmC*SmU*SmC SRnXSSSSS WV-15363 mU*SGeom5Ceom5CeomA*SG*SG*SC*ST*SG*SG 3187 SOOOSSSSSSS *ST*ST*RA*STn001mG*SmA*SmC*SmU*SmC SRSnXSSSS WV-14556 mUn001Geon001m5Ceon001m5CeomA*SG*SG*RC*ST 3188 nXnXnXOSSRS *SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU SRSSRSSSSSS *SmC WV-14557 mUn001Geon001m5Ceom5Ceon001mA*SG*SG*RC*ST 3189 nXnXOnXSSRS *SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU SRSSRSSSSSS *SmC WV-14558 mUn001Geon001m5Ceom5CeomAn001G*SG*RC*ST* 3190 nXnXOOnXSRS SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU* SRSSRSSSSSS SmC WV-14559 mUn001Geom5Ceon001m5Ceon001mA*SG*SG*RC*ST 3191 nXOnXnXSSRSS *SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU RSSRSSSSSS *SmC WV-14560 mUn001Geom5Ceon001m5CeomAn001G*SG*RC*ST* 3192 nXOnXOnXSRSS SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU* RSSRSSSSSS SmC WV-14561 mUn001Geom5Ceom5Ceon001mAn001G*SG*RC*ST* 3193 nXOnXOnXSRSS SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU* RSSRSSSSSS SmC WV-11533 mU*SGeon001m5Ceon001m5Ceon001mA*SG*SG*RC* 3194 SnXnXnXSSRSS ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*S RSSRSSSSSS mU*SmC WV-14562 mU*SGeon001m5Ceon001m5CeomAn001G*SG*RC*ST 3195 SnXnXOnXSRSS *SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU RSSRSSSSSS *SmC WV-14563 mU*SGeon001m5Ceom5Ceon001mAn001G*SG*RC*ST 3196 SnXOnXnXSRSS *SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU RSSRSSSSSS *SmC WV-14564 mU*SGeom5Ceon001m5Ceon001mAn001G*SG*RC*ST 3197 SOnXnXnXSRSS *SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*SmU RSSRSSSSSS *SmC WV-14349 Mod098L001mU*SGeom5Ceom5CeomA*SG*SG*RC* 3198 OSOOOSSRSSRS ST*SG*RG*ST*ST*RA*ST*SmG*SmA*SmC*S SRSSSSSS mU*SmC
All of the oligonucleotides in this table have the base sequence of UGCCAGGCTGGTTATGACUC (SEQ ID NO: 3172).

(1366) TABLE-US-00072 TABLE 22D Data of Malat1 oligonucleotides 0.004 uM 0.02 uM 0.1 uM WV-8587 1.23 1.21 0.94 0.95 0.84 0.81 0.54 0.53 0.61 WV-14733 1.81 1.06 1.36 1.47 1.12 1.17 0.98 0.97 0.72 WV-15351 1.27 0.92 1.00 0.89 0.95 0.92 0.74 0.66 0.71 WV-15352 1.49 1.78 1.52 0.88 0.83 0.91 0.50 0.52 0.73 WV-15353 0.85 0.91 1.10 0.65 0.59 0.68 0.44 0.42 0.40 WV-15354 1.31 1.00 0.90 0.69 0.94 0.79 0.56 0.87 0.74 WV-15356 0.77 0.87 0.68 0.49 0.67 0.63 0.30 0.35 0.31 WV-15357 0.91 1.02 1.13 0.66 0.75 0.79 0.37 0.32 0.36 WV-15358 0.80 0.82 0.90 0.83 0.85 0.85 0.36 0.45 0.43 WV-8582 1.11 1.06 1.15 1.30 1.15 1.14 0.67 0.85 1.06 WV-15359 1.16 1.26 1.02 0.92 0.83 0.83 0.85 0.90 WV-15360 1.57 1.38 1.31 1.05 0.99 0.83 1.03 0.91 0.80 WV-15361 0.92 1.11 1.00 0.71 0.63 0.68 0.74 1.09 0.73 WV-15362 1.23 1.22 1.07 0.90 0.83 0.82 0.99 0.97 0.80 WV-15363 1.16 1.03 0.85 0.89 0.87 0.90 1.10 1.18 1.01 WV-14556 0.81 0.84 0.91 0.46 0.42 0.58 0.15 0.23 0.17 WV-14557 0.75 1.10 0.96 0.46 0.40 0.54 0.19 0.19 0.21 WV-14558 0.96 1.11 0.90 0.77 1.08 0.78 1.27 0.40 0.45 WV-14559 0.80 0.62 0.75 0.35 0.36 0.37 0.12 0.17 0.13 WV-14560 1.11 0.99 1.03 0.44 0.48 0.60 0.29 0.31 0.15 WV-14561 0.71 0.73 1.04 0.47 0.41 0.48 0.22 0.24 0.16 WV-11533 0.74 0.75 0.87 0.40 0.37 0.41 0.14 0.14 0.09 WV-14562 0.79 0.60 0.60 0.53 0.45 0.64 0.22 0.33 0.24 WV-14563 0.76 0.96 0.79 0.57 0.51 0.53 0.23 0.23 0.24 WV-14564 0.72 0.65 0.70 0.58 0.47 0.50 0.17 0.20 0.21 WV-9491 1.02 0.96 1.28 0.82 0.93 1.27 0.88 0.91 1.06 WV-14349 1.07 1.34 1.03 0.86 0.77 1.11 0.63 0.60 0.79
Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown. WV-9491 is a negative control that is not designed to target Malat1.

(1367) Various Malat1 oligonucleotides were designed, constructed and tested which comprise one or more non-negatively charged internucleotidic linkages in a core. In various embodiments of a Malat1 oligonucleotide, a phosphorothioate in the Rp configuration is replaced by a non-negatively charged internucleotidic linkage.

(1368) TABLE-US-00073 TABLE 22E Data of Malat1 oligonucleotides WV- WV- WV- WV- WV- WV- 8587 15351 15352 15353 15354 9491 0.004 uM 1.23 1.27 1.49 0.85 1.31 1.02 1.21 0.92 1.78 0.91 1.00 0.96 0.94 1.00 1.52 1.10 0.90 1.28 0.02 uM 0.95 0.89 0.88 0.65 0.69 0.82 0.84 0.95 0.83 0.59 0.94 0.93 0.81 0.92 0.91 0.68 0.79 1.27 0.1 uM 0.54 0.74 0.50 0.44 0.56 0.88 0.53 0.66 0.52 0.42 0.87 0.91 0.61 0.71 0.73 0.40 0.74 1.06
Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

(1369) Various Malat1 oligonucleotides were designed, constructed and tested which comprise a non-negatively charged internucleotidic linkage. Various Malat1 oligonucleotides comprise 1 or more non-negatively charged internucleotidic linkages.

(1370) TABLE-US-00074 TABLE 22F Data of certain oligonucleotides. WV- WV- WV- WV- WV- 8587 15356 15357 15358 9491 0.004 uM 1.23 0.77 0.91 0.80 1.02 1.21 0.87 1.02 0.82 0.96 0.94 0.68 1.13 0.90 1.28 0.02 uM 0.95 0.49 0.66 0.83 0.82 0.84 0.67 0.75 0.85 0.93 0.81 0.63 0.79 0.85 1.27 0.1 uM 0.54 0.30 0.37 0.36 0.88 0.53 0.35 0.32 0.45 0.91 0.61 0.31 0.36 0.43 1.06
Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

(1371) Various Malat1 oligonucleotides were designed, constructed and tested which comprise a non-negatively charged internucleotidic linkage. Various Malat1 oligonucleotides comprise 1 or more non-negatively charged internucleotidic linkages. In various tables and throughout the text herein, the presence or absence of a hyphen in the designation of an oligonucleotide is irrelevant. For example, WV8582 is equivalent to WV-8582.

(1372) TABLE-US-00075 TABLE 22G Data of certain oligonucleotides. WV- WV- WV- WV- WV- WV- WV- 8582 15359 15360 15361 15362 15363 9491 0.004 uM 1.11 1.16 1.57 0.92 1.23 1.16 1.02 1.06 1.26 1.38 1.11 1.22 1.03 0.96 1.15 1.02 1.31 1.00 1.07 0.85 1.28 0.02 uM 1.30 0.92 1.05 0.71 0.90 0.89 0.82 1.15 0.83 0.99 0.63 0.83 0.87 0.93 1.14 0.83 0.83 0.68 0.82 0.90 1.27 0.1 uM 0.67 0.85 1.03 0.74 0.99 1.10 0.88 0.85 0.91 1.09 0.97 1.18 0.91 1.06 0.90 0.80 0.73 0.80 1.01 1.06
Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

(1373) Various Malat1 oligonucleotides were designed, constructed and tested which comprise a non-negatively charged internucleotidic linkage. Various Malat1 oligonucleotides comprise 1 or more non-negatively charged internucleotidic linkages.

(1374) TABLE-US-00076 TABLE 22H Data of certain oligonucleotides. 0.004 uM 0.02 uM WV-11533 0.74 0.75 0.87 0.40 0.37 0.41 WV-14556 0.81 0.84 0.91 0.46 0.42 0.58 WV-14557 0.75 1.10 0.96 0.46 0.40 0.54 WV-14558 0.96 1.11 0.90 0.77 1.08 0.78 WV-14559 0.80 0.62 0.75 0.35 0.36 0.37 WV-14560 1.11 0.99 1.03 0.44 0.48 0.60 WV-14561 0.71 0.73 1.04 0.47 0.41 0.48 WV-14562 0.79 0.60 0.60 0.53 0.45 0.64 WV-14563 0.76 0.96 0.79 0.57 0.51 0.53 WV-14564 0.72 0.65 0.70 0.58 0.47 0.50 WV-9491 1.02 0.96 1.28 0.82 0.93 1.27 0.1 uM WV-11533 0.14 0.14 0.09 WV-14556 0.15 0.23 0.17 WV-14557 0.19 0.19 0.21 WV-14558 1.27 0.40 0.45 WV-14559 0.12 0.17 0.13 WV-14560 0.29 0.31 0.15 WV-14561 0.22 0.24 0.16 WV-14562 0.22 0.33 0.24 WV-14563 0.23 0.23 0.24 WV-14564 0.17 0.20 0.21 WV-9491 0.88 0.91 1.06
Numbers represent knockdown of Malat1 mRNA relative to HPRT1, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

(1375) In some embodiments, oligonucleotides were designed, constructed and tested in vitro against suitable reference oligonucleotides which do not comprise any non-negatively charged internucleotidic linkages, e.g., in iCell Astrocytes, at several suitable doses (e.g., 0, 0.014, 0.041, 0.123, 0.37, 1.11, 3.33, 10 uM) gymnotic for a suitable period of time, e.g., 2 days.

(1376) Tables 23, 24 and 25 present experimental results.

(1377) TABLE-US-00077 TABLE 23 Data of certain oligonucleotides. Oliogonucleotide tested Dose (Relative fold change Malat1/HPRT1) (uM) WV-8587 WV-9696 0 0.924 0.970 1.106 1.162 1.040 0.799 0.013717 0.833 0.930 0.730 0.997 0.844 0.918 0.041152 1.186 0.868 0.874 1.076 0.957 0.844 0.123457 0.772 0.827 0.658 0.970 0.756 0.821 0.37037 0.610 0.610 0.553 0.821 0.520 0.681 1.111111 0.394 0.360 0.425 0.431 0.419 0.402 3.333333 0.157 0.136 0.162 0.225 0.214 0.220 10 0.051 0.052 0.065 0.090 0.086 0.091 Oliogonucleotide tested Dose (Relative fold change Malat1/HPRT1) (uM) WV-11114 WV-11533 0 0.761 0.881 1.212 0.958 0.985 1.056 0.013717 1.048 1.027 1.187 0.900 0.932 1.020 0.041152 0.912 0.958 1.108 0.453 0.503 0.479 0.123457 0.971 1.063 1.238 0.356 0.387 0.332 0.37037 0.706 0.846 0.692 0.105 0.107 0.096 1.111111 0.429 0.486 0.574 0.048 0.051 0.049 3.333333 0.181 0.196 0.203 0.033 0.032 0.030 10 0.080 0.075 0.087 0.026 0.034 0.031
Numbers represent knockdown of Malat1 mRNA, wherein 1.000 would represent no (0.0%) knockdown and 0.000 represents 100.0% knockdown; results from replicate experiments are shown.

(1378) TABLE-US-00078 TABLE 24 IC50 of certain Malat1 oligonucleotides. Oligonucleotide IC50 WV-8587 757 nM WV-9696 806 nM WV-11114 894 nM WV-11533 49 nM

(1379) Among other things, the present disclosure demonstrates that oligonucleotides comprising one or more non-negatively charged internucleotidic linkages can provide dramatically improved activitiesas illustrated in Table 24, more than 15-fold improvement can be achieved in terms of IC50.

(1380) In another experiment, several Malat1 oligonucleotides including WV-11533, which comprises three neutral internucleotidic linkages, were assessed for knockdown of Malat1, measured by a decrease in the abundance of a Malat1 RNA, WV-7772, which is complementary to the tested oligonucleotides, in the presence of RNaseH.

(1381) TABLE-US-00079 Oligo- SEQID Linkage/ nucleotide Description NO: NakedSequence Stereochemistry WV-11533 mU*SGeon001m5Ceon001m5Ceon001mA 3199 UGCCAGGCTG SnXnXnXSSRSSRS *SG*SG*RC*ST*SG*RG*ST*ST* GTTATGACUC SRSSSSSS RA*ST*SmG*SmA*SmC*SmU*SmC WV-8556 mU*Geom5Ceom5CeomA*G*G*C*T 3200 UGCCAGGCTGG XOOOXXXXXX *G*G*T*T*A*T*mG*mA*mC* TTATGACUC XXXXXXXXX mU*mC WV-8587 mU*SGeom5Ceom5CeomA*SG*SG* 3201 UGCCAGGCTGG SOOOSSRSSRSS RC*ST*SG*RG*ST*ST*RA*ST* TTATGACUC RSSSSSS SmG*SmA*SmC*SmU*SmC WV-7772 rCrUrGrArGrUrCrArUrArArCrCrA 3202 CUGAGUCAUAAC OOOOOOOOOOOO rGrCrCrUrGrGrCrA CAGCCUGGCA OOOOOOOOO WV-9696 L001mU*SGeom5Ceom5CeomA*SG*SG OSOOOSSRSSRS *RC*ST*SG*RG*ST*ST*RA*ST* 3203 UGCCAGGCT SRSSSSSS SmG*SmA*SmC*SmU*SmC GGTTATGACUC WV-11114 Mod091L001mU*SGeom5Ceom5CeomA* OSOOOSSRSSRS SG*SG*RC*sT*SG*RG*ST*ST* 3204 UGCCAGGCT SRSSSSSS RA*ST*SmG*SmA*SmC*SmU*SmC GGTTATGACUC

(1382) At a time point of 45 minutes, less than 20% of the Malat1 RNA remained in the presence of RNase H and WV-11533 or WV-8587, indicating greater than 80% knockdown; and about 60% of the Malat1 RNA remained in the presence of RNase H and WV-8556, which is stereorandom and does not comprise a neutral backbone. Among other things, the present disclosure demonstrates that oligonucleotides comprising non-negatively charged internucleotidic linkages and/or chirally controlled internucleotidic linkages showed significantly improved activities in reducing levels of target nucleic acids, e.g., through RNase H-mediated knockdown.

(1383) Certain oligonucleotides were also tested for stability in rat liver homogenate at 0, 1 and 2 days. For both WV-11533 and WV-8587, over 80% of the full-length oligonucleotide remained at 2 days; about 40% of the stereorandom WV-8556 remained.

(1384) Oligonucleotides were also tested for Tm with the Malat1 RNA, WV-7772. One example set of test conditions: 1 M Duplex in 1PBS (pH 7.2); Temperature Range: 15 C.-90 C.; Temperature Rate: 0.5 C./min; Measurement Interval: 0.5 C. The results showed the following duplex Tm ( C.) with WV-7772: WV-8556, 73.52; WV-8587, 69.57; and WV-11533, 68.67.

(1385) In some embodiments, oligonucleotides comprising non-negatively charged internucleotidic linkages provide improved splicing modulation activities. Various oligonucleotides for mediating skipping of an exon in DMD were prepared and/or tested, wherein the oligonucleotides comprise non-negatively charged internucleotidic linkages. Certain oligonucleotides comprising non-negatively charged internucleotidic linkages are listed in Table A1.

(1386) TABLE-US-00080 TABLE 25 A Example data of certain oligonucleotides. Oligonucleotide 10 uM 3 uM WV-9898 27.13 13.38 11.27 9.69 WV-9897 33.61 31.46 11.82 9.52 WV-9517 20.21 12.08 6.72 6.89 WV-11342 44.84 41.17 19.22 18.43 WV-11341 38.85 44.85 18.95 20.63 WV-11340 41.51 43.08 17.79 16.4 PMO 3.89 4.05 2.08 1.52 Mock 0.49 0.53 0.45 0.52
Numbers indicate the level of exon skipping; e.g., 27.13 in column 2, row 2, represents 27.13% skipping of a DMD exon. Oligonucleotides were tested in vitro on cells at 10 or 3 uM.

(1387) TABLE-US-00081 TABLE 25B Example data of certain oligonucleotides. Mock WV-11237 WV-3152 WV-3516 PMO 10 um 1 49 35 7 3 3 uM 1 22 16 3 2
Numbers indicate the level of exon skipping relative to control; numbers are approximate.
Oligonucleotides were tested in vitro on cells at 10 or 3 uM.
PMO indicates an all-PMO oligonucleotide.

(1388) Various DMD oligonucleotides for skipping exon 23 in mouse were constructed, several of which comprise a non-negatively charged internucleotidic linkage, including WV-11343, WV-11344, WV-11345, WV-11346, and WV-11347. These oligonucleotides were tested and demonstrated skipping of exon 23, as shown in the table below.

(1389) TABLE-US-00082 TABLE 25C.1 Example data of certain oligonucleotides. 10 uM 3.3 uM WV-7684 5 2 WV-10256 25 13 WV-11343 44 33 WV-10257 16 10 WV-11344 42 29 WV-10258 22 20 WV-11345 48 39 WV-10259 24 10 WV-11346 43 32 WV-10260 23 14 WV-11347 43 32
Numbers represent exon 23 skipping level relative to control.

(1390) In some experiments, del45-52 cells (patient derived myoblasts) were treated with various oligonucleotides, including WV-13405 (PMO), WV-9517 and WV-9898, in muscle differentiation medium at 15, 10, 3.3, 1.1, 0.3, 0.1 and 0 uM under free uptake conditions for 6 days before being collected and analyzed for dystrophin protein restoration by Western blot. WV-9517 and WV-9898 demonstrated significant DMD production at concentrations of 3.3 uM and higher; WV-13405 did not show significant DMD product at a concentration of 3.3 uM, but did show DMD production at concentrations of 10 and 15 uM. Control was Vinculin.

(1391) As shown in Table 25D, additional oligonucleotides were constructed which were capable of mediating skipping of exon 53 and which comprise at least one neutral internucleotidic linkage.

(1392) Various additional DMD oligonucleotides for skipping exon 23 in mouse were constructed. These oligonucleotides were tested and demonstrated skipping of exon 23, as shown in the table below.

(1393) TABLE-US-00083 TABLE 25C.2 Example data of certain oligonucleotides. WV-11345 WV-24092 WV-24098 Mock 10 uM 37.8 39.8 30.2 32.4 41.5 40.2 0 0 3.3 uM 22.4 22.9 13.4 14.5 24.3 23.5 0 0 1.1 uM 9.2 8.1 3 3.1 10.5 9.9 0 0
DMD oligonucleotides were tested in vitro for their ability to skip DMD exon 23 in H2K murine cells. Oligonucleotide delivery was gymnotic, and 4 day treatment was used.
Numbers represent exon 23 skipping level relative to control. 100.0 would represent 100% of transcripts skipped; 0 would represent 0% of transcripts skipped. Data from replicates are shown.

(1394) TABLE-US-00084 TABLE 25C.3 Example data of certain oligonucleotides. 10 uM 3.3 uM 1.1 uM WV-10258 22.9 11.6 3.8 WV-12885 34.2 17.8 6.1 32.4 18.6 6.9 WV-23576 23.7 10.6 3.8 25.6 11.5 3.3 WV-23577 23.3 13.9 6.6 WV-23578 22 11.8 4.9 16.1 13.9 7.1 WV-23579 19.2 8.3 6.7 20.7 29.8 5.5 WV-23937 18.8 9.2 3.5 6.3 4.2 1.3 WV-23938 26.4 16 6.9 30.3 16.7 7.3 WV-23939 35.2 23.3 11.8 33.6 22 12.9 Mock 0 0 0 0 0 0
DMD oligonucleotides were tested in vitro for their ability to skip DMD exon 23 in H2K murine cells. Oligonucleotide delivery was gymnotic, and 4 day treatment was used.
Numbers represent exon 23 skipping level relative to control. 100.0 would represent 100% of transcripts skipped; 0 would represent 0% of transcripts skipped. Data from replicates are shown.

(1395) TABLE-US-00085 TABLE 25C.4 Example data of certain oligonucleotides. WV- WV- WV- WV- 10258 25536 25537 25539 Mock 10 uM 22.9 2.3 10.7 11.8 15.1 12.5 8.1 0 0 3.3 uM 11.6 1.5 3.6 7.3 9.9 5.6 3.8 0 0 1.1 uM 3.8 1.1 1.3 2.7 4.2 1.8 2.3 0 0
DMD oligonucleotides were tested in vitro for their ability to skip DMD exon 23 in H2K murine cells. Oligonucleotide delivery was gymnotic, and 4 day treatment was used. Some of the tested oligonucleotides comprise one or more LNA.
Numbers represent exon 23 skipping level relative to control. 100.0 would represent 100% of transcripts skipped; 0 would represent 0% of transcripts skipped. Data from replicates are shown.

(1396) TABLE-US-00086 TABLE 25C.5 Example data of certain oligonucleotides. 10 uM 3.3 uM 1.1 uM WV- 22.9 11.6 3.8 10258 WV- 37.8 22.4 9.2 11345 39.8 22.9 8.1 WV- 34.2 17.8 6.1 12885 32.4 18.6 6.9 WV- 23.7 10.6 3.8 23576 25.6 11.5 3.3 WV- 23.3 13.9 6.6 23577 WV- 22 11.8 4.9 23578 16.1 13.9 7.1 WV- 19.2 8.3 6.7 23579 20.7 29.8 5.5 WV- 18.8 9.2 3.5 23937 6.3 4.2 1.3 WV- 26.4 16 6.9 23938 30.3 16.7 7.3 WV- 35.2 23.3 11.8 23939 33.6 22 12.9 WV- 30.2 13.4 3 24092 32.4 14.5 3.1 WV- 41.5 24.3 10.5 24098 40.2 23.5 9.9 WV- 2.3 1.5 1.1 25536 10.7 3.6 1.3 WV- 11.8 7.3 2.7 25537 15.1 9.9 4.2 WV- 12.5 5.6 1.8 25539 8.1 3.8 2.3 Mock 0 0 0 0 0 0
DMD oligonucleotides were tested in vitro for their ability to skip DMD exon 23 in H2K murine cells. Oligonucleotide delivery was gymnotic, and 4 day treatment was used. Some of the tested oligonucleotides comprise one or more non-negatively charged internucleotidic linkage.
Numbers represent exon 23 skipping level relative to control. 100.0 would represent 100% of transcripts skipped; 0 would represent 0% of transcripts skipped. Data from replicates are shown.

(1397) TABLE-US-00087 TABLE 25C.6 Example data of certain oligonucleotides. Conc. WV-24104 WV-24109 4.70927 0.891 0.837 0.814 1.059 4.40824 0.942 1.052 0.765 1.208 4.10721 0.948 1.030 0.754 1.104 3.80618 0.855 1.143 0.792 1.059 3.50515 1.067 1.234 0.831 0.891 3.20412 0.797 0.968 0.760 1.045 2.90309 0.968 0.825 0.675 1.067 2.60206 0.825 1.016 0.765 1.135 2.30103 1.059 0.872 0.648 0.613 2 0.988 1.067 0.413 0.548 1.70927 0.754 0.955 0.357 0.362 1.69897 0.922 0.797 0.313 0.340 1.40824 0.666 0.739 0.220 0.227 1.10721 0.548 0.604 0.162 0.170 0.80618 0.404 0.427 0.096 0.098 0.50515 0.352 0.427 0.062 0.053 0.20412 0.272 0.206 0.027 0.027 0.09691 0.132 0.103 0.013 0.014 0.39794 0.061 0.058 0.008 0.011 0.69897 0.028 0.032 0.007 0.008 1 0.018 0.019 0.008 0.009 1.30103 0.016 0.015 0.009 0.010
Oligonucleotides targeting Malat-1, wherein the oligonucleotides comprise a non-negatively charged internucleotidic linkage, were tested for their ability to knock down Malat-1 in GABA neurons in vitro, with 4 day treatment. Numbers represent Malat-1 level relative to HPRT1 control and water, wherein 1.0 would represent 100% Malat-1 level (0% knockdown) and 0 would represent 0% Malat-1 level (100% knockdown). Concentrations (Conc.) tested are provided as [Log (dose uM)].
Data from replicates are shown.
IC50 of WV-24104 was 132 nM; and IC50 of WV-24109 was 12 nM.

(1398) TABLE-US-00088 TABLE 25D Example data of certain oligonucleotides. 10 uM 3 uM mock 0.9 1.0 0.5 0.8 0.9 0.9 1.0 1.0 WV-9517 20.1 18.9 18.3 19.3 9.0 8.9 7.7 7.6 WV-11340 28.9 29.4 26.7 26.7 12.8 12.6 11.5 11.4 WV-11342 18.7 17.9 20.4 20.0 8.3 8.3 7.6 7.7 WV-12553 17.0 19.2 20.0 18.6 8.1 8.1 7.8 8.3 WV-12123 21.7 22.7 21.6 22.4 9.5 9.6 9.9 9.6 WV-12124 17.6 17.5 16.5 17.6 6.7 6.9 7.2 7.0 WV-12125 39.5 38.6 40.6 39.4 18.5 16.8 17.9 17.6 WV-12126 31.2 31.1 32.3 32.2 14.7 14.3 14.1 14.7 WV-12127 36.8 38.0 37.0 38.3 17.4 16.9 17.0 16.9 WV-12128 27.0 26.3 26.3 26.8 10.1 10.8 10.1 10.0 WV-12129 32.9 33.5 35.1 35.3 14.8 14.9 16.0 16.0 Mock 1.6 1.5 1.8 1.8 1.7 1.6 1.5 1.7 WV-9517 30.3 31.1 32.4 29.2 14.1 13.9 13.5 14.5 WV-11340 48.7 50.3 45.1 44.6 24.0 25.8 23.8 23.3 WV-12553 28.7 27.8 27.5 27.0 13.5 13.6 13.1 13.8 WV-9897 39.7 38.5 37.3 35.6 18.8 19.1 18.0 17.7 WV-11341 47.1 47.4 21.8 22.5 22.5 23.1 WV-12555 55.7 54.7 55.7 54.6 27.1 27.7 26.0 26.0 WV-12558 36.0 35.8 49.9 47.3 21.2 19.8 22.1 22.1 WV-9898 43.6 41.7 38.0 38.8 21.1 20.6 WV-11342 43.7 44.3 42.1 41.8 22.5 20.9 19.0 20.1 WV-12556 46.1 46.4 45.6 44.0 24.2 23.1 21.3 21.0 WV-12559 47.4 45.1 45.6 47.2 21.0 21.7 24.5 22.6 Mock 1.7 1.6 1.8 1.7 1.7 1.7 1.6 1.5 WV-9517 29.8 29.8 28.7 29.2 15.6 15.4 16.0 16.2 WV-11340 45.7 44.5 46.1 47.3 25.7 24.0 23.8 24.4 WV-11342 44.6 46.6 45.3 44.2 21.5 21.0 19.8 20.3 WV-12876 42.4 43.3 41.2 41.0 26.2 26.3 24.5 26.0 WV-12877 53.7 53.8 52.4 52.3 37.8 36.5 34.3 32.9 WV-12878 48.5 48.3 45.1 46.2 31.4 30.9 29.3 30.0 WV-12879 34.1 34.9 33.2 34.0 19.7 19.8 21.4 21.1 WV-12880 50.4 50.1 51.4 52.1 33.0 32.5 32.9 32.0 WV-12881 41.6 42.9 38.8 39.4 26.1 25.6 24.3 22.7 WV-12882 29.6 29.7 32.3 31.3 15.3 15.1 15.5 15.2 WV-12129 57.8 57.0 55.5 55.6 33.1 32.2
D45-52 myoblasts were treated for 4 days with 10 and 3 uM oligonucleotide.
Numbers in this and various other tables indicate amount of skipping relative to control.

(1399) Various DMD oligonucleotides comprising a chirally controlled neutral backbone were constructed, including WV-12555, which comprises a neutral internucleotidic linkage in the Rp configuration, and WV-12558, which comprises a neutral internucleotidic linkage in the Sp configuration. These were also tested for skipping a DMD exon, as shown in Table 25E.

(1400) TABLE-US-00089 TABLE 25E Example data of certain oligonucleotides. WV- WV- WV- WV- WV- WV- MOCK 9517 11340 9897 11341 12555 12558 10 uM 1.6 30.3 48.7 39.7 47.1 55.7 36.0 1.5 31.1 50.3 38.5 47.4 54.7 35.8 1.8 32.4 45.1 37.3 55.7 49.9 1.8 29.2 44.6 35.6 54.6 47.3 3 uM 1.7 14.1 24.0 18.8 21.8 27.1 21.2 1.6 13.9 25.8 19.1 22.5 27.7 19.8 1.5 13.5 23.8 18.0 22.5 26.0 22.1 1.7 14.5 23.3 17.7 23.1 26.0 22.1
D45-52 myoblasts were treated for 4 days with 10 and 3 uM oligonucleotide. Oligonucleotides were delivered gymnotically. Numbers represent amount of skipping relative to control.

(1401) In some embodiments, >2 fold increase in exon skipping efficiency was achieved.

(1402) TABLE-US-00090 TABLE 25F Example data of certain oligonucleotides. MDX mouse Human Human Human Muscle Liver Muscle Kidney WV-9517 82.4 77.8 84 73.7 3.08 7.9 2.01 3.59 WV-9897 88.3 82 96.1 75.2 9.12 4.2 5.5 3.8 WV-9898 74 75.8 96.8 81.5 5.07 6.4 8.9 5 WV-3473 69.8 69.8 ND 24 5.91 5.91 ND 0.15
Various DMD oligonucleotides for skipping exon 53 or 51 were incuted in tissue lysate for 5-days; full length oligonucleotides detected by LC-MS. Numbers represent percentage of full-length oligonucleotide remaining. Greater than 75% oligonucleotide remains in human and MDX muscle lysates at 5d incubation. Data was from a previous experiment performed for WV-3473, with 2d incubation in MDX muscle lysate. ND: Not determined; WV-3473 stability in human muscle lysate was not performed.

(1403) In some embodiments, an oligonucleotide comprising a neutral internucleotidic linkage (e.g., a cyclic guanidine type) demonstrated a higher level of exon skipping than a corresponding oligonucleotide which did not comprise such a neutral internucleotidic linkage.

(1404) In some embodiments, the present disclosure pertains to an oligonucleotide or an oligonucleotide composition which is capable of mediating single-stranded RNA interference, wherein the oligonucleotide or oligonucleotide composition comprises a non-negatively charged internucleotidic linkage.

(1405) As described herein, various oligonucleotides comprising a non-negatively charged internucleotidic linkage and targeting any of several different genes, with different base sequences, patterns of sugar modifications, backbone chemistry, and patterns of stereochemistry of backbone internucleotidic linkages were constructed, including but not limited to various oligonucleotides which target C9orf72 (a different gene than DMD, or Malat1).

(1406) Described herein are various non-limiting examples of oligonucleotides which target C9orf72 (which is a gene different from the other genes mentioned herein) and which comprise a non-negatively charged internucleotidic linkage.

(1407) A hexanucleotide repeat expansion in the C9orf72 gene (Chromosome 9, open reading frame 72) is reportedly the most frequent genetic cause of amyotrophic lateral sclerosis (ALs) and frontotemporal dementia (FTD). C9orf72 gene variants comprising the repeat expansion and/or products thereof are also associated with other C9orf72-related disorders, such as corticobasal degeneration syndrome (CBD), atypical Parkinsonian syndrome, olivopontocerebellar degeneration (OPCD), primary lateral sclerosis (PLs), progressive muscular atrophy (PMA), Huntington's disease (HD) phenocopy, Alzheimer's disease (AD), bipolar disorder, schizophrenia, and other non-motor disorders. Various oligonucleotides were designed and constructed which comprise a neutral internucleotidic linkage and which target a C9orf72 target (e.g., a C9orf72 oligonucleotide) and are capable of knocking down or decreasing expression, level and/or activity of the C9orf72 target gene and/or a gene product thereof (a transcript, particularly a repeat expansion containing transcript, a protein, etc.).

(1408) Various oligonucleotides designed to target C9orf72 and comprising a non-negatively charged internucleotidic linkage include, but are not limited to: WV-11532, WV-13305, WV-13307, WV-13309, WV-13311, WV-13312, WV-13313, WV-13803, WV-13804, WV-13805, WV-13806, WV-13807, WV-13808, WV-14553, and WV-14555. These are described below in Table 25G.

(1409) TABLE-US-00091 TABLE25G OligonucleotidestargetingC9orf72comprisinganeutraiintemucleotidiclinkage. Oligo- nucleo- SEQID tide Sequence NO: NakedSequence Stereochemistry WV- mC*Sm5Ceon001Teon001m5Ceon001 3205 CCTCACTCACCC SnXnXnXSSSRSSR 11532 mA*SC*ST*SC*RA*SC*SC*RC ACTCGCCA SSSSSSSS *SA*Se*ST*SmC*SmG*SmC* SmC*SmA WV- m5Ceo*Rm5Ceon001Teon001 3206 CCTCACTCACCC RnXnXnXRSSRSSR 13305 m5Ceon001Aeo*RC*ST*sC*RA* ACTCGCCA SSSSSSSS SC*SC*RC*SA*SC*ST*SmC* SmG*SmC*SmC*SmA WV_ m5Ceo*Sm5Ceon001Teon001 3207 CCTCACTCACCC SnXnXnXRSSRSSR 13307 m5Ceon001Aeo*RC*ST*SC*RA* ACTCGCCA SSSSSSSS SC*SC*RC*SA*Sc*ST*SmC* SmG*SmC*SmC*SmA WV_ m5Ceo*Rm5Ceon001Teon001 3208 CCTCACTCACCC RnXnXnXRSSRSSS 13309 m5Ceon001Aeo*RC*ST*SC*RA* ACTCGCCA RSSSSSSS SC*Sc*SC*RA*SC*ST*SmC* SmG*SmC*SmC*SmA WV- m5Ceo*Sm5Ceon001Teon001 3209 CCTCACTCACCC SnXnXnX.RSSRSSS 13311 m5Ceon001Aeo*RC*ST*SC*RA* ACTCGCCA RSSSSSSS SC*SC*SC*RA*SC*ST*SmC* SmG*SmC*SmC*SmA WV- mC*Sm5Ceon001Teon001m5Ceon001 3210 CCTCACTCACCC SnXnXnXSSSR 13312 mA*SC*ST*SC*RA*SC*SC*SC ACTCGCCA SSSSSSSSSSS *SA*SC*ST*SmC*SmG*SmC* SmC*SmA WV- m5Ceo*Rm5Ceon001Teon001 3211 CCTCACTCACCC RnXnXnXRSSR 13313 m5Ceon001Aeo*RC*ST*SC*RA* ACTCGCCA SSSSSSSSSSS Sc*SC*SC*SA*SC*ST*SmC* SmG *SmC*SmC* SmA WV- Teo*Geon001m5Ceon001m5Ceon001 3212 TGCCGCCTCCT XnXnXnXXXXXXX 13803 Geo*C*C*T*C*C*I*C*A* CACTCACCC XXXXXXXXX T*mC*mA*mC*mC*mC WV- Teo*Geom5Ccom5CcoGeo*C*C*T 3213 TGCCGCCTCCT XOOOXXXXXXXXX 13804 *C*C*T*C*A*C*T*mCn001 CACTCACCC XXnXnXnXX mAn001mCn001mC*mC WV- Teo*Geon001m5Ceon001m5Ceon001 3214 TGCCGCCTCCT XnXnXnXXXXXXXX 13805 Geo*C*C*T*C*C*T*C*A*C* CACTCACCC XXXXnXnXnXX T*mCn001mAn001mCn001mC*mC WV- Geo*m5Ceon001Geon001m5Ceon001 3215 GCGCGACTCCT XnXnXnXXXXXXXX 13806 Geo*A*C*T*C*C*T*G*A*G GAGTTCCAG XXXXOOOX *T*Teom5Ceom5CeoAeo*Geo WV- Geo*m5CeoGeom5CeoGeo*A*C*T 3216 GCGCGACTCCT XOOOXXXXXXXXXX 13807 *C*C*T*G*A*G*T*Teon001 GAGTTCCAG XnXnXnXX m5Ceon001m5Ceon001Aeo*Geo WV- Geo*m5Ceon001Geon001m5Ceon001 3217 GCGCGACTCCT XnXnXnXXXXXXXXX 13808 Geo*A*C*T*C*C*T*G*A*G GAGTTCCAG XXXnXnXnXX *T*Teon001m5Ceon001m5Ceon001 Aeo*Geo WV- m5Ceo*Rm5Ceon001Teon001 3218 CCTCACTCACCC RnXnXnXRSSRSSR 14553 m5Ceon001Aeo*RC*ST*SC*RA* ACTCGCCA SSSRSSSS SC*SC*RC*SA*SC*ST*Rm5Ceo *SmG*SmC*SmC*SmA WV- m5Ceo*Rm5Ceon001Teon001 3219 CCTCACTCACCC RnXnXnXRSSRSSS 14555 m5Ceon001Aeo*RC*ST*SC*RA* ACTCGCCA RSSRSSSS SC*SC*SC*RA*SC*ST*Rm5Ceo *SmG*SmC*SmC*SmA
Several variants of a C9orf72 mRNA are produced from the C9orf72 gene: V2 (which does not comprise the deleterious hexanucleotide repeat and which comprises about 90% of all transcripts); V3 (which comprises the hexanucleotide repeat and comprises about 9% of all transcripts); and V1 (which comprises the hexanucleotide repeat and comprises about 1% of all transcripts).
Hexanucleotide repeats reportedly elicit gain of function toxicities, at least partially mediated by the dipeptide repeat proteins and foci formation by, for example, repeat-expansion containing transcripts and/or spliced-out repeat-expansion containing introns and/or antisense transcription of the repeat-expansion containing region and various nucleic-acid binding proteins.
Both WV-8008 and WV-11532 have the same base sequence (or naked sequence), CCTCACTCACCCACTCGCCA (SEQ ID NO: 3220). They differ, inter alia, in that the latter comprises 3 contiguous neutral internucleotidic linkages (Xn), but the former does not comprise any neutral internucleotic linkages. The structures of these oligonucleotides is provided below, in Table 25H.

(1410) TABLE-US-00092 TABLE25H C9orf72oligonucleotides. Oligo- nucleotide Sequence Stereochemistry WV-8008 m5Ceo*Rm5CeoTeom5CeoAeo*RC*ST*SC*RA*SC*SC ROOORSSRSSRS *RC*SA*SC*ST*SmC*SmG*SmC*SmC*SmA SSSSSSS (SEQIDNO:3221) WV-11532 mC*Sm5Ceon001Teon001m5Ceon001mA*SC*ST*SC*RA SnXnXnXSSSRSS *SC*SC*RC*SA*SC*ST*SmC*SmG*SmC*SmC* RSSSSSSSS SmA(SEQIDNO:3222) ,
WV-8008 and WV-11532 were tested for their ability to knock down expression of hexanucleotide-comprising (i.e., disease-associated) transcript V3 compared to total transcripts (all V), as shown below in Table 25I.
Table 25I and J. Activity of various c9orf72 oligonucleotides.
In Tables 25I to 25J, various c9orf72 oligonucleotides were tested in motor neurons, with oligonucleotides delivered gymnotically at concentrations from 0.003 to 10 M (Concentrations are provided as exp10). Tested c9orf72 oligonucleotide WV-11532 comprises three neutral internucleotidic linkages. In Tables 14A and 14B, shown are residual levels of c9orf72 transcriptions [e.g., all transcripts (all V) or only V3] relative to HPRT1, after treatment with c9orf72 oligonucleotides, wherein 1.000 would represent 100% relative transcript level (no knockdown) and 0.000 would represent 0% relative transcript level (e.g., 100% knockdown). Results from replicate experiments are shown.

(1411) TABLE-US-00093 TABLE 25I Activity of various c9orf72 oligonucleotides (residual level of all V C9orf72 transcripts) Conc. WV-8008 WV-11532 2.495 0.999 0.958 0.913 1.006 0.894 0.900 1.796 0.965 0.864 0.882 0.972 0.829 0.858 1.097 1.006 0.900 0.932 0.907 0.888 0.858 0.398 0.800 0.742 0.806 0.795 0.747 0.742 0.301 0.624 0.611 0.687 0.562 0.554 0.554 1 0.524 0.500 0.521 0.409 0.411 0.387

(1412) TABLE-US-00094 TABLE 25J Activity of various c9orf72 oligonucleotides (residual level of V3 C9orf72 transcripts) Conc. WV-8008 WV-11532 2.495 0.947 0.871 1.014 0.927 0.853 0.908 1.796 0.877 0.841 0.908 0.836 0.769 0.841 1.097 0.665 0.743 0.871 0.620 0.633 0.717 0.398 0.555 0.427 0.707 0.421 0.415 0.427 0.301 0.210 0.178 0.304 0.096 0.105 0.094 1 0.056 0.071 0.083 0.012 0.015 0.015

(1413) As described herein and in data not shown, various oligonucleotides comprising a non-negatively charged internucleotidic linkage and targeting different genes, with different base sequences, patterns of sugar modifications, backbone chemistries, and patterns of stereochemistry of backbone internucleotidic linkages were constructed, including but not limited to various oligonucleotides which target DMD, Malat1, or C9orf72.

(1414) Oligonucleotides comprising a non-negatively charged internucleotidic linkage were also constructed to target six other genes not described herein (wherein the six genes were not DMD, Malat1, or C9orf72); these oligonucleotides include oligonucleotides designed to target these genes and reduce the expression, level and/or activity of the gene or its gene product. These and various oligonucleotides comprising a neutral internucleotidic linkage described herein are capable of performing various functions, including reducing the level, expression and/or activity of a gene or its gene product (e.g., via a RNaseH- or steric-hindrance-mediated mechanism, or via a single-stranded RNA interference-mediated mechanism) and inducing skipping of an exon (e.g., skipping modulation).

(1415) Without wishing to be bound by any particular theory, Applicant notes that a non-negatively charged and/or neutral internucleotidic linkage can improve an oligonucleotide's entry into a cell and/or escape from an endosome.

(1416) Oligonucleotides which Comprise a Non-Negatively Charged Internucleotidic Linkage can Provide Desired Levels of TLR9 Activation

(1417) Among other things, oligonucleotides comprising non-negatively charged internucleotidic linkages can provide desired levels of properties and/or activities, e.g., TLR9 antagonist or agonist activities. In some embodiments, oligonucleotides comprising non-negatively charged internucleotidic linkages demonstrate lower levels of TLR9 activation in human and/or an animal model (e.g., a mouse) compared to certain comparable oligonucleotides of the same base sequences but having no non-negatively charged internucleotidic linkages. In some embodiments, oligonucleotides comprising non-negatively charged internucleotidic linkages have lower toxicity compared to certain oligonucleotides of the same base sequences but having no non-negatively charged internucleotidic linkages. In some embodiments, a non-negatively charged internucleotidic linkage is within a CpG motif and is the internucleotidic linkage between the C and G.

(1418) In an experiment, several oligonucleotides to target gene C were constructed. Gene C is a different gene than DMD, or SMalat-1. The sequence of these oligonucleotides comprises a CpG, a motif known to activate TLR9.

(1419) Table 25K.

(1420) This experiment represents a test of induction of human TLR9 or mouse TLR9 in HEK293 cells. Numbers represent relative inductive relative to negative control, water. Concentrations tested: 0.93 uM, 2.77 uM, 8.33 uM, 25 uM, 75 uM. Positive control: WV-BZ21. The experiment was performed in biological duplicates.

(1421) TABLE-US-00095 TABLE25K Oligonucleotidesusedinthisstudy Oligo- SEQID nucleotide Sequence NO: Stereochemistry WV-HZ12 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3223 SOOOSSSRSS RN*SN*SN*SN*SmC*SmG*SmN*SmN*SmN RSSSSSSSS WV-BZ761 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3224 SOOOSSSRSS RN*SN*SN*SN*SmCmG*SmN*SmN*SmN RSSSSOSSS WV-BZ762 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3225 SOOOSSSRSS RN*SN*SN*SN*Sm5CeomG*SmN*SmN*SmN RSSSSOSSS WV-BZ763 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3226 SOOOSSSRSS RN*SN*SN*SN*Sm5Ceo*SmG*SmN*SmN*SmN RSSSSSSSS WV-BZ764 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3227 SOOOSSSRSS RN*SN*SN*SN*Rm5CeomG*SmN*SmN*SmN RSSSROSSS WV-BZ765 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3228 SOOOSSSRSS RN*SN*SN*SN*Rm5Ceo*SmG*SmN*SmN*SmN RSSSRSSSS WV-BZ766 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3229 SOOOSSSRSS RN*SN*SN*SN*Sm5mC*StnG*SmN*SmN*SmN RSSSSSSSS WV-BA207 mN*Sm5NeoNeom5NeomN*SN*SN*SN*RN*SN*SN* 3230 SOOOSSSRSS SN*RN*SN*SN*SmCn001mG*SmN*SmN*SmN SRSSSnXSSS WV-BA208 m5Neo*Rm5NeoNeom5NeoNeo*RN*SN*SN*RN*SN* 3231 ROOORSSRSS SN*RN*SN*SN*SN*SmCn001mG*SmN*SmN*SmN RSSSSnXSSS WV-BA209 m5Neo*Rm5NeoNeom5NeoNeo*RN*SN*SN*RN*SN* 3232 ROOORSSRSS SN*SN*RN*SN*SN*SmCn001mG*SmN*SmN*SmN SRSSSnXSSS WV-BZ21 T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T 3233 XXXXXXXXXX *T*G*T*C*G*T*T XXXXXXXXXX XXX

(1422) TABLE-US-00096 TABLE 25L Activity of certain oligonucleotides. 0.93 uM 2.77 uM 8.33 uM 25 uM 75 uM WV-HZ12 1.0 1.0 1.0 1.0 0.9 1.1 1.0 1.1 1.0 1.0 WV-BZ761 1.0 1.0 1.0 1.0 1.0 1.1 1.0 1.1 1.0 0.9 WV-BZ762 1.0 1.0 1.0 1.1 1.0 1.0 1.1 1.0 1.0 1.0 WV-BZ763 1.0 1.0 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.0 WV-BZ764 1.0 1.0 1.0 0.9 1.0 1.0 1.0 1.0 1.0 1.0 WV-BZ765 1.0 0.9 1.1 1.0 1.0 1.0 1.1 1.0 0.9 0.9 WV-BZ766 1.1 1.3 1.5 1.5 1.5 1.2 1.3 1.3 1.4 1.4 WV-BA207 1.0 1.0 1.0 1.0 1.0 1.1 1.1 1.0 1.0 1.0 WV-BA208 1.0 1.0 1.0 1.0 1.0 1.0 1.1 1.0 0.9 1.0 WV-BA209 1.0 1.0 1.0 0.9 1.0 1.1 1.0 0.9 1.0 1.0 WV-BZ21 10.0 12.0 12.0 11.4 11.0 (positive 9.4 10.4 11.4 11.5 11.1 control)
All the tested oligonucleotides (WV-HZ12, WV-BZ761, WV-BZ762, WV-BZ763, WV-BZ764, WV-BZ765, WV-BZ766, WV-BA207, WV-BA208, and WV-BA209) target gene C and all have the same base sequence, wherein each base is indicated generically by N, except that the single CpG motif is indicated. WV-BZ21, positive control, has a base sequence of TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 3234), which comprises several CpG motifs, and is not designed to target gene C. Numbers indicate relative induction of hTLR9 activity relative to water.

(1423) TABLE-US-00097 TABLE 25M Activity of certain oligonucleotides. 0.93 uM 2.77 uM 8.33 uM 25 uM 75 uM WV-HZ12 2.9 4.4 4.7 5.0 4.9 3.0 4.1 4.8 5.1 5.2 WV-BZ761 1.2 1.5 1.8 2.1 2.1 1.2 1.4 1.8 2.1 2.2 WV-BZ762 1.0 1.0 1.0 1.0 1.0 1.0 1.1 1.1 0.9 1.0 WV-BZ763 1.0 1.1 1.1 1.1 1.0 1.1 1.0 1.1 1.1 1.1 WV-BZ764 1.0 1.1 1.1 1.1 1.1 1.0 1.1 1.1 1.1 1.1 WV-BZ765 1.0 1.2 1.3 1.3 1.2 1.1 1.2 1.3 1.3 1.3 WV-BZ766 1.1 1.3 1.4 1.6 1.6 1.1 1.2 1.4 1.6 1.6 WV-BA207 1.1 1.1 1.1 1.1 1.1 1.0 1.0 1.1 1.1 1.2 WV-BA208 1.0 1.1 1.1 1.2 1.1 1.0 1.0 1.1 1.2 1.2 WV-BA209 1.0 1.2 1.1 1.2 1.1 1.0 1.1 1.2 1.2 1.3 WV-BZ21 21.4 22.4 22.9 21.2 18.1 (positive 22.9 24.0 23.8 22.3 18.9 control)
These oligonucleotides were also tested for induction of mouse TLR9.
Numbers indicate relative induction of mTLR9 activity relative to water.

(1424) In some embodiments, it was observed that in some instances certain oligonucleotides that did not induce appreciable TLR9 activation, or induced very low level of TLR9 activation above mock against human or mouse TLR9.

(1425) Example Oligonucleotides Comprising Additional Moieties

(1426) In some embodiments, the present disclosure provides oligonucleotides comprising one or more additional moieties, e.g., targeting moieties, carbohydrate moieties, etc. In some embodiments, the present disclosure provides oligonucleotides comprising one or more sulfonamide moieties. In some embodiments, a provided oligonucleotide comprise one or two or more sulfonamide moieties. In some embodiments, the present disclosure provides oligonucleotides that can modulate splicing, e.g., DMD oligonucleotides that can modulate exon skipping, wherein the oligonucleotides comprise one or more sulfonamide moieties. In some embodiments, the present disclosure provides oligonucleotides that mediate skipping of DMD exon 23, 45, 51 or 53, or multiple DMD exons, wherein the oligonucleotides comprise one or more sulfonamide moieties.

(1427) In some embodiments, a sulfonamide moiety has or comprises the structure of -L-SO.sub.2N(R.sup.1).sub.2. In some embodiments, a sulfonamide moiety has or comprises the structure of SO.sub.2N(R.sup.1).sub.2. In some embodiments, a sulfonamide moiety has or comprises the structure of CySO.sub.2N(R.sup.1).sub.2. In some embodiments, Cy is aromatic. In some embodiments, Cy is an optionally substituted phenyl ring. In some embodiments, Cy is

(1428) ##STR00509##
In some embodiments, Cy is an optionally substituted heteroaryl ring. In some embodiments, Cy is an optionally substituted 5-6 membered heteroaryl ring having 1-4 heteroatoms. In some embodiments, Cy is

(1429) ##STR00510##
In some embodiments, each R.sup.1 is H.

(1430) A sulfonamide moiety can be connected to an oligonucleotide chain via various suitable linkers in accordance with the present disclosure, such as those described herein and/or in WO/2017/062862, linkers of which is incorporated herein by reference. Example sulfonamides moieties, including mono-, bi-, and tri-sulfonamide moieties, are described below:

(1431) ##STR00511##

(1432) In some embodiments, an oligonucleotide comprise a modified internucleotidic linkage and a sulfonamide moiety optionally through a linker. In some embodiments, an oligonucleotide comprising a modified internucleotidic linkage and a sulfonamide moiety is a siRNA, double-straned siRNA, single-stranded siRNA, gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir, microRNA, pre-microRNs, antimir, supermir, ribozyme, Ul adaptor, RNA activator, RNAi agent, decoy oligonucleotide, triplex forming oligonucleotide, aptamer or adjuvant. In some embodiments, the present disclosure provides an oligonucleotide which comprises a modified internucleotidic linkage which comprises a sulfonamide. In some embodiments, an oligonucleotide comprises a sulfonamide and a chirally controlled internucleotidic linkage. In some embodiments, an oligonucleotide comprises a sulfonamide and a chirally controlled internucleotidic linkage which is a phosphorothioate internucleotidic linkage.

(1433) In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a sulfonamide moiety or a derivative or variant thereof. In some embodiments, the present disclosure pertains to an oligonucleotide composition, wherein the oligonucleotide comprises a sulfonamide moiety or a derivative or variant thereof and the oligonucleotide comprises at least one chirally controlled internucleotidic linkage.

(1434) In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a sulfonamide moiety or a derivative or variant thereof, wherein the oligonucleotide is capable of mediating a decrease in the expression, level and/or activity of a target gene or gene product thereof.

(1435) In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a sulfonamide moiety or a derivative or variant thereof, wherein the oligonucleotide is capable of mediating modulation of exon skipping of a target gene. In some embodiments, the present disclosure pertains to an oligonucleotide which comprises a sulfonamide moiety or a derivative or variant thereof, wherein the oligonucleotide is capable of increasing skipping of an exon of a target gene.

(1436) Example oligonucleotides that can be utilized for splicing modulation, e.g., exon skipping, that comprise a sulfonamide moiety include WV-3548, WV-3366, etc. Other oligonucleotides comprising a sulfonamide moiety were designed, constructed and/or tested for various activities. For example, oligonucleotides comprising a mono-sulfonamide moiety, such as WV-2836, WV-7419, WV-7421, WV-7422, WV-7408, WV-7409, WV-7427, WV-7863, and WV-7864; oligonucleotide comprising a bi-sulfonamide, WV-7423; and oligonucleotide comprising a tri-sulfonamide, WV-7417.

(1437) TABLE-US-00098 TABLE26A CertainMalat1oligonucleotides. Oligo- SEQID Linkage/ nucleotide Description NO: NakedSequence Stereochemistry WV-2735 Geo*Geo*Geo*Teo*m5Ceo*A* 3235 GGGTCAGCTG XXXXXXXXXXX G*C*T*G*C*C*A*A*T*Geo CCAATGCTAG XXXXXXXX *m5Ceo*Teo*Aeo*Geo WV-2835 Mod027L001*Geo*Geo*Geo*Teo* 3236 GGGTCAGCTGC XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-2836 Mod028L001*Geo*Geo*Geo*Teo* 3237 GGGTCAGCTGC XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-3174 mU*mG*mC*mC*mA*G*G*C 3238 UGCCAGGCTGG XXXXXXXXXXX *T*G*G*T*T*A*T*mG*mA TTATGACUC XXXXXXXX *mC*mU*mC WV-7301 Teo*Geo*m5Ceo*m5Ceo*Aeo*G 3239 TGCCAGGCTGG XXXXXXXXXXX *G*C*T*G*G*T*T*A*T* TTATGACTC XXXXXXXX Geo*Aeo*m5Ceo*Teo*m5Ceo WV-7408 Mod027L00lGeo*Geo*Geo*Teo* 3240 GGGTCAGCTGC OXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7409 Mod028L001Geo*Geo*Geo*Teo* 3241 GGGTCAGCTGC OXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7417 Mod029L001*Geo*Geo*Geo*Teo* 3242 GGGTCAGCTGC XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7419 Mod045L001*Geo*Geo*Geo*Teo* 3243 GGGTCAGCTGC XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CAATGCTAG XXXXXXXXX A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7421 Mod047L001*Geo*Geo*Geo*Teo* 3244 GGGTCAGCTGC XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7422 Mod048L001*Geo*Geo*Geo*Teo* 3245 GGGTCAGCTG XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CCAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7423 Mod049L001*Geo*Geo*Geo*Teo* 3246 GGGTCAGCTG XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CCAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7427 Mod045L001Geo*Geo*Geo*Teo* 3247 GGGTCAGCTG OXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CCAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7863 Mod046L001Geo*Geo*Geo*Teo* 3248 GGGTCAGCTG OXXXXXXXXXX m5Ceo*A*G*C*T*G*C*CA CCAATGCTAG XXXXXXXXX A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-7864 Mod054L001Geo*Geo*Geo*Teo* 3249 GGGTCAGCTG OXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CCAATGCTAG XXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo WV-9430 Mod029L001mU*mG*mC*mC* 3250 UGCCAGGCTG OXXXXXXXXX mA*G*G*C*T*G*G*T*T*A GTTATGACUC XXXXXXXXXX *T*mG*mA*mC*mU*mC WV-7420 Mod046L001*Geo*Geo*Geo*Teo* 3251 GGGTCAGCTG XXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A CCAATGCTAG XXXXXXXXXXX *A*T*Geo*m5Ceo*Teo*Aeo* Geo
For this Table, descriptions match those of Table A1, and

(1438) ##STR00512##
In these Mods, C(O) connects to NH of a linker (e.g., L001).

(1439) Oligonucleotides comprising a sulfonamide moiety were tested for their ability to knockdown Malat1. Tested oligonucleotides were gymnotically delivered to A48-50 patient derived myotubes, which were dosed at 3,1, 0.3 and 0.1 M concentrations. Cells were allowed to differentiate for 4 days (e.g., this experiment was 4 days post-differentiation). qPCR was used to evaluate knockdown of Malat-1. The results are shown in Table 26B.

(1440) TABLE-US-00099 TABLE 26B Example data of Malat1 oligonucleotides. WV- WV- WV- WV- WV- WV- WV- WV- 3174 8927 8929 8930 8931 8934 9385 9390 Mock .sup.3 M 10 11 10 11 9 8 33 95 .sup.1 M 18 2.8 24 22 19 20 49 100 0.3 M 39 56 50 67 46 42 43 67 95 0.1 M 63 73 68 81 68 69 56 81 100
Numbers represent relative Malat-1 mRNA level.
Various Malat1 oligonucleotides, many comprising a sulfonamide moiety, were tested for their ability to knockdown Malat1 in pre-differentiated myotubes. Certain data are shown in Table 26C. 48-50 patient derived myoblasts were differentiated for 4 days prior to dosing with at 1 and 0.1 M concentrations. RNA was harvested 48 hours post-treatment for measurement.

(1441) TABLE-US-00100 TABLE 26C Example data of Malat1 oligonucleotides. WV- WV- WV- WV- WV- WV- WV- WV- 3174 8927 8929 8930 8931 8934 9385 9390 .sup.1 M 31 25 25 36 24 18 45 0.1 M 62 70 79 72 78 55 59 66 WV- WV- WV- WV- 8448 7558 7559 7560 MOCK .sup.1 M 33 34 22 23 98 0.1 M 68 72 69 82 98
Numbers represent relative Malat-1 mRNA level. Numbers are approximate.

(1442) In some experiments, animals were dosed with oligonucleotides, including some which comprise a sulfonamide moiety, and the animals were later sacrificed and their tissues tested for the level of the ligonucleotides.

(1443) In some experiments, the following protocol was used: Animals: 32 male Mdx mice and 32 male C57BL/6 mice (all 8-10 week-old). Test animals were acclimated to the facility for at least 3 days upon arrival. Dosing: S.C. (subcutaneous) dosing on days 1, 3 and 5 (5 mL/kg). Necropsy: animals were euthanized 72 hours after the last SC injection. All animals were perfused with PBS. The following tissues were collected: brain, sciatic nerves, spinal cord, eyes, liver, kidney, spleen, heart, diaphragm, gastrocnemius, quadriceps and triceps, white fat, brown fat. Fresh tissues will be rinsed briefly with PBS, gently blotted dry, weighed and snap frozen in Liquid Nitrogen in 2-mL tubes and stored at 80C(on dry ice). Histology: Quadricep and Kidney postfixed in 10% Formalin and processed to slides (paraffin embedded sections). In some experiments, suitable variants of this protocol were used.

(1444) Certain results are shown in Tables 27, 28 and 29.

(1445) TABLE-US-00101 TABLE 27 Knock-down and oligonucleotide presence in various tissues. Heart pK Malat1 Quadriceps pD Triceps pD Gastro pD Diaphragm pD Heart pD Mean SD Sequence Mean SD Mean SD Mean SD Mean SD Mean SD (ug/g) PBS 1.000 1.000 1.000 1.000 1.000 0.000 0.142 0.265 0.042 0.276 0.074 0.000 WV-2735 0.776 0.699 0.731 0.879 0.707 1.631 0.122 0.150 0.107 0.158 0.173 0.692 WV-2835 0.639 0.588 0.417 0.895 0.510 1.987 0.119 0.036 0.065 0.116 0.066 0.203 WV-2836 0.621 0.834 0.616 0.769 0.619 7.001 0.124 0.206 0.169 0.229 0.389 1.331
Numbers indicate Malat1 mRNA levels relative to mHprt (mHPRT or mHPRT1), and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 5-6 wks MDX mice; Route: Subcutaneous; # Doses: QD for 3 days; Time Point Post Last Dose: 2 days; Daily Dose Level (ug): 12.5 mg/kg.

(1446) TABLE-US-00102 TABLE 28 Knock-down and oligonucleotide presence in various tissues. Oligo- Quadriceps pD Triceps pD Gastro pD Diaphragm pD Heart pD nucleotide Mean SD Mean SD Mean SD Mean SD Mean SD PBS 1.000 0.266 1.000 0.207 1.000 0.138 1.000 0.191 1.000 0.221 WV-2735 0.952 0.232 0.876 0.180 0.998 0.072 0.651 0.046 1.032 0.541 WV-2835 0.593 0.167 0.877 0.180 0.645 0.124 0.563 0.091 1.032 0.240 WV-2836 0.556 0.172 0.739 0.047 0.695 0.102 0.614 0.120 0.544 0.109 WV-3174 0.610 0.109 1.009 0.047 0.809 0.137 0.698 0.069 0.588 0.258 WV-7301 0.624 0.074 0.846 0.172 0.837 0.141 0.453 0.031 0.887 0.142 Quadriceps pK Diaphragm pK Heart pK Oligo- Mean SD Mean SD Mean SD nucleotide (ug/g) (ug/g) (ug/g) PBS 0.000 0.000 0.096 0.015 0.000 0.000 WV-2735 5.616 2.724 3.207 1.465 0.342 0.169 WV-2835 8.421 3.374 5.734 1.465 0.777 0.203 WV-2836 11.221 7.877 6.142 1.006 0.664 0.441 WV-3174 9.792 8.339 4.609 1.006 0.619 0.122 WV-7301 6.659 3.858 5.728 2.092 0.707 0.191
Numbers indicate Malat1 mRNA levels relative to mHprt, and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 10-12 wks MDX mice; Route: Subcutaneous; # Doses: QD for 3 days; Time Point Post Last Dose: 3 days; and Daily Dose Level (ug): 12 mg/kg.

(1447) TABLE-US-00103 TABLE 29 Knock-down and oligonucleotide presence in various tissues. Oligo- Quadriceps pD Triceps pD Gastro pD Diaphragm pD Heart pD nucleotide Mean SD Mean SD Mean SD Mean SD Mean SD PBS 1.000 0.266 1.000 0.191 1.000 0.249 1.000 0.191 1.000 0.147 WV-2735 0.753 0.230 0.667 0.132 0.756 0.136 0.651 0.046 0.596 0.140 WV-2835 0.611 0.165 0.549 0.077 0.656 0.101 0.563 0.091 0.546 0.092 WV-2836 0.640 0.186 0.596 0.114 0.812 0.216 0.614 0.120 0.774 0.168 WV-3174 0.796 0.142 0.610 0.111 0.870 0.081 0.698 0.069 0.703 0.099 WV-7301 0.456 0.116 0.498 0.097 0.753 0.113 0.453 0.031 0.368 0.031 Quadriceps pK Diaphragm pK Heart pK Oligo- Mean SD Mean SD Mean SD nucleotide (ug/g) (ug/g) (ug/g) PBS 0.000 0.000 0.108 0.016 0.000 0.000 WV-2735 2.787 0.734 9.219 3.234 0.428 0.084 WV-2835 2.700 0.891 9.895 2.466 0.726 0.207 WV-2836 2.273 0.621 9.751 6.912 0.670 0.242 WV-3174 2.142 0.778 7.568 1.807 0.612 0.172 WV-7301 2.868 0.334 6.174 2.456 0.975 0.216
Numbers indicate Malat1 mRNA levels relative to mHprt, and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 10-12 wks wt mice; Route: Subcutaneous; # Doses: QD for 3 days; Time Point Post Last Dose: 3 days; and Daily Dose Level (ug): 12 mg/kg.

(1448) TABLE-US-00104 TABLE 30 Knock-down and oligonucleotide presence in various tissues. Malat1 Quadriceps pD Gastro pD Diaphragm pD Heart pD Sequence Mean SD Mean SD Mean SD Mean SD PBS 1.000 0.256 1.000 0.309 1.000 0.345 1.000 0.432 WV-3174 0.752 0.118 0.833 0.160 0.647 0.058 0.599 0.120 WV-3174 0.603 0.118 0.678 0.145 0.421 0.092 0.582 0.185 WV-3174 0.454 0.112 0.523 0.104 0.380 0.081 0.415 0.062 WV-3174 0.342 0.033 0.505 0.119 0.322 0.077 0.340 0.055 Quadriceps pK Gastro pK Diaphragm pK Heart pK Malat1 Mean SD Mean SD Mean SD Mean SD Sequence (ug/g) (ug/g) (ug/g) (ug/g) PBS 0.011 0.025 0.000 0.000 0.000 0.000 0.000 0.000 WV-3174 1.388 0.677 1.704 0.524 2.502 0.919 1.781 0.668 WV-3174 6.651 5.930 4.563 1.705 7.366 3.939 2.532 0.487 WV-3174 12.374 4.081 14.574 8.235 12.075 3.739 4.611 1.050 WV-3174 15.227 4.925 14.124 2.285 22.734 4.484 12.660 2.437
Numbers indicate Malat1 mRNA levels relative to mHprt, and presence of oligonucleotide (ug/g). Experimental procedure: Study Species: 5-6 wks wt mice; Route: Subcutaneous; # Doses: QD for 1 days; Time Point Post Last Dose: 3 days; and Daily Dose Level (ug): 200 mg/kg.
Example Methods for Preparing Oligonucleotides and Compositions

(1449) Among other things, the present disclosure provides technologies (methods, reagents, conditions, purification processes, etc.) for prepaing oligonucleotides and oligonucleotide compositions, including chirally controlled oligonucleotides and chirally controlled oligonucleotide nucleotides. Various technologies (methods, reagents, conditions, purification processes, etc.), as described herein, can be utilized to prepare provided oligonucleotides and compositions thereof in accordance with the present disclosure, including but not limited to those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the preparation technologies of each of which are incorporated herein by reference.

(1450) In some embodiments, the present disclosure provides chirally controlled oligonucleotides. In some embodiments, a provided chirally controlled oligonucleotide is over 50% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 55% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 60% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 65% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 70% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 75% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 80% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 85% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 90% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 91% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 92% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 93% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 94% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 95% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 96% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 97% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 98% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.5% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.6% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.7% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.8% pure. In some embodiments, a provided chirally controlled oligonucleotide is over about 99.9% pure. In some embodiments, a provided chirally controlled oligonucleotide is over at least about 99% pure.

(1451) In some embodiments, a chirally controlled oligonucleotide composition is a composition designed to comprise a single oligonucleotide type. In certain embodiments, such compositions are about 50% diastereomerically pure. In some embodiments, such compositions are about 50% diastereomerically pure. In some embodiments, such compositions are about 50% diastereomerically pure. In some embodiments, such compositions are about 55% diastereomerically pure. In some embodiments, such compositions are about 60% diastereomerically pure. In some embodiments, such compositions are about 65% diastereomerically pure. In some embodiments, such compositions are about 70% diastereomerically pure. In some embodiments, such compositions are about 75% diastereomerically pure. In some embodiments, such compositions are about 80% diastereomerically pure. In some embodiments, such compositions are about 85% diastereomerically pure. In some embodiments, such compositions are about 90% diastereomerically pure. In some embodiments, such compositions are about 91% diastereomerically pure. In some embodiments, such compositions are about 92% diastereomerically pure. In some embodiments, such compositions are about 93% diastereomerically pure. In some embodiments, such compositions are about 94% diastereomerically pure. In some embodiments, such compositions are about 95% diastereomerically pure. In some embodiments, such compositions are about 96% diastereomerically pure. In some embodiments, such compositions are about 97% diastereomerically pure. In some embodiments, such compositions are about 98% diastereomerically pure. In some embodiments, such compositions are about 99% diastereomerically pure. In some embodiments, such compositions are about 99.5% diastereomerically pure. In some embodiments, such compositions are about 99.6% diastereomerically pure. In some embodiments, such compositions are about 99.7% diastereomerically pure. In some embodiments, such compositions are about 99.8% diastereomerically pure. In some embodiments, such compositions are about 99.9% diastereomerically pure. In some embodiments, such compositions are at least about 99% diastereomerically pure.

(1452) Among other things, the present disclosure recognizes the challenge of stereoselective (rather than stereorandom or racemic) preparation of oligonucleotides. Among other things, the present disclosure provides methods and reagents for stereoselective preparation of oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotidic linkages, and particularly for oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotidic linkages. In some embodiments, in a stereorandom or racemic preparation of oligonucleotides, at least one chiral internucleotidic linkage is formed with less than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 90:10, 95:5, 96:4, 97:3, or 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 95:5 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 96:4 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 97:3 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 98:2 diastereoselectivity. In some embodiments, for a stereoselective or chirally controlled preparation of oligonucleotides, each chiral internucleotidic linkage is formed with greater than 99:1 diastereoselectivity. In some embodiments, diastereoselectivity of a chiral internucleotidic linkage in an oligonucleotide may be measured through a model reaction, e.g. formation of a dimer under essentially the same or comparable conditions wherein the dimer has the same internucleotidic linkage as the chiral internucleotidic linkage, the 5-nucleoside of the dimer is the same as the nucleoside to the 5-end of the chiral internucleotidic linkage, and the 3-nucleoside of the dimer is the same as the nucleoside to the 3-end of the chiral internucleotidic linkage.

(1453) In some embodiments, a chirally controlled oligonucleotide composition is a composition designed to comprise multiple oligonucleotide types. In some embodiments, methods of the present disclosure allow for the generation of a library of chirally controlled oligonucleotides such that a pre-selected amount of any one or more chirally controlled oligonucleotide types can be mixed with any one or more other chirally controlled oligonucleotide types to create a chirally controlled oligonucleotide composition. In some embodiments, the pre-selected amount of an oligonucleotide type is a composition having any one of the above-described diastereomeric purities.

(1454) In some embodiments, the present disclosure provides methods for making a chirally controlled oligonucleotide comprising steps of: (1) coupling; (2) capping; (3) optionally modifying; (4) deblocking; and (5) repeating steps (1)-(4) until a desired length is achieved.

(1455) In some embodiments, the present disclosure provides a method, e.g., for preparing an oligonucleotide, comprising one or more cycles, each of which independently comprises: (1) a coupling step; (2) optionally a pre-modification capping step; (3) a modification step; (4) optionally a post-modification capping step; and (5) optionally a de-blocking step.

(1456) In some embodiments, a cycle comprises one or more pre-modification capping steps. In some embodiments, a cycle comprises one or more post-modification capping steps. In some embodiments, a cycle comprises one or more pre- and post-modification capping steps. In some embodiments, a cycle comprises one or more de-blocking steps. In some embodiments, a cycle comprises a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a de-blocking step. In some embodiments, a cycle comprises a coupling step, a pre-modification capping step, a modification step, and a de-blocking step. In some embodiments, a cycle comprises a coupling step, a modification step, a post-modification capping step and a de-blocking step. In some embodiments, comprise a coupling step, a pre-modification capping step, a modification step, a post-modification capping step, and a de-blocking step. In some embodiments, one or more cycles comprise a coupling step, a pre-modification capping step, a modification step, and a de-blocking step. In some embodiments, one or more cycles comprise a coupling step, a modification step, a post-modification capping step and a de-blocking step.

(1457) When describing the provided methods, the word cycle has its ordinary meaning as understood by a person of ordinary skill in the art. In some embodiments, one round of steps (1)-(4) is referred to as a cycle. In some embodiments, some cycles comprise modifying. In some embodiments, some cycles do not comprise modifying. In some embodiments, some cycles comprise and some cycles do not comprise modifying. In some embodiments, each cycle independently comprises a modifying step. In some embodiments, each cycle does not comprise a cycling step.

(1458) In some embodiments, to form a chirally controlled internucleotidic linkage, a chirally pure phosphoramidite comprising a chiral auxiliary is utilized to stereoselectively form the chirally controlled internucleotidic linkage. Various phosphoramidite and chiral auxiliaries, e.g., those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the phosphoramidite and chiral auxiliaries of each of which are incorporated herein by reference, may be utilized in accordance with the present disclosure.

(1459) In some embodiments, a coupling step provides an oligonucleotide comprises an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof, wherein P.sup.L is P. In some embodiments, such an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, such an internucleotidic linkage comprises a chiral auxiliary moiety.

(1460) In some embodiments, a modifying step provides an oligonucleotide comprises an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, etc., or a salt form thereof, wherein P.sup.L is PW. In some embodiments, a modifying step provides an oligonucleotide comprises an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof, wherein P.sup.L is PW. In some embodiments, W is S. In some embodiments, W is O. In some embodiments, such an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, such an internucleotidic linkage comprises a chiral auxiliary moiety. In some embodiments, a modifying step provides a non-negatively charged internucleotidic linkage. In some embodiments, a non-negatively charged internucleotidic linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In some embodiments, such an internucleotidic linkage is a neutral internucleotidic linkage. In some embodiments, such an internucleotidic linkage is a chirally controlled internucleotidic linkage. In some embodiments, such an internucleotidic linkage comprises a chiral auxiliary moiety. In some embodiments, such an internucleotidic linkage comprises no chiral auxiliary moiety. In some embodiments, a chiral auxiliary moiety falls off during modification.

(1461) Provided technologies provide various advantages. Among other things, as demonstrated herein, provided technologies can greatly improve oligonucleotide synthesis crude purity and yield, particularly for modified and/or chirally pure oligonucleotides that provide a number of properties and activities that are critical for therapeutic purposes. With the capability to provide unexpectedly high crude purity and yield for therapeutically important oligonucleotides, provided technologies can significantly reduce manufacturing costs (through, e.g., simplified purification, greatly improved overall yields, etc.). In some embodiments, provided technologies can be readily scaled up to produce oligonucleotides in sufficient quantities and qualities for clinical purposes. In some embodiments, provided technologies comprising chiral auxiliaries that comprise electron-withdrawing groups in G.sup.2 (e.g., PSM chiral auxiliaries) are particularly useful for preparing chirally controlled internucleotidic linkages comprising PN bonds (e.g., non-negatively charged internucleotidic linkages such as n001, n002, n003, n004, n005, n006, n007, n008, n009, n010, etc.) and can significantly simplify manufacture operations, reduce cost, and/or facilitate downstream formation.

(1462) In some embodiments, provided technologies provides improved reagents compatibility. For example, as demonstrated in the present disclosure, provided technologies provide flexibility to use different reagent systems for oxidation, sulfurization and/or azide reactions, particularly for chirally controlled oligonucleotide synthesis.

(1463) Among other things, the present disclosure provides oligonucleotide compositions of high crude purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotide composition of high crude purity. In some embodiments, the present disclosure provides chirally controlled oligonucleotide of high crude purity. In some embodiments, the present disclosure provides oligonucleotide of high crude purity and/or high stereopurity.

(1464) Support and Linkers

(1465) In some embodiments, oligonucleotides can be prepared in solution. In some embodiments, oligonucleotides can be prepared using a support. In some embodiments, oligonucleotides are prepared using a solid support. Suitable support that can be utilized in accordance with the present disclosure include, e.g., solid support described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the solid support of each of which is incorporated herein by reference.

(1466) In some embodiments, a linker moiety is utilized to connect an oligonucleotide chain to a support during synthesis. Suitable linkers are widely utilized in the art, and include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the linker of each of which is incorporated herein by reference

(1467) In some embodiments, the linking moiety is a succinamic acid linker, or a succinate linker (COCH.sub.2CH.sub.2CO), or an oxalyl linker (COCO). In some embodiments, the linking moiety and the nucleoside are bonded together through an ester bond. In some embodiments, a linking moiety and a nucleoside are bonded together through an amide bond. In some embodiments, a linking moiety connects a nucleoside to another nucleotide or nucleic acid. Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991, Chapter 1 and Solid-Phase Supports for Oligonucleotide Synthesis, Pon, R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28. In some embodiments, a universal linker (UnyLinker) is used to attached the oligonucleotide to the solid support (Ravikumar et al., Org. Process Res. Dev., 2008, 12 (3), 399-410). In some embodiments, other universal linkers are used (Pon, R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28). In some embodiments, various orthogonal linkers (such as disulfide linkers) are used (Pon, R. T., Curr. Prot. Nucleic Acid Chem., 2000, 3.1.1-3.1.28).

(1468) Among other things, the present disclosure recognizes that a linker can be chosen or designed to be compatible with a set of reaction conditions employed in oligonucleotide synthesis. In some embodiments, to avoid degradation of oligonucleotides and to avoid desulfurization, auxiliary groups are selectively removed before de-protection. In some embodiments, DPSE group can selectively be removed by F.sup. ions. In some embodiments, the present disclosure provides linkers that are stable under a DPSE de-protection condition, e.g., 0.1M TBAF in MeCN, 0.5M HF-Et.sub.3N in THE or MeCN, etc. In some embodiments, a provided linker is a linker as exemplified below:

(1469) ##STR00513##
Solvents

(1470) Syntheses of provided oligonucleotides are generally performed in aprotic organic solvents. In some embodiments, a solvent is a nitrile solvent such as, e.g., acetonitrile. In some embodiments, a solvent is a basic amine solvent such as, e.g., pyridine. In some embodiments, a solvent is an ethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, a solvent is a halogenated hydrocarbon such as, e.g., dichloromethane. In some embodiments, a mixture of solvents is used. In certain embodiments a solvent is a mixture of any one or more of the above-described classes of solvents.

(1471) In some embodiments, when an aprotic organic solvent is not basic, a base is present in the reacting step. In some embodiments where a base is present, the base is an amine base such as, e.g., pyridine, quinoline, or N,N-dimethylaniline. Example other amine bases include pyrrolidine, piperidine, N-methyl pyrrolidine, pyridine, quinoline, N,N-dimethylaminopyridine (DMAP), or N,N-dimethylaniline.

(1472) In some embodiments, a base is other than an amine base.

(1473) In some embodiments, an aprotic organic solvent is anhydrous. In some embodiments, an anhydrous aprotic organic solvent is freshly distilled. In some embodiments, a freshly distilled anhydrous aprotic organic solvent is a basic amine solvent such as, e.g., pyridine. In some embodiments, a freshly distilled anhydrous aprotic organic solvent is an ethereal solvent such as, e.g., tetrahydrofuran. In some embodiments, a freshly distilled anhydrous aprotic organic solvent is a nitrile solvent such as, e.g., acetonitrile.

(1474) Chiral Reagents/Chiral Auxiliaries

(1475) In some embodiments, chiral reagents (may also be referred to as chiral auxiliaries) are used to confer stereoselectivity in the production of chirally controlled oligonucleotides. Many chiral reagents, also referred to by those of skill in the art and herein as chiral auxiliaries, may be used in accordance with methods of the present disclosure. Examples of such chiral reagents are described herein and in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the chiral auxiliaries of each of which is incorporated by reference.

(1476) In some embodiments, a chiral reagent for use in accordance with the methods of the present disclosure is of Formula 3-I, below:

(1477) ##STR00514##
wherein: W.sup.1 and W.sup.2 are any of O, S, NG.sup.5-, or NG.sup.5-O; U.sub.1 and U.sub.3 are carbon atoms which are bonded to U.sub.2 if present, or to each other if r is 0, via a single, double or triple bond; U.sub.2 is C, CG.sup.8-, CG.sup.8G.sup.8-, NG.sup.8-, N, O, or S where r is an integer of 0 to 5; and each of G.sup.1, G.sup.2, G.sup.3, G.sup.4, G.sup.5, and G.sup.8 is independently R.sup.1 as described in the present disclosure.

(1478) In some embodiments, W.sup.1 and W.sup.2 are any of O, S, or NG.sup.5-, U.sub.1 and U.sub.3 are carbon atoms which are bonded to U.sub.2 if present, or to each other if r is 0, via a single, double or triple bond. U.sub.2 is C, CG.sup.8-, CG.sup.8G.sup.8-, NG.sup.8-, N, O, or S where r is an integer of 0 to 5 and no more than two heteroatoms are adjacent. When any one of U.sub.2 is C, a triple bond must be formed between a second instance of U.sub.2, which is C, or to one of U.sub.1 or U.sub.3. Similarly, when any one of U.sub.2 is CG.sup.8, a double bond is formed between a second instance of U.sub.2 which is CG.sup.8- or N, or to one of U.sub.1 or U.sub.3.

(1479) In some embodiments, U.sub.1G.sup.3G.sup.4-(U.sub.2).sub.rU.sub.3G.sup.1G.sup.2- is CG.sup.3G.sup.4-CG.sup.1G.sup.2-. In some embodiments, U.sub.1(U.sub.2).sub.rU.sub.3 is CG.sup.3=CG.sup.1-. In some embodiments, U.sub.1(U.sub.2).sub.rU.sub.3 is CC. In some embodiments, U.sub.1(U.sub.2).sub.rU.sub.3 is CG.sup.3-CG.sup.8-CG.sup.1G.sup.2-. In some embodiments, U.sub.1(U.sub.2).sub.rU.sub.3 is CG.sup.3G.sup.4-OCG.sup.1G.sup.2-. In some embodiments, U.sub.1(U.sub.2).sub.rU.sub.3 is CG.sup.3G.sup.4-NG.sup.8-CG.sup.1G.sup.2-. In some embodiments, U.sub.1(U.sub.2).sub.rU.sub.3 is CG.sup.3G.sup.4-NCG.sup.2-. In some embodiments, U.sub.1(U.sub.2).sub.rU.sub.3 is CG.sup.3G.sup.4-NCG.sup.8-CG.sup.1G.sup.2-.

(1480) In some embodiments, G.sup.1, G.sup.2, G.sup.3, G.sup.4, G.sup.5, and G.sup.8 are independently R.sup.1 as described in the present disclosure. In some embodiments, G.sup.1, G.sup.2, G.sup.3, G.sup.4, G.sup.5, and G.sup.8 are independently R as described in the present disclosure. In some embodiments, G.sup.1, G.sup.2, G.sup.3, G.sup.4, G.sup.5, and G.sup.8 are independently hydrogen, or an optionally substituted group selected from aliphatic, alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaliphatic, heterocyclyl, heteroaryl, and aryl; or two of G.sup.1, G.sup.2, G.sup.3, G.sup.4, and G.sup.5 are G.sup.6 (taken together to form an optionally substituted, saturated, partially unsaturated or unsaturated carbocyclic or heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, and is fused or unfused). In some embodiments, a ring so formed is substituted by oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments, when a ring formed by taking two G.sup.6 together is substituted, it is substituted by a moiety which is bulky enough to confer stereoselectivity during the reaction.

(1481) In some embodiments, a ring formed by taking two of G.sup.6 together is optionally substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl, tetrahydropyranyl, or piperazinyl. In some embodiments, a ring formed by taking two of G.sup.6 together is optionally substituted cyclopentyl, pyrrolyl, cyclopropyl, cyclohexenyl, cyclopentenyl, tetrahydropyranyl, pyrrolidinyl, or piperazinyl.

(1482) In some embodiments, G.sup.1 is optionally substituted phenyl. In some embodiments, G.sup.1 is phenyl. In some embodiments, G.sup.2 is methyl or hydrogen. In some embodiments, G.sup.2 is hydrogen. In some embodiments, G.sup.1 is optionally substituted phenyl and G.sup.2 is methyl. In some embodiments, G.sup.1 is phenyl and G.sup.2 is methyl. In some embodiments, G.sup.1 is CH.sub.2Si(R).sub.3, wherein one R is optionally substituted C.sub.1-6 aliphatic, and the other two R are each independently an optionally substituted 3-20 membered, monocyclic or polycyclic, saturated, partially unsaturated or aromatic ring having 0-5 heteroatoms. In some embodiments, the other two R are each independently optionally substituted phenyl. In some embodiments, G.sup.1 is CH.sub.2SiMePh.sub.2.

(1483) In some embodiments, r is 0.

(1484) In some embodiments, W.sup.1 is NG.sup.5-O. In some embodiments, W.sup.1 is NG.sup.5-O, wherein the O is bonded to H. In some embodiments, W.sup.1 is NG.sup.5-. In some embodiments, one of G.sup.3 and G.sup.4 is taken together with G.sup.5 to form an optionally substituted 3-10 membered ring. In some embodiments, one of G.sup.3 and G.sup.4 is taken together with G.sup.5 to form an optionally substituted pyrrolidinyl ring. In some embodiments, one of G.sup.3 and G.sup.4 is taken together with G.sup.5 to form a pyrrolidinyl ring. In some embodiments, G.sup.5 is optionally substituted C.sub.1-6 aliphatic. In some embodiments, G.sup.5 is methyl. In some embodiments, one of G.sup.1 and G.sup.2 and one of G.sup.3 and G.sup.4 are taken together with their intervening atoms to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms. In some embodiments, a formed ring 3-membered. In some embodiments, a formed ring 4-membered. In some embodiments, a formed ring 5-membered. In some embodiments, a formed ring 6-membered. In some embodiments, a formed ring 7-membered. In some embodiments, a formed ring is substituted. In some embodiments, a formed ring is unsubstituted. In some embodiments, a formed ring has no heteroatom. In some embodiments, a formed ring is saturated. For example compounds, see WV-CA-293 and WV-CA-294.

(1485) In some embodiments, W.sup.2 is O.

(1486) In some embodiments, a chiral reagent is a compound of Formula 3-AA:

(1487) ##STR00515##
wherein each variable is independently as defined above and described herein.

(1488) In some embodiments of Formula 3AA, W.sup.1 and W.sup.2 are independently NG.sup.5-, O, or S; G.sup.1, G.sup.2, G.sup.3, G.sup.4, and G.sup.5 are independently hydrogen, or an optionally substituted group selected from alkyl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaliphatic, heterocyclyl, heteroaryl, or aryl; or two of G.sup.1, G.sup.2, G.sup.3, G.sup.4, and G.sup.5 are G.sup.6 (taken together to form an optionally substituted saturated, partially unsaturated or unsaturated carbocyclic or heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused), and no more than four of G.sup.1, G.sup.2, G.sup.3, G.sup.4, and G.sup.5 are G.sup.6. Similarly to the compounds of Formula 3-I, any of G.sup.1, G.sup.2, G.sup.3, G.sup.4, or G.sup.5 are optionally substituted by oxo, thioxo, alkyl, alkenyl, alkynyl, heteroaryl, or aryl moieties. In some embodiments, such substitution induces stereoselectivity in chirally controlled oligonucleotide production. In some embodiments, a heteroatom-containing moiety, e.g., heteroaliphatic, heterocyclyl, heteroaryl, etc., has 1-5 heteroatoms. In some embodiments, the heteroatoms are selected from nitrogen, oxygen, sulfure and silicon. In some embodiments, at least one heteroatom is nitrogen.

(1489) In some embodiments, W.sup.1 is NG.sup.5-O. In some embodiments, W.sup.1 is NG.sup.5-O, wherein the O is bonded to H. In some embodiments, W.sup.1 is NG.sup.5-. In some embodiments, G.sup.5 and one of G.sup.3 and G.sup.4 are taken together to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms in addition to the nitrogen atom of W.sup.1. In some embodiments, G.sup.5 and G.sup.3 are taken together to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms in addition to the nitrogen atom of W.sup.1. In some embodiments, G.sup.5 and G.sup.4 are taken together to form an optionally substituted 3-10 membered ring having 0-3 heteroatoms in addition to the nitrogen atom of W.sup.1. In some embodiments, a formed ring is an optionally substituted 4, 5, 6, 7, or 8 membered ring. In some embodiments, a formed ring is an optionally substituted 4-membered ring. In some embodiments, a formed ring is an optionally substituted 5-membered ring. In some embodiments, a formed ring is an optionally substituted 6-membered ring. In some embodiments, a formed ring is an optionally substituted 7-membered ring.

(1490) In some embodiments, a provided chiral reagent has the structure of

(1491) ##STR00516##
In some embodiments, a provided chiral reagent has the structure of

(1492) ##STR00517##
In some embodiments, a provided chiral reagent has the structure of

(1493) ##STR00518##
In some embodiments, a provided chiral reagent has the structure of

(1494) ##STR00519##
In some embodiments, a provided chiral reagent has the structure of

(1495) ##STR00520##
In some embodiments, a provided chiral reagent has the structure of

(1496) ##STR00521##
In some embodiments, a provided chiral reagent has the structure of

(1497) ##STR00522##
In some embodiments, a provided chiral reagent has the structure of

(1498) ##STR00523##

(1499) In some embodiments, W.sup.1 is NG.sup.5, W.sup.2 is O, each of G.sup.1 and G.sup.3 is independently hydrogen or an optionally substituted group selected from C.sub.1-10 aliphatic, heterocyclyl, heteroaryl and aryl, G.sup.2 is C(R).sub.2Si(R).sub.3, and G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused. In some embodiments, each R is independently hydrogen, or an optionally substituted group selected from C.sub.1-C.sub.6 aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, G.sup.2 is C(R).sub.2Si(R).sub.3, wherein C(R).sub.2 is optionally substituted CH.sub.2, and each R of Si(R).sub.3 is independently an optionally substituted group selected from C.sub.1-10 aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, at least one R of Si(R).sub.3 is independently optionally substituted C.sub.1-10 alkyl. In some embodiments, at least one R of Si(R).sub.3 is independently optionally substituted phenyl. In some embodiments, one R of Si(R).sub.3 is independently optionally substituted phenyl, and each of the other two R is independently optionally substituted C.sub.1-10 alkyl. In some embodiments, one R of Si(R).sub.3 is independently optionally substituted C.sub.1-10 alkyl, and each of the other two R is independently optionally substituted phenyl. In some embodiments, G.sup.2 is optionally substituted CH.sub.2Si(Ph)(Me).sub.2. In some embodiments, G.sup.2 is optionally substituted CH.sub.2Si(Me)(Ph).sub.2. In some embodiments, G.sup.2 is CH.sub.2Si(Me)(Ph).sub.2. In some embodiments, G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated 5-6 membered ring containing one nitrogen atom (to which G.sup.5 is attached). In some embodiments, G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated 5-membered ring containing one nitrogen atom. In some embodiments, G.sup.1 is hydrogen. In some embodiments, G.sup.3 is hydrogen. In some embodiments, both G.sup.1 and G.sup.3 are hydrogen.

(1500) In some embodiments, W.sup.1 is NG.sup.5, W.sup.2 is O, each of G.sup.1 and G.sup.3 is independently R.sup.1, G.sup.2 is R.sup.1, and G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused. In some embodiments, each of G.sup.1 and G.sup.3 is independently R. In some embodiments, each of G.sup.1 and G.sup.3 is independently H. In some embodiments, G.sup.2 is connected to the rest of the molecule through a carbon atom, and the carbon atom is substituted with one or more electron-withdrawing groups. In some embodiments, G.sup.2 is methyl substituted with one or more electron-withdrawing groups. In some embodiments, G.sup.2 is methyl substituted with one and no more than one electron-withdrawing group. In some embodiments, G.sup.2 is methyl substituted with two or more electron-withdrawing groups. Among other things, a chiral auxiliary having G.sup.2 comprising an electron-withdrawing group can be readily removed by a base (base-labile, e.g., under an anhydrous condition substantially free of water; in many instances, preferably before oligonucleotides comprising internucleotidic linkages comprising such chiral auxiliaries are exposed to conditions/reagent systems comprising a substantial amount of water, particular in the presence of a base (e.g., cleavage conditions/reagent systems using NH.sub.4OH)) and provides various advantages as described herein, e.g., high crude purity, high yield, high stereoselectivity, more simplified operation, fewer steps, further reduced manufacture cost, and/or more simplified downstream formulation (e.g., low amount of salt(s) after cleavage), etc. In some embodiments, as described in the Examples, such auxiliaries may provide alternative or additional chemical compatibility with other functional and/or protection groups. In some embodiments, as demonstrated in the Examples, base-labile chiral auxiliaries are particularly useful for construction of chirally controlled non-negatively charged internucleotidic linkages (e.g., neutral internucleotidic linkages such as n001); in some instances, as demonstrated in the Examples, they can provide significantly improved yield and/or crude purity with high stereoselectivity, e.g., when utilized with removal using a base under an anhydrous condition. In some embodiments, such a chiral auxiliary is bonded to a linkage phosphorus via an oxygen atom (e.g., which corresponds to a OH group in a corresponding chiral auxiliary compound, e.g., a compound of formula I), the carbon atom in the chiral auxiliary to which the oxygen is bonded (the alpha carbon) also bonds to H (in addition to other groups; in some embodiments, a secondary carbon), and the next carbon atom (the beta carbon) in the chiral auxiliary is boned to one or two electron-withdrawing groups. In some embodiments, W.sup.2H is OH. In some embodiments, G.sup.1 is H. In some embodiments, G.sup.2 comprises one or two electron-withdrawing groups or can otherwise facilitate remove of the chiral auxiliary by a base. In some embodiments, G.sup.1 is H, G.sup.2 comprises one or two electron-withdrawing groups, W.sup.2His OH. In some embodiments, G.sup.1 is H, G.sup.2 comprises one or two electron-withdrawing groups, W.sup.2H is OH, W.sup.1H is NG.sup.5-H, and one of G.sup.3 and G.sup.4 is taken together with G.sup.5 to form with their intervening atoms a ring as described herein (e.g., an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having in addition to the nitrogen atom to which G.sup.5 is on, 0-5 heteroatoms (e.g., an optionally substituted 3, 4, 5, or 6-membered monocyclic saturated ring having in addition to the nitrogen atom to which G.sup.5 is on no other heteroatoms)).

(1501) As appreciated by those skilled in the art, various electron-withdrawing groups are known in the art and can be utilized in accordance with the present disclosure. In some embodiments, an electronic-withdrawing group comprises and/or is connected to the carbon atom through, e.g., S(O), S(O).sub.2, P(O)(R.sup.1), P(S)R.sup.1, or C(O). In some embodiments, an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2. In some embodiments, an electron-withdrawing group is aryl or heteroaryl, e.g., phenyl, substituted with one or more of CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.

(1502) In some embodiments, G.sup.2 is -L-R. In some embodiments, G.sup.2 is -L-L-R, wherein L is C(R).sub.2 or optionally substituted CH.sub.2, and L is P(O)(R), P(O)(R)O, P(O)(OR), P(O)(OR)O, P(O)[N(R)], P(O)[N(R)]O, P(O)[N(R)][N(R)], P(S)(R), S(O).sub.2, S(O).sub.2, S(O).sub.2O, S(O), C(O), C(O)N(R), or S. In some embodiments, L is C(R).sub.2. In some embodiments, L is optionally substituted CH.sub.2.

(1503) In some embodiments, L is C(R).sub.2. In some embodiments, each R is independently hydrogen, or an optionally substituted group selected from C.sub.1-C.sub.6 aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, L is CH.sub.2. In some embodiments, L is P(O)(R), P(S)(R), S(O).sub.2. In some embodiments, G.sup.2 is -LC(O)N(R).sub.2. In some embodiments, G.sup.2 is -LP(O)(R).sub.2. In some embodiments, G.sup.2 is -LP(S)(R).sub.2. In some embodiments, each R is independently optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl as described in the present disclosure (e.g., those embodiments described for R). In some embodiments, each R is independently optionally substituted phenyl. In some embodiments, each R is independently optionally substituted phenyl wherein one or more substituents are independently selected from CN, OMe, Cl, Br, and F. In some embodiments, each R is independently substituted phenyl wherein one or more substituents are independently selected from CN, OMe, Cl, Br, and F. In some embodiments, each R is independently substituted phenyl wherein the substituents are independently selected from CN, OMe, Cl, Br, and F. In some embodiments, each R is independently mono-substituted phenyl, wherein the substituent is independently selected from CN, OMe, Cl, Br, and F. In some embodiments, two R are the same. In some embodiments, two R are different. In some embodiments, G.sup.2 is -LS(O)R. In some embodiments, G.sup.2 is -LC(O)N(R).sub.2. In some embodiments, G.sup.2 is -LS(O).sub.2R. In some embodiments, R is optionally substituted aliphatic, heteroaliphatic, aryl, or heteroaryl as described in the present disclosure (e.g., those embodiments described for R). In some embodiments, R is optionally substituted phenyl. In some embodiments, R is optionally substituted phenyl wherein one or more substituents are independently selected from CN, OMe, Cl, Br, and F. In some embodiments, R is substituted phenyl wherein one or more substituents are independently selected from CN, OMe, Cl, Br, and F. In some embodiments, R is substituted phenyl wherein each substituent is independently selected from CN, OMe, Cl, Br, and F. In some embodiments, R is mono-substituted phenyl. In some embodiments, R is mono-substituted phenyl, wherein the substituent is independently selected from CN, OMe, Cl, Br, and F. In some embodiments, a substituent is an electron-withdrawing group. In some embodiments, an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.

(1504) In some embodiments, G.sup.2 is optionally substituted CH.sub.2-L-R, wherein each of L and R is independently as described in the present disclosure. In some embodiments, G.sup.2 is optionally substituted CH(-L-R).sub.2, wherein each of L and R is independently as described in the present disclosure. In some embodiments, G.sup.2 is optionally substituted CH(SR).sub.2. In some embodiments, G.sup.2 is optionally substituted CH.sub.2SR. In some embodiments, the two R groups are taken together with their intervening atoms to form a ring. In some embodiments, a formed ring is an optionally substituted 5,6,7-membered ring having 0-2 heteroatoms in addition to the intervening heteroatoms. In some embodiments, G.sup.2 is optionally substituted

(1505) ##STR00524##
In some embodiments, G.sup.2 is

(1506) ##STR00525##
In some embodiments, S may be converted to S(O) or S(O).sub.2, e.g., by oxidation, e.g., to facilitate removal by a base.

(1507) In some embodiments, G.sup.2 is -L-R, wherein each variable is as described in the present disclosure. In some embodiments, G.sup.2 is CH.sub.2R. In some embodiments, G.sup.2 is CH(R).sub.2. In some embodiments, G.sup.2 is C(R).sub.3. In some embodiments, R is optionally substituted aryl or heteroaryl. In some embodiments, R is substituted aryl or heteroaryl wherein one or more substituents are independently an electron-withdrawing group. In some embodiments, -L- is optionally substituted CH.sub.2, and R is R, wherein R is optionally substituted aryl or heteroaryl. In some embodiments, R is substituted aryl or heteroaryl wherein one or more substituents are independently an electron-withdrawing group. In some embodiments, R is substituted aryl or heteroaryl wherein each substituent is independently an electron-withdrawing group. In some embodiments, R is aryl or heteroaryl substituted with two or more substituents, wherein each substituent is independently an electron-withdrawing group. In some embodiments, an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2. In some embodiments, R is

(1508) ##STR00526##

(1509) In some embodiments, R is p-NO.sub.2Ph-. In some embodiments, R is

(1510) ##STR00527##
In some embodiments, R is

(1511) ##STR00528##
In some embodiments, R is

(1512) ##STR00529##
In some embodiments, R is

(1513) ##STR00530##
In some embodiments, R is

(1514) ##STR00531##
In some embodiments, G.sup.2 is

(1515) ##STR00532##
In some embodiments, R is

(1516) ##STR00533##
In some embodiments, R is

(1517) ##STR00534##
In some embodiments, R is 2,4,6-trichlorophenyl. In some embodiments, R is 2,4,6-trifluorophenyl. In some embodiments, G.sup.2 is CH(4-chlorophenyl).sub.2. In some embodiments, G.sup.2 is CH(R).sub.2, wherein each R is

(1518) ##STR00535##
In some embodiments, G.sup.2 is CH(R).sub.2, wherein each R is

(1519) ##STR00536##
In some embodiments, R is C(O)R. In some embodiments, R is CH.sub.3C(O).

(1520) In some embodiments, G.sup.2 is -LS(O).sub.2R, wherein each variable is as described in the present disclosure. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2R. In some embodiments, G.sup.2 is -LS(O)R, wherein each variable is as described in the present disclosure. In some embodiments, G.sup.2 is CH.sub.2S(O)R. In some embodiments, G.sup.2 is -LC(O).sub.2R, wherein each variable is as described in the present disclosure. In some embodiments, G.sup.2 is CH.sub.2C(O).sub.2R. In some embodiments, G.sup.2 is -LC(O)R, wherein each variable is as described in the present disclosure. In some embodiments, G.sup.2 is CH.sub.2C(O)R. In some embodiments, -L- is optionally substituted CH.sub.2, and R is R. In some embodiments, R is optionally substituted aryl or heteroaryl. In some embodiments, R is optionally substituted aliphatic. In some embodiments, R is optionally substituted heteroaliphatic. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is not phenyl, or mono-, di- or tri-substituted phenyl, wherein each substituent is selected from NO.sub.2, halogen, CN, C.sub.1-3 alkyl, and C.sub.1-3 alkyloxy. In some embodiments, R is substituted aryl or heteroaryl wherein one or more substituents are independently an electron-withdrawing group. In some embodiments, R is substituted aryl or heteroaryl wherein each substituent is independently an electron-withdrawing group. In some embodiments, R is aryl or heteroaryl substituted with two or more substituents, wherein each substituent is independently an electron-withdrawing group. In some embodiments, an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2. In some embodiments, R is phenyl. In some embodiments, R is substituted phenyl. In some embodiments, R is

(1521) ##STR00537##
In some embodiments, R is

(1522) ##STR00538##
In some embodiments, R is

(1523) ##STR00539##
In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is t-butyl. In some embodiments, R is isopropyl. In some embodiments, R is methyl. In some embodiments, G.sup.2 is CH.sub.2C(O)OMe. In some embodiments, G.sup.2 is CH.sub.2C(O)Ph. In some embodiments, G.sup.2 is CH.sub.2C(O)-tBu.

(1524) In some embodiments, G.sup.2 is -LNO.sub.2. In some embodiments, G.sup.2 is CH.sub.2NO.sub.2. In some embodiments, G.sup.2 is -LS(O).sub.2N(R).sub.2. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(R).sub.2. In some embodiments, G.sup.2 is -LS(O).sub.2NHR. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2NHR. In some embodiments, R is methyl. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2NH(CH.sub.3). In some embodiments, R is CH.sub.2Ph. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2NH(CH.sub.2Ph). In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.2Ph).sub.2. In some embodiments, R is phenyl. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2NHPh. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.3)Ph. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.3).sub.2. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2NH(CH.sub.2Ph). In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2NHPh. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2NH(CH.sub.2Ph). In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.3).sub.2. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.3)Ph. In some embodiments, G.sup.2 is -LS(O).sub.2N(R)(OR). In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(R)(OR). In some embodiments, each R is methyl. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.3)(OCH.sub.3). In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(Ph)(OCH.sub.3). In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.2Ph)(OCH.sub.3). In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2N(CH.sub.2Ph)(OCH.sub.3). In some embodiments, G.sup.2 is -LS(O).sub.2OR. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2OR. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2OPh. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2OCH.sub.3. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2OCH.sub.2Ph.

(1525) In some embodiments, G.sup.2 is -LP(O)(R).sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)(R).sub.2. In some embodiments, G.sup.2 is -LP(O)[N(R).sub.2].sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)[N(R).sub.2].sub.2. In some embodiments, G.sup.2 is -LP(O)[O(R).sub.2].sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)[O(R).sub.2].sub.2. In some embodiments, G.sup.2 is -LP(O)(R)[N(R).sub.2].sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)(R)[N(R).sub.2]. In some embodiments, G.sup.2 is -LP(O)(R)[O(R)]. In some embodiments, G.sup.2 is CH.sub.2P(O)(R)[O(R)]. In some embodiments, G.sup.2 is -LP(O)(OR)[N(R).sub.2]. In some embodiments, G.sup.2 is CH.sub.2P(O)(OR)[N(R).sub.2]. In some embodiments, G.sup.2 is -LC(O)N(R).sub.2, wherein each variable is as described in the present disclosure. In some embodiments, G.sup.2 is CH.sub.2C(O)N(R).sub.2. In some embodiments, each R is independently R. In some embodiments, one R is optionally substituted aliphatic, and one R is optionally substituted aryl. In some embodiments, one R is optionally substituted C.sub.1-6 aliphatic, and one R is optionally substituted phenyl. In some embodiments, each R is independently optionally substituted C.sub.1-6 aliphatic. In some embodiments, G.sup.2 is CH.sub.2P(O)(CH.sub.3)Ph. In some embodiments, G.sup.2 is CH.sub.2P(O)(CH.sub.3).sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)(Ph).sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)(OCH.sub.3).sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)(CH.sub.2Ph).sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)[N(CH.sub.3)Ph].sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)[N(CH.sub.3).sub.2].sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)[N(CH.sub.2Ph).sub.2].sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)(OCH.sub.3).sub.2. In some embodiments, G.sup.2 is CH.sub.2P(O)(OPh).sub.2.

(1526) In some embodiments, G.sup.2 is -LSR. In some embodiments, G.sup.2 is CH.sub.2SR. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl.

(1527) In some embodiments, a provided chiral reagent has the structure of

(1528) ##STR00540##
wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, a provided chiral reagent has the structure of

(1529) ##STR00541##
wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, each R.sup.1 is independently R as described in the present disclosure. In some embodiments, each R.sup.1 is independently R, wherein R is optionally substituted aliphatic, aryl, heteroaliphatic, or heteroaryl as described in the present disclosure. In some embodiments, each R.sup.1 is phenyl. In some embodiments, R.sup.1 is -L-R. In some embodiments, R.sup.1 is -L-R, wherein L is O, S, or N(R). In some embodiments, a provided chiral reagent has the structure of

(1530) ##STR00542##
wherein each X.sup.1 is independently H, an electron-withdrawing group, NO.sub.2, CN, OR, Cl, Br, or F, and W is O or S. In some embodiments, a provided chiral reagent has the structure of

(1531) ##STR00543##
wherein each X.sup.1 is independently H, an electron-withdrawing group, NO.sub.2, CN, OR, Cl, Br, or F, and W is O or S. In some embodiments, each X.sup.1 is independently CN, OR, Cl, Br, or F, wherein R is not H. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is CH.sub.3. In some embodiments, one or more X.sup.1 are independently electron-withdrawing groups (e.g., CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, P(S)(R.sup.1).sub.2, etc.).

(1532) In some embodiments, a provided chiral reagent has the structure of

(1533) ##STR00544##
wherein R.sup.1 is as described in the present disclosure. In some embodiments, a provided chiral reagent has the structure of

(1534) ##STR00545##
wherein R.sup.1 is as described in the present disclosure. In some embodiments, R.sup.1 is R as described in the present disclosure. In some embodiments, R.sup.1 is R, wherein R is optionally substituted aliphatic, aryl, heteroaliphatic, or heteroaryl as described in the present disclosure. In some embodiments, R.sup.1 is -L-R. In some embodiments, R.sup.1 is -L-R, wherein L is O, S, or N(R). In some embodiments, a provided chiral reagent has the structure of

(1535) ##STR00546##
wherein X.sup.1 is H, an electron-withdrawing group, NO.sub.2, CN, OR, Cl, Br, or F, and W is O or S. In some embodiments, a provided chiral reagent has the structure of

(1536) ##STR00547##
wherein X.sup.1 is H, an electron-withdrawing group, NO.sub.2, CN, OR, Cl, Br, or F, and W is O or S. In some embodiments, X.sup.1 is CN, OR, Cl, Br, or F, wherein R is not H. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is CH.sub.3. In some embodiments, X.sup.1 is an electron-withdrawing group (e.g., CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, P(S)(R.sup.1).sub.2, etc.). In some embodiments, X.sup.1 is an electron-withdrawing group that is not CN, NO.sub.2, or halogen. In some embodiments, X.sup.1 is not H, CN, NO.sub.2, halogen, or C.sub.1-3 alkyloxy.

(1537) In some embodiments, G.sup.2 is CH(R.sup.21)CH(R.sup.22)C(R.sup.23)(R.sup.24), wherein each of R.sup.21, R.sup.22, R.sup.23, and R.sup.24 is independently R. In some embodiments, R.sup.22 and R.sup.23 are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted aryl or heteroaryl ring as described herein. In some embodiments, one or more substituents are independently electron-withdrawing groups. In some embodiments, R.sup.21 and R.sup.24 are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted ring as described herein. In some embodiments, R.sup.21 and R.sup.24 are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted saturated or partially saturated ring as described herein. In some embodiments, R.sup.22 and R.sup.23 are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted aryl or heteroaryl ring as described herein, and R.sup.21 and R.sup.24 are both R, and the two R groups are taken together with their intervening atoms to form an optionally substituted partially saturated ring as described herein. In some embodiments, R.sup.21 is H. In some embodiments, R.sup.24 is H. In some embodiments, G.sup.2 is optionally substituted

(1538) ##STR00548##
In some embodiments, G.sup.2 is optionally substituted

(1539) ##STR00549##
wherein each Ring A.sup.2 is independently a 3-15 membered monocyclic, bicyclic or polycyclic ring as described herein. In some embodiments, Ring A.sup.2 is an optionally substituted 5-10 membered monocyclic aryl or heteroaryl ring having 1-5 heteroatoms as described herein. In some embodiments, Ring A.sup.2 is an optionally substituted phenyl ring as described herein. In some embodiments, In some embodiments, G.sup.2 is optionally substituted

(1540) ##STR00550##
In some embodiments, G.sup.2 is

(1541) ##STR00551##
In some embodiments, G.sup.2 is

(1542) ##STR00552##
In some embodiments, G.sup.2 is

(1543) ##STR00553##

(1544) Certain useful example compounds for chiral auxiliaries are presented in, e.g., Tables CA-1 to CA-13. In some embodiments, a useful compound is an enantiomer of a compound in, e.g., Tables CA-1 to CA-13. In some embodiments, a useful compound is a diastereomer of a compound in, e.g., Tables CA-1 to CA-13. In some embodiments, a compound useful for chiral auxiliaries for removal under basic conditions (e.g., by a base under an anhydrous condition) is a compound of Tables CA-1 to CA-13, or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-1 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-2 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-3 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-4 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-5 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-6 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-7 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-8 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-9 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-10 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-11 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-12 or an enantiomer or a diastereomer thereof. In some embodiments, such a compound is a compound of Table CA-13 or an enantiomer or a diastereomer thereof.

(1545) In some embodiments, when contacted with a base, a chiral auxiliary moiety, e.g., of an internucleotidic linkage, whose corresponding compound is a compound of Formula 3-I or 3-AA may be released as an alkene, which has the same structure as a product formed by elimination of a water molecule from the corresponding compound (elimination of W.sup.2H=OH and an alpha-H of G.sup.2). In some embodiments, such an alkene has the structure of (electron-withdrawing group).sub.2=C(R.sup.1)-L-N(R.sup.5)(R.sup.6), (electron-withdrawing group)HC(R.sup.1)-L-N(R.sup.5)(R.sup.6), CH(-L-R)C(R.sup.1)-L-N(R.sup.5)(R.sup.6) wherein the CH group is optionally substituted, or C.sup.xC(R.sup.1)-L-N(R.sup.5)(R.sup.6), wherein C.sup.x is optionally substituted

(1546) ##STR00554##
and may be optionally fused with one or more optionally substituted rings, and each other variable is independently as described herein. In some embodiments, C.sup.x is optionally substituted

(1547) ##STR00555##
In some embodiments, C.sup.x is

(1548) ##STR00556##
In some embodiments, such an alkene is

(1549) ##STR00557##
In some embodiments, such an alkene is

(1550) ##STR00558##
In some embodiments, such an alkene is

(1551) ##STR00559##

(1552) In some embodiments, a chiral reagent is an aminoalcohol. In some embodiments, a chiral reagent is an aminothiol. In some embodiments, a chiral reagent is an aminophenol. In some embodiments, a chiral reagent is (S)- and (R)-2-methylamino-1-phenylethanol, (1R,2S)-ephedrine, or (1R,2S)-2-methylamino-1,2-diphenylethanol.

(1553) In some embodiments of the disclosure, a chiral reagent is a compound of one of the following formulae:

(1554) ##STR00560##

(1555) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer (e.g., WV-CA-237 is a related stereoisomer of WV-CA-236 (a related diastereomer, having the same constitution, the same configuration at one chiral center but not the other); WV-CA-108 is a related enantiomer of WV-CA-236 (mirror image of each other)):

(1556) TABLE-US-00105 TABLE CA-1 Example chiral auxiliaries. WV-CA-231 WV-CA-232 WV-CA-233 WV-CA-234 WV-CA-235 WV-CA-236 WV-CA-237 WV-CA-238 WV-CA-239 WV-CA-240 0 WV-CA-241 WV-CA-242 WV-CA-243 WV-CA-244 WV-CA-245 WV-CA-246 WV-CA-247 WV-CA-248 WV-CA-249 WV-CA-250 0 WV-CA-251 WV-CA-252 WV-CA-253 WV-CA-254 WV-CA-255 WV-CA-256 WV-CA-257 WV-CA-258 WV-CA-259 WV-CA-260 0 WV-CA-261 WV-CA-262 WV-CA-263 WV-CA-264 WV-CA-265 WV-CA-266 WV-CA-267 WV-CA-268 WV-CA-269 WV-CA-270 00 WV-CA-271 01 WV-CA-272 02 WV-CA-273 03 WV-CA-274 04 WV-CA-275 05 WV-CA-276 06 WV-CA-277 07 WV-CA-278 08 WV-CA-279 09 WV-CA-280 0 WV-CA-281 WV-CA-282 WV-CA-283 WV-CA-284 WV-CA-285 WV-CA-286 WV-CA-287 WV-CA-288 WV-CA-289 WV-CA-290 0 WV-CA-291 WV-CA-293 WV-CA-294

(1557) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-1 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-1 or a salt thereof.

(1558) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1559) TABLE-US-00106 TABLE CA-2 Example chiral auxiliaries. WV-CA-231 WV-CA-239 WV-CA-249 WV-CA-272 WV-CA-273 WV-CA-274 WV-CA-275 0 WV-CA-276 WV-CA-277 WV-CA-278 WV-CA-279 WV-CA-280 WV-CA-281 WV-CA-282 WV-CA-283 WV-CA-284 WV-CA-285 0

(1560) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-2 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-2 or a salt thereof.

(1561) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1562) TABLE-US-00107 TABLE CA-3 Example chiral auxiliaries. WV-CA-236 WV-CA-237 WV-CA-238 WV-CA-240 WV-CA-241 WV-CA-242 WV-CA-243 WV-CA-252 WV-CA-290 WV-CA-291 0 WV-CA-108 WV-CA-183

(1563) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-3 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-3 or a salt thereof.

(1564) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1565) TABLE-US-00108 TABLE CA-4 Example chiral auxiliaries. WV-CA-251 WV-CA-253 WV-CA-255 WV-CA-257 WV-CA-258 WV-CA-263

(1566) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-4 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-4 or a salt thereof.

(1567) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1568) TABLE-US-00109 TABLE CA-5 Example chiral auxiliaries. WV-CA-254 WV-CA-256 0 WV-CA-259

(1569) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-5 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-5 or a salt thereof.

(1570) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1571) TABLE-US-00110 TABLE CA-6 Example chiral auxiliaries. WV-CA-260 WV-CA-261 WV-CA-262

(1572) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-6 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-6 or a salt thereof.

(1573) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1574) TABLE-US-00111 TABLE CA-7 Example chiral auxiliaries. WV-CA-245 WV-CA-264 WV-CA-265 WV-CA-266

(1575) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-7 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-7 or a salt thereof.

(1576) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1577) TABLE-US-00112 TABLE CA-8 Example chiral auxiliaries. WV-CA-267 WV-CA-269 0 WV-CA-271

(1578) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-8 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-8 or a salt thereof.

(1579) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1580) TABLE-US-00113 TABLE CA-9 Example chiral auxiliaries. WV-CA-268 WV-CA-270

(1581) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-9 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-9 or a salt thereof.

(1582) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1583) TABLE-US-00114 TABLE CA-10 Example chiral auxiliaries. WV-CA-244 WV-CA-246

(1584) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-10 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-10 or a salt thereof.

(1585) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1586) TABLE-US-00115 TABLE CA-11 Example chiral auxiliaries. WV-CA-247 WV-CA-248

(1587) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-11 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-11 or a salt thereof.

(1588) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1589) TABLE-US-00116 TABLE CA-12 Example chiral auxiliaries. WV-CA-250 WV-CA-286 WV-CA-287 0 WV-CA-288 WV-CA-289

(1590) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-12 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-12 or a salt thereof.

(1591) In some embodiments, a useful chiral reagent is a compound selected from the compounds below, or its related stereoisomer, particularly enantiomer:

(1592) TABLE-US-00117 TABLE CA-13 Example chiral auxiliaries. WV-CA-110 WV-CA-315 WV-CA-110b WV-CA-324

(1593) In some embodiments, a provided compound is an enantiomer of a compound selected from Table CA-13 or a salt thereof. In some embodiments, a provided compound is a diastereomer of a compound selected from Table CA-13 or a salt thereof.

(1594) As appreciated by those skilled in the art, chiral reagents are typically stereopure or substantially stereopure, and are typically utilized as a single stereoisomer substantially free of other stereoisomers. In some embodiments, compounds of the present disclosure are stereopure or substantially stereopure.

(1595) As demonstrated herein, when used for preparing a chiral internucleotidic linkage, to obtain stereoselectivity generally stereochemically pure chiral reagents are utilized. Among other things, the present disclosure provides stereochemically pure chiral reagents, including those having structures described.

(1596) The choice of chiral reagent, for example, the isomer represented by Formula Q or its stereoisomer, Formula R, permits specific control of chirality at a linkage phosphorus. Thus, either an Rp or Sp configuration can be selected in each synthetic cycle, permitting control of the overall three dimensional structure of a chirally controlled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide has all Rp stereocenters. In some embodiments of the disclosure, a chirally controlled oligonucleotide has all Sp stereocenters. In some embodiments of the disclosure, each linkage phosphorus in the chirally controlled oligonucleotide is independently Rp or Sp. In some embodiments of the disclosure, each linkage phosphorus in the chirally controlled oligonucleotide is independently Rp or Sp, and at least one is Rp and at least one is Sp. In some embodiments, the selection of Rp and Sp centers is made to confer a specific three dimensional superstructure to a chirally controlled oligonucleotide. Examples of such selections are described in further detail herein.

(1597) In some embodiments, a provided oligonucleotide comprise a chiral auxiliary moiety, e.g., in an internucleotidic linkage. In some embodiments, a chiral auxiliary is connected to a linkage phosphorus. In some embodiments, a chiral auxiliary is connected to a linkage phosphorus through W.sup.2. In some embodiments, a chiral auxiliary is connected to a linkage phosphorus through W.sup.2, wherein W.sup.2 is 0. Optionally, W.sup.1, e.g., when W.sup.1 is NG.sup.5-, is capped during oligonucleotide synthesis. In some embodiments, W.sup.1 in a chiral auxiliary in an oligonucleotide is capped, e.g., by a capping reagent during oligonucleotide synthesis. In some embodiments, W.sup.1 may be purposeful capped to modulate oligonucleotide property. In some embodiments, W.sup.1 is capped with R.sup.1. In some embodiments, R.sup.1 is C(O)R. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is methyl.

(1598) In some embodiments, a chiral reagent for use in accordance with the present disclosure is selected for its ability to be removed at a particular step in the above-depicted cycle. For example, in some embodiments it is desirable to remove a chiral reagent during the step of modifying the linkage phosphorus. In some embodiments, it is desirable to remove a chiral reagent before the step of modifying the linkage phosphorus. In some embodiments, it is desirable to remove a chiral reagent after the step of modifying the linkage phosphorus. In some embodiments, it is desirable to remove a chiral reagent after a first coupling step has occurred but before a second coupling step has occurred, such that a chiral reagent is not present on the growing oligonucleotide during the second coupling (and likewise for additional subsequent coupling steps). In some embodiments, a chiral reagent is removed during the deblock reaction that occurs after modification of the linkage phosphorus but before a subsequent cycle begins. Example methods and reagents for removal are described herein.

(1599) In some embodiments, removal of chiral auxiliary is achieved when performing the modification and/or deblocking step, as illustrated in Scheme I. It can be beneficial to combine chiral auxiliary removal together with other transformations, such as modification and deblocking. A person of ordinary skill in the art would appreciate that the saved steps/transformation could improve the overall efficiency of synthesis, for instance, with respect to yield and product purity, especially for longer oligonucleotides. One example wherein the chiral auxiliary is removed during modification and/or deblocking is illustrated in Scheme I.

(1600) In some embodiments, a chiral reagent for use in accordance with methods of the present disclosure is characterized in that it is removable under certain conditions. For instance, in some embodiments, a chiral reagent is selected for its ability to be removed under acidic conditions. In certain embodiments, a chiral reagent is selected for its ability to be removed under mildly acidic conditions. In certain embodiments, a chiral reagent is selected for its ability to be removed by way of an E1 elimination reaction (e.g., removal occurs due to the formation of a cation intermediate on the chiral reagent under acidic conditions, causing the chiral reagent to cleave from the oligonucleotide). In some embodiments, a chiral reagent is characterized in that it has a structure recognized as being able to accommodate or facilitate an E1 elimination reaction. One of skill in the relevant arts will appreciate which structures would be envisaged as being prone toward undergoing such elimination reactions.

(1601) In some embodiments, a chiral reagent is selected for its ability to be removed with a nucleophile. In some embodiments, a chiral reagent is selected for its ability to be removed with an amine nucleophile. In some embodiments, a chiral reagent is selected for its ability to be removed with a nucleophile other than an amine.

(1602) In some embodiments, a chiral reagent is selected for its ability to be removed with a base. In some embodiments, a chiral reagent is selected for its ability to be removed with an amine. In some embodiments, a chiral reagent is selected for its ability to be removed with a base other than an amine.

(1603) In some embodiments, chirally pure phosphoramidites comprising chiral auxiliaries may be isolated before use. In some embodiments, chirally pure phosphoramidites comprising chiral auxiliaries may be used without isolationin some embodiments, they may be used directly after formation.

(1604) Activation

(1605) As appreciated by those skilled in the art, oligonucleotide preparation may use various conditions, reagents, etc. to active a reaction component, e.g., during phosphoramidite preparation, during one or more steps during in the cycles, during post-cycle cleavage/deprotection, etc. Various technologies for activation can be utilized in accordance with the present disclosure, including but not limited to those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US 20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the activation technologies of each of which are incorporated by reference. Certain activation technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

(1606) Coupling

(1607) In some embodiments, cycles of the present disclosure comprise stereoselective condensation/coupling steps to form chirally controlled internucleotidic linkages. For condensation, often an activating reagent is used, such as 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, 1-phenylimidazolium triflate (PhIMT), benzimidazolium triflate (BIT), benztriazole, 3-nitro-1,2,4-triazole (NT), tetrazole, 5-ethylthiotetrazole (ETT), 5-benzylthiotetrazole (BTT), 5-(4-nitrophenyl) tetrazole, N-cyanomethylpyrrolidinium triflate (CMPT), N-cyanomethylpiperidinium triflate, N-cyanomethyldimethylammonium triflate, etc. Suitable conditions and reagents, including chiral phosphoramidites, include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the condensation reagents, conditions and methods of each of which are incorporated by reference. Certain coupling technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

(1608) In some embodiments, a phosphoramidite for coupling has the structure of

(1609) ##STR00687##
wherein each variable is independently as described in the present disclosure. In some embodiments, each R is independently optionally substituted C.sub.1-6 aliphatic. A person skill in the art will appreciate that two R groups in any structure or formula can either be the same or different. In some embodiments, each R is independently optionally substituted C.sub.1-6 alkyl. In some embodiments, each R is independently optionally substituted C.sub.1-6 alkenyl. In some embodiments, each R is independently optionally substituted C.sub.1-6 alkynyl. In some embodiments, each R is indenpendtly isopropyl. In some embodiments, X-L-R.sup.1 comprises an optionally substituted triazole group. In some embodiments, X is a covalent bond. In some embodiments, L is a covalent bond. In some embodiments, X-L-R.sup.1 is R.sup.1. In some embodiments, R.sup.1 comprise an optionally substituted ring. In some embodiments, R.sup.1 is R as described herein. In some embodiments, R.sup.1 is optionally substituted

(1610) ##STR00688##
In some embodiments, R.sup.1 is

(1611) ##STR00689##
In some embodiments, R.sup.1 is

(1612) ##STR00690##
In some embodiments, R.sup.1 is

(1613) ##STR00691##
In some embodiments, -L-comprises C.sub.1-6 alkylene. In some embodiments, -L-comprises C.sub.1-6 alkenylene. In some embodiments, -L- comprises

(1614) ##STR00692##
In some embodiments, R.sup.1 is R as described herein. In some embodiments, -L- is

(1615) ##STR00693##
and R.sup.1 is H. In some embodiments, -L-R.sup.1 is

(1616) ##STR00694##
In some embodiments, X-L-R is

(1617) ##STR00695##
In some embodiments, X-L-R.sup.1 is OCH.sub.2CH.sub.2CN.

(1618) In some embodiments, a chiral phosphoramidite for coupling has the structure of

(1619) ##STR00696##
wherein each variable is independently as described in the present disclosure. In some embodiments, a chiral phosphoramidite for coupling has the structure of

(1620) ##STR00697## ##STR00698##
In some embodiments, a chiral phosphoramidite for coupling has the structure of

(1621) ##STR00699##
wherein each variable is independently as described in the present disclosure. In some embodiments, G.sup.1 or G.sup.2 comprises an electron-withdrawing group as described in the present disclosure. In some embodiments, a chiral phosphoramidite for coupling has the structure of

(1622) ##STR00700## ##STR00701##
wherein each variable is independently as described in the present disclosure. In some embodiments, R.sup.1 is R as described in the present disclosure. In some embodiments, R.sup.1 is R as described in the present disclosure. In some embodiments, R is optionally substituted phenyl as described in the present disclosure. In some embodiments, R is phenyl. In some embodiments, R is 4-methyl phenyl. In some embodiments, R is 4-methoxy phenyl. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic as described in the present disclosure. In some embodiments, R is optionally substituted C.sub.1-6 alkyl as described in the present disclosure. For example, in some embodiments, R is methyl; in some embodiments, R is isopropyl; in some embodiments, R is t-butyl; etc.

(1623) In some embodiments, R.sup.5s-L.sup.s- is RO. In some embodiments, RO is DMTrO. In some embodiments, R.sup.4s is H. In some embodiments, R.sup.4s and R.sup.2s are taken together to form a bridge -L-O as described in the present disclosure. In some embodiments, the O is connected to the carbon at the 2 position. In some embodiments, L is CH.sub.2. In some embodiments, L is CH(Me)-. In some embodiments, L is (R)CH(Me)-. In some embodiments, L is (S)CH(Me)-. In some embodiments, R.sup.2s is H. In some embodiments, R.sup.2s is F. In some embodiments, R.sup.2s is OR. In some embodiments, R.sup.2s is OMe. In some embodiments, R.sup.2s is -MOE. As appreciated by those skilled in the art, BA may be suitably protected during synthesis.

(1624) In some embodiments, an internucleotidic linkage formed in a coupling step has the structure of formula I or a salt form thereof. In some embodiments, P.sup.L is P. In some embodiments, X-L-R.sup.1 is

(1625) ##STR00702##
wherein each variable is independently in accordance with the present disclosure. In some embodiments, X-L-R.sup.1 is CH.sub.2CH.sub.2CN.

(1626) In some embodiments, a coupling forms an internucleotidic linkage with a stereoselectivity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 90% or more. In some embodiments, the stereoselectivity is 91% or more. In some embodiments, the stereoselectivity is 92% or more. In some embodiments, the stereoselectivity is 93% or more. In some embodiments, the stereoselectivity is 94% or more. In some embodiments, the stereoselectivity is 95% or more. In some embodiments, the stereoselectivity is 96% or more. In some embodiments, the stereoselectivity is 97% or more. In some embodiments, the stereoselectivity is 98% or more. In some embodiments, the stereoselectivity is 99% or more.

(1627) Capping

(1628) If the final nucleic acid is larger than a dimer, the unreacted-OH moiety is generally capped with a blocking/capping group. Chiral auxiliaries in oligonucleotides may also be capped with a blocking group to form a capped condensed intermediate. Suitable capping technologies (e.g., reagents, conditions, etc.) include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the capping technologies of each of which are incorporated by reference. In some embodiments, a capping reagent is a carboxylic acid or a derivate thereof. In some embodiments, a capping reagent is RCOOH. In some embodiments, a capping step introduces RCOO to unreacted 5-OH group and/or amino groups in chiral auxiliaries. In some embodiments, a cycle may comprise two or more capping steps. In some embodiments, a cycle comprises a first capping before modification of a coupling product (e.g., converting P(III) to P(V)), and another capping after modification of a coupling product. In some embodiments, a first capping is performed under an amidation condition, e.g., which comprises an acylating reagent (e.g., an anhydride having the structure of (RC(O)).sub.2O, (e.g., Ac.sub.2O)) and a base (e.g., 2,6-lutidine). In some embodiments, a first capping caps an amino group, e.g., that of a chiral auxiliary in an internucleotidic linkage. In some embodiments, an internucleotidic linkage formed in a coupling step has the structure of formula I or a salt form thereof. In some embodiments, P.sup.L is P. In some embodiments, X-L-R.sup.1 is

(1629) ##STR00703##
wherein each variable is independently in accordance with the present disclosure. In some embodiments, R.sup.1 is RC(O). In some embodiments, R is CH.sub.3. In some embodiments, each chirally controlled coupling (e.g., using a chiral auxiliary) is followed with a first capping. Typically, cycles for non-chirally controlled coupling using traditional phosphoramidite to construct natural phosphate linkages do not contain a first capping. In some embodiments, a second capping is performed, e.g., under an esterification condition (e.g., capping conditions of traditional phosphoramidite oligonucleotide synthesis) wherein free 5-OH are capped.

(1630) Certain capping technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

(1631) Modifying

(1632) In some embodiments, an internucleotidic linkage wherein its linkage phosphorus exists as P(III) is modified to form another modified internucleotidic linkage (e.g., one of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof) or a natural phosphate linkage. In many embodiments, P(III) is modified by reaction with an electrophile. Various types of reactions suitable for P(III) may be utilized in accordance with the present disclosure. Suitable modifying technologies (e.g., reagents (e.g., sulfurization reagent, oxidation reagent, etc.), conditions, etc.) include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the modifying technologies of each of which are incorporated by reference.

(1633) In some embodiments, as illustrated in the Examples, the present disclosure provides modifying reagents for introducing non-negatively charged internucleotidic linkages including neutral internucleotidic linkages.

(1634) In some embodiments, modifying is within a cycle. In some embodiments, modifying can be outside of a cycle. For example, in some embodiments, one or more modifying steps can be performed after the oligonucleotide chain has been reached to introduce modifications simultaneously at one or more internucleotidic linkages and/or other locations.

(1635) In some embodiments, modifying comprises use of click chemistry, e.g., wherein an alkyne group of an oligonucleotide, e.g., of an internucleotidic linkage, is reacted with an azide. Various reagents and conditions for click chemistry can be utilized in accordance with the present disclosure. In some embodiments, an azide has the structure of R.sup.1N.sub.3, wherein R.sup.1 is as described in the present disclosure. In some embodiments, R.sup.1 is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.1 is isopropyl.

(1636) In some embodiments, as demonstrated in the examples, a P(III) linkage can be converted into a non-negatively charged internucleotidic linkage by reacting the P(III) linkage with an azide or an azido imidazolinium salt (e.g., a compound comprising

(1637) ##STR00704##
in some embodiments, referred to as an azide reaction) under suitable conditions. In some embodiments, an azido imidazolinium salt is a salt of PF.sub.6.sup.. In some embodiments, an azido imidazolinium salt is a salt of

(1638) ##STR00705##
In some embodiments, a useful reagent, e.g., an azido imidazolinium salt, is a salt of

(1639) ##STR00706##
In some embodiments, a useful reagent is a salt of

(1640) ##STR00707##
In some embodiments, a useful reagent is a salt of

(1641) ##STR00708##
In some embodiments, a useful reagent is a salt of

(1642) ##STR00709##
Such reagents comprising nitrogen cations also contain counter anions (e.g., Q.sup. as described in the present disclosure), which are widely known in the art and are contained in various chemical reagents. In some embodiments, a useful reagent is Q.sup.+Q.sup., wherein Q.sup.+ is

(1643) ##STR00710##
and Q.sup. is a counter anion. In some embodiments, Q.sup.+ is

(1644) ##STR00711##
In some embodiments, Q.sup.+ is

(1645) ##STR00712##
In some embodiments, Q.sup.+ is

(1646) ##STR00713##
In some embodiments, Q.sup.+ is

(1647) ##STR00714##
In some embodiments, Q.sup.+ is

(1648) ##STR00715##
As appreciated by those skilled in the art, in a compound having the structure of Q.sup.+Q.sup., typically the number of positive charges in Q.sup.+ equals the number of negative charges in Q.sup.. In some embodiments, Q.sup.+ is a monovalent cation and Q.sup. is a monovalent anion. In some embodiments, Q.sup. is F.sup., Cl.sup., Br.sup., BF.sub.4.sup., PF.sub.6.sup., TfO.sup., Tf.sub.2N.sup., AsF.sub.6.sup., ClO.sub.4.sup., or SbF.sub.6.sup.. In some embodiments, Q.sup. is PF.sub.6.sup.. Those skilled in the art readily appreciate that many other types of counter anions are available and can be utilized in accordance with the present disclosure. In some embodiments, an azido imidazolinium salt is 2-azido-1,3-dimethylimidazolinium hexafluorophosphate. In some embodiments, an azide is

(1649) ##STR00716##
In some embodiments, an azido imidazolinium salt is

(1650) ##STR00717##
In some embodiments, an azido imidazolinium salt is

(1651) ##STR00718##
In some embodiments, an azide is

(1652) ##STR00719##
In some embodiments, an azide is

(1653) ##STR00720##
In some embodiments, an azide is

(1654) ##STR00721##
In some embodiments, an azido imidazolinium salt is

(1655) ##STR00722##
In some embodiments, an azido imidazolinium salt is

(1656) ##STR00723##
In some embodiments, an azido imidazolinium salt is

(1657) ##STR00724##
In some embodiments, an azido imidazolinium salt is

(1658) ##STR00725##

(1659) In some embodiments, a P(III) linkage is reacted with an electrophile having the structure of R-G.sup.Z, wherein R is as described in the present disclosure, and G.sup.Z is a leaving group, e.g., Cl, Br, I, OTf, Oms, OTosyl, etc. In some embodiments, R is CH.sub.3. In some embodiments, R is CH.sub.2CH.sub.3. In some embodiments, R is CH.sub.2CH.sub.2CH.sub.3. In some embodiments, R is CH.sub.2OCH.sub.3. In some embodiments, R is CH.sub.3CH.sub.2OCH.sub.2. In some embodiments, R is PhCH.sub.2OCH.sub.2. In some embodiments, R is HCCCH.sub.2. In some embodiments, R is H.sub.3CCCCH.sub.2. In some embodiments, R is CH.sub.2CHCH.sub.2. In some embodiments, R is CH.sub.3SCH.sub.2. In some embodiments, R is CH.sub.2COOCH.sub.3. In some embodiments, R is CH.sub.2COOCH.sub.2CH.sub.3. In some embodiments, R is CH.sub.2CONHCH.sub.3.

(1660) In some embodiments, after a modifying step, a P(III) linkage phosphorus is converted into a P(V) internucleotidic linkage. In some embodiments, a P(III) linkage phosphorus is converted into a P(V) internucleotidic linkage, and all groups bounded to the linkage phosphorus remain unchanged. In some embodiments, a linkage phosphorus is converted from P into P(O). In some embodiments, a linkage phosphorus is converted from P into P(S). In some embodiments, a linkage phosphorus is converted from P into P(N-L-R.sup.5). In some embodiments, a linkage phosphorus is converted from P into

(1661) ##STR00726##
wherein each variable is independently as described in the present disclosure. In some embodiments, P is converted into

(1662) ##STR00727##
In some embodiments, P is converted into

(1663) ##STR00728##
In some embodiments, P is converted into

(1664) ##STR00729##
In some embodiments, P is converted into

(1665) ##STR00730##
In some embodiments, P is converted into

(1666) ##STR00731##
As appreciated by those skilled in the art, for each cation there typically exists a counter anion so that the total number of positive charges equals the total number of negative charges in a system (e.g., compound, composition, etc.). In some embodiments, a counter anion is Q.sup. as described in the present disclosure (e.g., F.sup., Cl.sup., Br.sup., BF.sub.4.sup., PF.sub.6.sup., TfO.sup., Tf.sub.2N.sup., AsF.sub.6.sup., ClO.sub.4.sup., SbF.sub.6.sup., etc.). In some embodiments, an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein P.sup.L is P, is converted into an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof, wherein P.sup.L is P(W) or P.fwdarw.B(R).sub.3 or P.sup.N. In some embodiments, an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein P.sup.L is P, is converted into an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein P.sup.L is P(W) or P.fwdarw.B(R).sub.3. In some embodiments, a linkage phosphorus P, which is P.sup.L in an internucleotidic linkage having the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof is converted into P.sup.L which is P(W) or P.fwdarw.B(R).sub.3. In some embodiments, a linkage phosphorus P, which is P.sup.L in an internucleotidic linkage having the structure of formula I or a salt form thereof is converted into P.sup.L which is P(W) or P.fwdarw.B(R).sub.3. In some embodiments, W is O(e.g., for an oxidation reaction). In some embodiments, W is S (e.g., for a sulfurization reaction). In some embodiments, W is N-L-R.sup.5 (e.g., for an azide reaction). In some embodiments, an internucleotidic linkage having the structure of formula I or a salt form thereof (e.g., wherein P.sup.L is P) is converted into an internucleotidic linkage having the structure of formula III or a salt form thereof:

(1667) ##STR00732##
wherein: P.sup.N is P(N-L-R.sup.5),

(1668) ##STR00733## Q.sup. is an anion, and each other variables is independently as described in the present disclosure.

(1669) In some embodiments, P.sup.N is P(N-L-R.sup.5). In some embodiments, P.sup.N is

(1670) ##STR00734##

(1671) In some embodiments, P.sup.N is

(1672) ##STR00735##
In some embodiments, P.sup.N is

(1673) ##STR00736##
In some embodiments, P.sup.N is

(1674) ##STR00737##
In some embodiments, P.sup.N is

(1675) ##STR00738##
In some embodiments, internucleotidic linkages of the present disclosure may exist in a salt form. In some embodiments, internucleotidic linkages of formula III may exist in a salt form. In some embodiments, in a salt form of an internucleotidic linkage of formula III P.sup.N is

(1676) ##STR00739##
In some embodiments, P.sup.N is PW.sup.N, wherein W.sup.N is as described herein.

(1677) In some embodiments, Y, Z, and X-L-R.sup.1 remains the same during the conversion. In some embodiments, each of X, Y and Z is independently O. In some embodiments, as described herein, X-L-R.sup.1 is of such a structure that HX-L-R.sup.1 is a chiral reagent described herein, or a capped chiral reagent described herein wherein an amino group of the chiral reagent (typically of W.sup.1H or W.sup.2H, which comprises an amino group NHG.sup.5-) is capped, e.g., with C(O)R (replacing a H, e.g., N [C(O)R]G.sup.5-). In some embodiments, X-L-R.sup.1 is

(1678) ##STR00740##
wherein each variable is independently in accordance with the present disclosure. In some embodiments, wherein R.sup.1 is C(O)R. In some embodiments, R.sup.1 is CH.sub.3C(O). In some embodiments, as described herein, G.sup.2 comprises an electron-withdrawing group. In some embodiments, G.sup.2 is CH.sub.2SO.sub.2Ph.

(1679) In some embodiments, an internucleotidic linkage (e.g., a modified internucleotidic linkage, a chiral internucleotidic linkage, a chirally controlled internucleotidic linkage, a non-negatively charged internucleotidic linkage, a neutral internucleotidic linkage, etc.) has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, wherein P.sup.L is P(N-L-R.sup.5), or of formula III or a salt form thereof. In some embodiments, such an internucleotidic linkage is chirally controlled. In some embodiments, all such internucleotidic linkages are chirally controlled. In some embodiments, linkage phosphorus of at least one of such internucleotidic linkages is Rp. In some embodiments, linkage phosphorus of at least one of such internucleotidic linkages is Sp. In some embodiments, linkage phosphorus of at least one of such internucleotidic linkages is Rp, and linkage phosphorus of at least one of such internucleotidic linkages is Sp. In some embodiments, oligonucleotides of the present disclosure comprises one or more (e.g., 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.) such internucleotidic linkages. In some embodiments, such oligonucleotide further comprise one or more other types of internucleotidic linkages, e.g., one or more natural phosphate linkages, and/or one or more phosphorothioate internucleotidic linkages (e.g., in some embodiments, one or more of which are independently chirally controlled; in some embodiments, each of which is independently chirally controlled; in some embodiments, at least one is Rp; in some embodiments, at least one is Sp; in some embodiments, at least one is Rp and at least one is Sp; etc.) In some embodiments, such oligonucleotides are stereopure (substantially free of other stereoisomers). In some embodiments, the present disclosure provides chirally controlled oligonucleotide compositions of such oligonucleotides. In some embodiments, the present disclosure provides chirally pure oligonucleotide compositions of such oligonucleotides.

(1680) In some embodiments, modifying proceeds with a stereoselectivity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 85% or more. In some embodiments, the stereoselectivity is 90% or more. In some embodiments, the stereoselectivity is 91% or more. In some embodiments, the stereoselectivity is 92% or more. In some embodiments, the stereoselectivity is 93% or more. In some embodiments, the stereoselectivity is 94% or more. In some embodiments, the stereoselectivity is 95% or more. In some embodiments, the stereoselectivity is 96% or more. In some embodiments, the stereoselectivity is 97% or more. In some embodiments, the stereoselectivity is 98% or more. In some embodiments, the stereoselectivity is 99% or more. In some embodiments, modifying is stereospecific.

(1681) Deblocking

(1682) In some embodiments, a cycle comprises a cycle step. In some embodiments, the 5 hydroxyl group of the growing oligonucleotide is blocked (i.e., protected) and must be deblocked in order to subsequently react with a nucleoside coupling partner.

(1683) In some embodiments, acidification is used to remove a blocking group. Suitable deblocking technologies (e.g., reagents, conditions, etc.) include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the deblocking technologies of each of which are incorporated by reference. Certain deblocking technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

(1684) Cleavage and Deprotection

(1685) At certain stage, e.g., after the desired oligonucleotide lengths have been achieved, cleavage and/or deprotection are performed to deprotect blocked nucleobases etc. and cleave the oligonucleotide products from support. In some embodiments, cleavage and deprotection are performed separately. In some embodiments, cleavage and deprotection are performed in one step, or in two or more steps but without separation of products in between. In some embodiments, cleavage and/or deprotection utilizes basic conditions and elevated temperature. In some embodiments, for certain chiral auxiliaries, a fluoride condition is required (e.g., TBAF, HF-ET.sub.3N, etc., optionally with additional base). Suitable cleavage and deprotection technologies (e.g., reagents, conditions, etc.) include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the cleavage and deprotection technologies of each of which are incorporated by reference. Certain cleavage and deprotection technologies, e.g., reagents, conditions, methods, etc. are illustrated in the Examples.

(1686) In some embodiments, certain chiral auxiliaries are removed under basic conditions. In some embodiments, oligonucleotides are contacted with a base, e.g., an amine having the structure of N(R).sub.3, to remove certain chiral auxiliaries (e.g., those comprising an electronic-withdrawing group in G.sup.2 as described in the present disclosure). In some embodiments, a base is NHR.sub.2. In some embodiments, each R is independently optionally substituted C.sub.1-6 aliphatic. In some embodiments, each R is independently optionally substituted C.sub.1-6 alkyl. In some embodiments, an amine is DEA. In some embodiments, an amine is TEA. In some embodiments, an amine is provided as a solution, e.g., an acetonitrile solution. In some embodiments, such contact is performed under anhydrous conditions. In some embodiments, such a contact is performed immediately after desired oligonucleotide lengths are achieved (e.g., first step post synthesis cycles). In some embodiments, such a contact is performed before removal of chiral auxiliaries and/or protection groups and/or cleavage of oligonucleotides from a solid support. In some embodiments, contact with a base may remove cyanoethyl groups utilized in standard oligonucleotide synthesis, providing an natural phosphate linkage which may exist in a salt form (with the cation being, e.g., an ammonium salt). In some embodiments, contact with a base provides an internucleotidic linkage of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, or a salt form thereof. In some embodiments, contact with a base removes a chiral auxiliary from an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, or a salt form thereof. In some embodiments, contact with a base removes a chiral auxiliary (e.g., X-L-R.sup.1) from an internucleotidic linkage of formula I or a salt form thereof (e.g., wherein P.sup.L is P(N-L-R.sup.5)). In some embodiments, contact with a base removes a chiral auxiliary (e.g., X-L-R.sup.1) from an internucleotidic linkage of formula III or a salt form thereof. In some embodiments, In some embodiments, contact with a base converts an internucleotidic linkage of formula I or a salt form thereof (e.g., wherein P.sup.L is P(N-L-R.sup.5)), or of formula III or a salt form thereof, into an internucleotidic linkage of formula II-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, or a salt form thereof.

(1687) Cycles

(1688) Suitable cycles for preparing oligonucleotides of the present disclosure include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647 (e.g., Schemes I, I-b, I-c, I-d, I-e, I-f, etc.), WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the cycles of each of which are incorporated by reference. For example, in some embodiments, an example cycle is Scheme I-f. Certain cycles are illustrated in the Examples (e.g., for preparation of natural phosphate linkages, utilizing other chiral auxiliaries, etc.).

(1689) ##STR00741##

(1690) In some embodiments, R.sup.2 is H or OR.sup.1, wherein R.sup.1 is not hydrogen. In some embodiments, R.sup.2s is H or OR.sup.1, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.2s is H. In some embodiments, R.sup.2s is OMe. In some embodiments, R.sup.2s is OCH.sub.2CH.sub.2OCH.sub.3. In some embodiments, R.sup.2s is F. In some embodiments, R.sup.4s is H. In some embodiments, R.sup.4s and R.sup.2s are taken together to form a bridge -L-O as described in the present disclosure. In some embodiments, the O is connected to the carbon at the 2 position. In some embodiments, L is CH.sub.2. In some embodiments, L is CH(Me)-. In some embodiments, L is (R)CH(Me)-. In some embodiments, L is (S)CH(Me)-.

(1691) Purification and Characterization

(1692) Various purification and/or characterization technologies (methods, instruments, protocols, etc.) can be utilized to purify and/or characterize oligonucleotides and oligonucleotide compositions in accordance with the present disclosure. In some embodiments, purification is performed using various types of HPLC/UPLC technologies. In some embodiments, characterization comprises MS, NMR, UV, etc. In some embodiments, purification and characterization may be performed together, e.g., HPLC-MS, UPLC-MS, etc. Example purification and characterization technologies include those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US 20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and/or WO 2019/055951, the purification and characterization technologies of each of which are incorporated by reference.

(1693) In some embodiments, the present disclosure provides methods for preparing provided oligonucleotide and oligonucleotide compositions. In some embodiments, a provided method comprises providing a provided chiral reagent having the structure of formula 3-I or 3-AA. In some embodiments, a provided method comprises providing a provided chiral reagent having the structure of

(1694) ##STR00742##
wherein W.sup.1 is NG.sup.5, W.sup.2 is O, each of G.sup.1 and G.sup.3 is independently hydrogen or an optionally substituted group selected from C.sub.1-10 aliphatic, heterocyclyl, heteroaryl and aryl, G.sup.2 is C(R).sub.2Si(R).sub.3, and G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated, partially unsaturated or unsaturated heteroatom-containing ring of up to about 20 ring atoms which is monocyclic or polycyclic, fused or unfused, wherein each R is independently hydrogen, or an optionally substituted group selected from C.sub.1-C.sub.6 aliphatic, carbocyclyl, aryl, heteroaryl, and heterocyclyl. In some embodiments, a provided chiral reagent has the structure of

(1695) ##STR00743##
wherein each variable is independently as described in the present disclosure. In some embodiments, a provided methods comprises providing a phosphoramidite comprising a moiety from a chiral reagent having the structure of

(1696) ##STR00744##
wherein W.sup.1H and W.sup.2H, or the hydroxyl and amino groups, form bonds with the phosphorus atom of the phosphoramidite. In some embodiments, W.sup.1H and W.sup.2H, or the hydroxyl and amino groups, form bonds with the phosphorus atom of the phosphoramidite, e.g., in

(1697) ##STR00745##
In some embodiments, a phosphoramidite has the structure of

(1698) ##STR00746## ##STR00747## ##STR00748## ##STR00749##
or wherein B.sup.PRO is BA as described in the present disclosure, and each other variable is as described in the present disclosure. In some embodiments, B.sup.PRO is a protected nucleobase. In some embodiments, B.sup.PRO is protected A, T, G, C, U or a tautomers thereof. In some embodiments, R is a protection group. In some embodiments, R is DMTr.

(1699) In some embodiments, G.sup.2 is C(R).sub.2Si(R).sub.3, wherein C(R).sub.2 is optionally substituted CH.sub.2, and each R of Si(R).sub.3 is independently an optionally substituted group selected from C.sub.1-10 aliphatic, heterocyclyl, heteroaryl and aryl. In some embodiments, at least one R of Si(R).sub.3 is independently optionally substituted C.sub.1-10 alkyl. In some embodiments, at least one R of Si(R).sub.3 is independently optionally substituted phenyl. In some embodiments, one R of Si(R).sub.3 is independently optionally substituted phenyl, and each of the other two R is independently optionally substituted C.sub.1-10 alkyl. In some embodiments, one R of Si(R).sub.3 is independently optionally substituted C.sub.1-10 alkyl, and each of the other two R is independently optionally substituted phenyl. In some embodiments, G.sup.2 is optionally substituted CH.sub.2Si(Ph)(Me).sub.2. In some embodiments, G.sup.2 is optionally substituted CH.sub.2Si(Me)(Ph).sub.2. In some embodiments, G.sup.2 is CH.sub.2Si(Me)(Ph).sub.2. In some embodiments, G.sup.2 is CH.sub.2SiMe.sub.3. In some embodiments, G.sup.2 is CH.sub.2Si(iPr).sub.3. In some embodiments, G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated 5-6 membered ring containing one nitrogen atom (to which G.sup.5 is attached). In some embodiments, G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated 5-membered ring containing one nitrogen atom. In some embodiments, G.sup.1 is hydrogen. In some embodiments, G.sup.3 is hydrogen. In some embodiments, both G.sup.1 and G.sup.3 are hydrogen. In some embodiments, both G.sup.1 and G.sup.3 are hydrogen, G.sup.2 is C(R).sub.2Si(R).sub.3, wherein C(R).sub.2 is optionally substituted CH.sub.2, and each R of Si(R).sub.3 is independently an optionally substituted group selected from C.sub.1-10 aliphatic, heterocyclyl, heteroaryl and aryl, and G.sup.4 and G.sup.5 are taken together to form an optionally substituted saturated 5-membered ring containing one nitrogen atom. In some embodiments, a provided method further comprises providing a fluoro-containing reagent. In some embodiments, a provided fluoro-containing reagent removes a chiral reagent, or a product formed from a chiral reagent, from oligonucleotides after synthesis. Various known fluoro-containing reagents, including those F-sources for removing SiR.sub.3 groups, can be utilized in accordance with the present disclosure, for example, TBAF, HF.sub.3Et.sub.3N etc. In some embodiments, a fluoro-containing reagent provides better results, for example, shorter treatment time, lower temperature, less de-sulfurization, etc, compared to traditional methods, such as concentrated ammonia. In some embodiments, for certain fluoro-containing reagent, the present disclosure provides linkers for improved results, for example, less cleavage of oligonucleotides from support during removal of chiral reagent (or product formed therefrom during oligonucleotide synthesis). In some embodiments, a provided linker is an SP linker. In some embodiments, the present disclosure demonstrated that a HF-base complex can be utilized, such as HFNR.sub.3, to control cleavage during removal of chiral reagent (or product formed therefrom during oligonucleotide synthesis). In some embodiments, HFNR.sub.3 is HFNEt.sub.3. In some embodiments, HFNR.sub.3 enables use of traditional linkers, e.g., succinyl linker.

(1700) In some embodiments, as described herein, G.sup.2 comprises an electron-withdrawing group, e.g., at its position. In some embodiments, G.sup.2 is methyl substituted with one or more electron-withdrawing groups. In some embodiments, an electronic-withdrawing group comprises and/or is connected to the carbon atom through, e.g., S(O), S(O).sub.2, P(O)(R.sup.1), P(S)R.sup.1, or C(O). In some embodiments, an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2. In some embodiments, an electron-withdrawing group is aryl or heteroaryl, e.g., phenyl, substituted with one or more of CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2. In some embodiments, G.sup.2 is CH.sub.2S(O)R. In some embodiments, G.sup.2 is CH.sub.2S(O).sub.2R. In some embodiments, G.sup.2 is CH.sub.2P(O)(R).sub.2. Additional example embodiments are described, e.g., as for chiral reagents/auxiliaries.

(1701) Confirmation that a stereocontrolled oligonucleotide (e.g., one prepared by a method described herein or in the art) comprises the intended stereocontrolled (chirally controlled) internucleotidic linkage can be performed using a variety of suitable technologies. A stereocontrolled (chirally controlled) oligonucleotide comprises at least one stereocontrolled internucleotidic linkage, which can be, e.g., a stereocontrolled internucleotidic linkage comprising a phosphorus, a stereocontrolled phosphorothioate internucleotidic linkage (PS) in the Rp configuration, a PS in the Sp configuration, etc. Useful technologies include, as non-limiting examples: NMR (e.g., 1D (one-dimensional) and/or 2D (two-dimensional) .sup.1H.sup.31P HETCOR (heteronuclear correlation spectroscopy)), HPLC, RP-HPLC, mass spectrometry, LC-MS, and/or stereospecific nucleases. In some embodiments, stereospecific nucleases include: benzonase, micrococcal nuclease, and svPDE (snake venome phosphodiesterase), which are specific for internucleotidic linkages in the Rp configuration (e.g., a PS in the Rp configuration); and nuclease P1, mung bean nuclease, and nuclease S1, which are specific for internucleotidic linkages in the Sp configuration (e.g., a PS in the Sp configuration).

(1702) In some embodiments, the present disclosure pertains to a method of confirming or identifying the stereochemistry pattern of the backbone of an oligonucleotide and/or stereochemistry of particular internucleotidic linkages. In some embodiments, an oligonucleotide comprises a stereocontrolled internucleotidic linkage comprising a phosphorus, a stereocontrolled phosphorothioate (PS) in the Rp configuration, or a PS in the Sp configuration. In some embodiments, an oligonucleotide comprises at least one stereocontrolled internucleotidic linkage and at least one internucleotidic linkage which is not stereocontrolled. In some embodiments, a method comprises digestion of an oligonucleotide with a stereospecific nuclease. In some embodiments, a stereospecific nuclease is selected from: benzonase, micrococcal nuclease, and svPDE (snake venom phosphodiesterase), which are specific for internucleotidic linkages in the Rp configuration (e.g., a PS in the Rp configuration); and nuclease P1, mung bean nuclease, and nuclease S1, which are specific for internucleotidic linkages in the Sp configuration (e.g., a PS in the Sp configuration). In some embodiments, an oligonucleotide or fragments thereof produced by digestion with a stereospecific nuclease are analyzed. In some embodiments, an oligonucleotide or fragments thereof (e.g., produced by digestion with a stereospecific nuclease) are analyzed by NMR, 1D (one-dimensional) and/or 2D (two-dimensional) .sup.1H.sup.31P HETCOR (heteronuclear correlation spectroscopy), HPLC, RP-HPLC, mass spectrometry, LC-MS, UPLC, etc. In some embodiments, an oligonucleotide or fragments thereof are compared with chemically synthesized fragments of the oligonucleotide having a known pattern of stereochemistry.

(1703) Without wishing to be bound by any particular theory, the present disclosure notes that, in at least some cases, stereospecificity of a particular nuclease may be altered by a modification (e.g., 2-modification) of a sugar, by a base sequence, or by a stereochemical context. For example, in some embodiments, benzonase and micrococcal nuclease, which are specific for Rp internucleotidic linkages, were both unable to cleave an isolated PS Rp internucleotidic linkage flanked by PS Sp internucleotidic linkages.

(1704) Various techniques and materials can be utilized. In some embodiments, the present disclosure provides useful combinations of technologies. For example, in some embodiments, stereochemistry of one or more particular internucleotidic linkages of an oligonucleotide can be confirmed by digestion of the oligonucleotide with a stereospecific nuclease and analysis of the resultant fragments (e.g., nuclease digestion products) by any of a variety of techniques (e.g., separation based on mass-to-charge ratio, NMR, HPLC, mass spectrometry, etc.). In some embodiments, stereochemistry of products of digesting an oligonucleotide with a stereospecific nuclease can be confirmed by comparison (e.g., NMR, HPLC, mass spectrometry, etc.) with chemically synthesized fragments (e.g., dimers, trimers, tetramers, etc.) produced, e.g., via technologies that control stereochemistry.

(1705) In one example, an oligonucleotide was confirmed to have the designed and intended pattern of stereochemistry in the backbone. The tested oligonucleotide comprises a core comprising 2-deoxy nucleosides, wherein all of the internucleotidic linkages were PS in the Sp configuration except for one PS in the Rp configuration; and two wings, each of which comprising 2-OMe nucleosides, wherein all the internucleotidic linkages in each wing were phosphodiester (PO) except for one PS in the Sp configuration in each wing. The oligonucleotide was digested with a stereospecific nuclease (e.g., nuclease P1). The various fragments were analyzed (e.g., by LC-MS and by comparison with chemically synthesized fragments of known stereochemistry). It was confirmed that the oligonucleotide had the intended pattern of stereochemistry in its backbone.

(1706) In another example, an oligonucleotide having a different sequence was confirmed to have the intended pattern of stereochemistry in its backbone, using digestion with a stereospecific nuclease and analysis of the resultant fragments. This oligonucleotide comprises a core comprising 2-deoxy nucleotides, wherein all of the internucleotidic linkages were PS in the Sp configuration except for one PS in the Rp configuration; and two wings, each of which comprising 2-OMe nucleotides, wherein all the internucleotidic linkages in each wing were phosphodiester (PO) except for one PS in the Sp configuration in each wing.

(1707) In yet another example, a different oligonucleotide was tested to confirm that the internucleotidic linkages were in the intended configurations. The oligonucleotide is capable of skipping exon 51 of DMD; the majority of the nucleotides in the oligonucleotide were 2-F and the remainder were 2-OMe; the majority of the internucleotidic linkages in the oligonucleotide were PS in the Sp configuration and the remainder were PO. This oligonucleotide was tested by digestion with stereospecific nucleases, and the resultant digestion fragments were analyzed (e.g., by LC-MS and by comparison with chemically synthesized fragments of known stereochemistry). The results confirmed that the oligonucleotide had the intended pattern of stereocontrolled internucleotidic linkages.

(1708) In some embodiments, NMR is useful for characterization and/or confirming stereochemistry. In a set of example experiments, a set of oligonucleotides comprising a stereocontrolled CpG motif were tested to confirm the intended stereochemistry of the CpG motif. Oligonucleotides of the set comprise a motif having the structure of pCpGp, wherein C is Cytosine, G is Guanine, and p is a phosphorothioate which is stereorandom or stereocontrolled (e.g., in the Rp or Sp configuration). For example, one oligonucleotide comprises a pCpGp structure, wherein the pattern of stereochemistry of the phosphorothioates (e.g., the ppp) was RRR; in another oligonucleotide, the pattern of stereochemistry of the ppp was RSS; in another oligonucleotide, the pattern of stereochemistry of the ppp was RSR; etc. In the set, all possible patterns of stereochemistry of the ppp were represented. In the portion of the oligonucleotide outside the pCpGp structure, all the internucleotidic linkages were PO; all nucleosides in the oligonucleotides were 2-deoxy. These various oligonucleotides were tested in NMR, without digestion with a stereospecific nuclease, and distinctive patterns of peaks were observed, indicating that each PS which was Rp or Sp produced a unique peak, and confirming that the oligonucleotides comprised stereocontrolled PS internucleotidic linkages of the intended stereochemistry.

(1709) Stereochemistry patterns of the internucleotidic linkages of various other stereocontrolled oligonucleotides were confirmed, wherein the oligonucleotides comprise a variety of chemical modifications and patterns of stereochemistry.

(1710) As those skilled in the art will appreciate, in some embodiments, a product oligonucleotide of a step, cycle or preparation is an oligonucleotide comprising O.sup.5P, O.sup.P, *.sup.PD, *.sup.PDS, *.sup.PDR, *.sup.N, *.sup.NS and/or *.sup.NR as described herein, which oligonucleotide is optionally linked to a support (e.g., CPG) optionally via a linker (e.g., a CAN linker). For example, in some embodiments, after coupling and/or pre-modification capping and before modification, O.sup.5P is

(1711) ##STR00750##
or a salt form thereof. In some embodiments, after modification O.sup.5P is L.sup.PO, L.sup.PA, L.sup.PB, or a salt form thereof.
Metabolites

(1712) In some embodiments, a DMD oligonucleotide corresponds to a fragment of a different, longer DMD oligonucleotide. In some embodiments, a DMD oligonucleotide corresponds to a metabolite produced by cleavage (e.g., enzymatic cleavage by a nuclease) of a longer DMD oligonucleotide, which produces a fragment or portion of the longer DMD oligonucleotide. In some embodiments, the present disclosure pertains to an DMD oligonucleotide which corresponds to a metabolite produced by the cleavage of a DMD oligonucleotide described herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which corresponds to a portion, or fragment of a DMD oligonucleotide disclosed herein.

(1713) Several experiments were performed wherein a DMD oligonucleotide was incubated in vitro in the presence of any of various substances comprising nucleases. In various experiments, such substances include brain homogenatem, cerebrospinal fluid or plasma from Sprague-Dawley rat or Cynomolgus monkey. Plasma was heparinized. Oligonucleotides were incubated for various time points (e.g., 0, 1, 2, 3, 4 or 5 days for brain tissue homogenate, with a pre-incubation period of 0, 1 or 2 days; 0, 1, 2, 4, 8, 16, 24 or 48 hrs for cerebrospinal fluid; or 0, 1, 2, 4, 8, 16 or 24 hrs for plasma). Pre-incubation indicates that the homogenate is incubated at 37 degrees C. for 0, 24 or 48 hrs to activate the enzymes before adding the oligonucleotide. Final concentration and volume of oligonucleotides was 20 M in 200 l. Products produced by cleavage of the oligonucleotides were analyzed by LC/MS.

(1714) For one DMD oligonucleotide, which is 20 bases long, tested in rat brain homogenate, the major metabolites represented the 3 end of the oligonucleotide, which were truncated by 4, 10, 11, 12, or 13 bases.

(1715) One test DMD oligonucleotide has a length of 20 bases and was tested in rat brain homogenate, yielding major metabolites which were truncated at the 5 end by 4, 10, 11, 12, or 13 bases, leaving metabolites representing the 3 end of the oligonucleotide and which were 16, 10, 9, 8 or 7 bases long, respectively. This oligonucleotide also produced a metabolite which was a 5 fragment which was 12 bases long (truncated at the 3 end by 8 bases).

(1716) A second test oligonucleotide has a length of 20 bases and was tested in rat brain homogenate, yielding major metabolites which were truncated at the 3 end by 4, 8, 9 or 10 bases, leaving metabolites representing the 5 end of the oligonucleotide and which were 16, 12, 11 or 10 bases long, respectively.

(1717) The two tested oligonucleotides comprise internucleotidic linkages which are phosphodiesters, phosphorothioate in the Rp configuration, and phosphorothioates in the Sp configuration. In some embodiments, phosphodiesters were more labile than the phosphorothioate in the Rp configuration or the phosphorothioate in the Sp configuration. In some cases, a metabolite of an oligonucleotide represents a product of a cleavage at a phosphodiester.

(1718) In some embodiments, the present disclosure pertains to a DMD oligonucleotide which corresponds to a metabolite of a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases shorter than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases shorter than that of a DMD oligonucleotide disclosed herein.

(1719) In some embodiments, a metabolite is designated as 3-N- #, or 5-N- #, wherein the # indicates the number of bases removed, and the 3 or 5 indicates which end of the molecule from which the bases were deleted. For example, 3-N-1 indicates a fragment or metabolite wherein 1 base was removed from the 3 end.

(1720) In some embodiments, the present disclosure perhaps to an oligonucleotide which corresponds to a fragment or metabolite of a DMD oligonucleotide disclosed herein, wherein the fragment or metabolite can be described as corresponding to 3-N-1, 3-N-2, 3-N-3, 3-N-4, 3-N-5, 3-N-6, 3-N-7, 3-N-8, 3-N-9, 3-N-10, 3-N-11, 3-N-12, 5-N-1, 5-N-2, 5-N-3, 5-N-4, 5-N-5, 5-N-6, 5-N-7, 5-N-8, 5-N-9, 5-N-10, 5-N-11, or 5-N-12 of a DMD oligonucleotide described herein.

(1721) In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases shorter on the 5 end than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases shorter on the 5 end than that of a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases shorter on the 3 end than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases shorter on the 3 end than that of a DMD oligonucleotide disclosed herein.

(1722) In some embodiments, the present disclosure pertains to a DMD which corresponds to a metabolite of a DMD oligonucleotide, wherein the metabolite is truncated on the 5 and/or 3 end relative to the DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD which corresponds to a metabolite of a DMD oligonucleotide, wherein the metabolite is truncated on both the 5 and 3 end relative to the DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more total bases shorter on the 5 and/or 3 end than a DMD oligonucleotide disclosed herein. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which has a base sequence which is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or more bases total shorter on the 5 and/or 3 end than that of a DMD oligonucleotide disclosed herein.

(1723) In some embodiments, the present disclosure pertains to a DMD oligonucleotide which would be represented by a product of cleavage of a DMD oligonucleotide disclosed herein, which is cleaved at a phosphodiester linkage. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which would be represented by a product of cleavage of a DMD oligonucleotide disclosed herein, if such an oligonucleotide were cleaved at a phosphorothioate linkage in the Rp configuration. In some embodiments, the present disclosure pertains to a DMD oligonucleotide which would be represented by a product of cleavage of a DMD oligonucleotide disclosed herein, if such an oligonucleotide were cleaved at one or more phosphodiester linkages and/or phosphorothioate linkages in the Rp configuration.

(1724) Biological Applications, Example Use, and Dosing Regimens

(1725) As described herein, provided compositions and methods are useful for various purposes, e.g., those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, and/or WO 2017/210647. Among other things, provided technologies can function and/or provide various benefits through a number of chemical and/or biological mechanisms, pathways, etc. (e.g., RNase H, RNAi, splicing modulation (exon skipping (e.g., for DMD in DMD subjects/samples), exon inclusion (e.g., for SMN2 in SMA subjects/samples)), etc.). In some embodiments, provided technologies reduce levels, activities, expressions, etc. of a nucleic acid and/or a product thereof. For example, in some embodiments, provided technologies reduce levels and/or activities of target transcripts and/or products encoded thereby (without the intention to be limited by any particular theory, in some embodiments, via RNase H pathway). In some embodiments, provided technologies increase levels and/or activities of target transcripts and/or products encoded thereby (without the intention to be limited by any particular theory, in some embodiments, via exon skipping). A number of oligonucleotides comprising various types of modified internucleotidic linkages, including many comprising non-negatively charged internucleotidic linkages (e.g., n001), which have various base sequences and/or target various nucleic acids (e.g., transcripts of various genes) were prepared, and various useful properties, activities, and/or advantages were demonstrated. Certain such oligonucleotides, including many comprising non-negatively charged internucleotidic linkages, target transcripts of PNPLA3, C9orf72, SMN2, etc. and have demonstrated various activities and/or benefits. Example oligonucleotides comprising non-negatively charged internucleotidic linkages and targeting various genes, and compositions and uses thereof, include those described in WO 2018/223056, WO 2019/032607, PCT/US18/55653, and WO 2019/032612, each of which is independently incorporated herein by reference.

(1726) In some embodiments, the present disclosure provides methods for modulating level of a transcript or a product encoded thereby in a system, comprising administering an effective amount of a provided oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides methods for modulating level of a transcript or a product encoded thereby in a system, comprising contacting the transcript a provided oligonucleotide or a composition thereof. In some embodiments, a system is an in vitro system. In some embodiments, a system is a cell. In some embodiments, a system is a tissue. In some embodiments, a system is an organ. In some embodiments, a system is an organism. In some embodiments, a system is a subject. In some embodiments, a system is a human. In some embodiments, modulating level of a transcript decreases level of the transcript. In some embodiments, modulating level of a transcript increases level of the transcript.

(1727) In some embodiments, the present disclosure provides methods for preventing or treating a condition, disease, or disorder associated with a nucleic acid sequence or a product encoded thereby, comprising administering to a subject suffering therefrom or susceptible thereto an effective amount of a provided oligonucleotide or composition thereof, wherein the oligonucleotide or composition thereof modulate level of a transcript of the nucleic acid sequence. In some embodiments, a nucleic acid sequence is a gene. In some embodiments, modulating level of a transcript decreases level of the transcript. In some embodiments, modulating level of a transcript increases level of the transcript.

(1728) In some embodiments, change of the level of a modulated transcript, e.g., through knock-down, exon skipping, etc., is at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, or 1000 fold.

(1729) In some embodiments, provided oligonucleotides and oligonucleotide compositions modulate splicing. In some embodiments, provided oligonucleotides and oligonucleotide compositions promote exon skipping, thereby produce a level of a transcript which has increased beneficial functions that the transcript prior to exon skipping. In some embodiments, a beneficial function is encoding a protein that has increased biological functions. In some embodiments, the present disclosure provides methods for modulating splicing, comprising administering to a splicing system a provided oligonucleotide or oligonucleotide composition, wherein splicing of at least one transcript is altered. In some embodiments, level of at least one splicing product is increased at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 200, 500, or 1000 fold. In some embodiments, the present disclosure provides methods for modulating DMD splicing, comprising administering to a splicing system a provided DMD oligonucleotide or composition thereof.

(1730) In some embodiments, the present disclosure provides methods for preventing or treating DMD, comprising administering to a subject susceptible thereto or suffering therefrom a pharmaceutical composition comprising an effective amount of a provided oligonucleotide or oligonucleotide composition.

(1731) In some embodiments, provided compositions and methods provide improved splicing patterns of transcripts compared to a reference pattern, which is a pattern from a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof. An improvement can be an improvement of any desired biological functions. In some embodiments, for example, in DMD, an improvement is production of an mRNA from which a dystrophin protein with improved biological activities is produced.

(1732) In some embodiments, particularly useful and effective are chirally controlled oligonucleotides and chirally controlled oligonucleotide compositions, wherein the oligonucleotides (or oligonucleotides of a plurality in chirally controlled oligonucleotide compositions) optionally comprises one or more non-negatively charged internucleotidic linkages. Among other things, such oligonucleotides and oligonucleotide compositions can provide greatly improved effects, better delivery, lower toxicity, etc.

(1733) For Duchenne muscular dystrophy, example mutations and/or suitable DMD exons for skipping are widely known in the art, including but not limited to those described in U.S. Pat. No. 8,759,507, US Patent No. U.S. Pat. No. 8,486,907, and reference cited therein.

(1734) In some embodiments, one or more skipped exons are selected from exon 2, 29, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 and 60. In some embodiments, exon 2 of DMD is skipped. In some embodiments, exon 29 of DMD is skipped. In some embodiments, exon 40 of DMD is skipped. In some embodiments, exon 41 of DMD is skipped. In some embodiments, exon 42 of DMD is skipped. In some embodiments, exon 43 of DMD is skipped. In some embodiments, exon 44 of DMD is skipped. In some embodiments, exon 45 of DMD is skipped. In some embodiments, exon 46 of DMD is skipped. In some embodiments, exon 47 of DMD is skipped. In some embodiments, exon 48 of DMD is skipped. In some embodiments, exon 49 of DMD is skipped. In some embodiments, exon 50 of DMD is skipped. In some embodiments, exon 51 of DMD is skipped. In some embodiments, exon 52 of DMD is skipped. In some embodiments, exon 53 of DMD is skipped. In some embodiments, exon 54 of DMD is skipped. In some embodiments, exon 50 of DMD is skipped. In some embodiments, exon 55 of DMD is skipped. In some embodiments, a skipped exon is any exon whose inclusion decreases a desired function of DMD. In some embodiments, a skipped exon is any exon whose skipping increased a desired function of DMD.

(1735) In some embodiments, more than one exon of DMD is skipped. In some embodiments, two or more exons of DMD are skipped. In some embodiments, two or more adjacent exons of DMD are skipped.

(1736) In some embodiments, for exon skipping of DMD transcript, or for treatment of DMD, a sequence of a provided plurality of oligonucleotides comprises a DMD sequence list herein. In some embodiments, a sequence comprises one of SEQ ID Nos 1-30 of U.S. Pat. No. 8,759,507. In some embodiments, a sequence comprises one of SEQ ID Nos 1-211 of US Patent No. U.S. Pat. No. 8,486,907. In some embodiments, for exon skipping of DMD transcript, or for treatment of DMD, a sequence of a provided plurality of oligonucleotides is a DMD sequence disclosed herein. In some embodiments, a sequence is one of SEQ ID Nos 1-30 of U.S. Pat. No. 8,759,507. In some embodiments, a sequence is one of SEQ ID Nos 1-211 of US Patent No. U.S. Pat. No. 8,486,907. In some embodiments, a sequence is, comprises or comprises at least 15 consecutive bases of the sequence of any oligonucleotide list herein, e.g., in Table A1. In some embodiments, a sequence is one described in Kemaladewi, et al., Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy, BMC Med Genomics. 2011 Apr. 20; 4:36. doi: 10.1186/1755-8794-4-36; or Malerba et al., Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy, Mol Ther Nucleic Acids. 2012 Dec. 18;1: e62. doi: 10.1038/mtna.2012.54.

(1737) In some embodiments, a provided oligonucleotide composition is administered at a dose and/or frequency lower than that of an otherwise comparable reference oligonucleotide composition with comparable effect in altering the splicing of a target transcript. In some embodiments, a stereocontrolled (chirally controlled) oligonucleotide composition is administered at a dose and/or frequency lower than that of an otherwise comparable stereorandom reference oligonucleotide composition with comparable effect in altering the splicing of the target transcript. If desired, a provided composition can also be administered at higher dose/frequency due to its lower toxicities.

(1738) In some embodiments, provided oligonucleotides, compositions and methods have low toxicities, e.g., when compared to a reference composition. As widely known in the art, oligonucleotides can induce toxicities when administered to, e.g., cells, tissues, organism, etc. In some embodiments, oligonucleotides can induce undesired immune response. In some embodiments, oligonucleotide can induce complement activation. In some embodiments, oligonucleotides can induce activation of the alternative pathway of complement. In some embodiments, oligonucleotides can induce inflammation. Among other things, the complement system has strong cytolytic activity that can damages cells and should therefore be modulated to reduce potential injuries. In some embodiments, oligonucleotide-induced vascular injury is a recurrent challenge in the development of oligonucleotides for e.g., pharmaceutical use. In some embodiments, a primary source of inflammation when high doses of oligonucleotides are administered involves activation of the alternative complement cascade. In some embodiments, complement activation is a common challenge associated with phosphorothioate-containing oligonucleotides, and there is also a potential of some sequences of phosphorothioates to induce innate immune cell activation. In some embodiments, cytokine release is associated with administration of oligonucleotides. For example, in some embodiments, increases in interleukin-6 (IL-6) monocyte chemoattractant protein (MCP-1) and/or interleukin-12 (IL-12) is observed. See, e.g., Frazier, Antisense Oligonucleotide Therapies: The Promise and the Challenges from a Toxicologic Pathologist's Perspective. Toxicol Pathol., 43:78-89, 2015; and Engelhardt, et al., Scientific and Regulatory Policy Committee Points-to-consider Paper: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides. Toxicol Pathol. 43:935-944, 2015.

(1739) Oligonucleotide compositions as provided herein can be used as agents for modulating a number of cellular processes and machineries, including but not limited to, transcription, translation, immune responses, epigenetics, etc. In addition, oligonucleotide compositions as provided herein can be used as reagents for research and/or diagnostic purposes. One of ordinary skill in the art will readily recognize that the present disclosure disclosure herein is not limited to particular use but is applicable to any situations where the use of synthetic oligonucleitides is desirable. Among other things, provided compositions are useful in a variety of therapeutic, diagnostic, agricultural, and/or research applications.

(1740) Various dosing regimens can be utilized to administer provided chirally controlled oligonucleotide compositions, e.g., those described in in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, and/or WO 2017/210647, the dosing regimens of each of which is incorporated herein by reference.

(1741) In some embodiments, with their low toxicity, provided oligonucleotides and compositions can be administered in higher dosage and/or with higher frequency. In some embodiments, with their improved delivery (and other properties), provided compositions can be administered in lower dosages and/or with lower frequency to achieve biological effects, for example, clinical efficacy.

(1742) A single dose can contain various amounts of oligonucleotides. In some embodiments, a single dose can contain various amounts of a type of chirally controlled oligonucleotide, as desired suitable by the application. In some embodiments, a single dose contains about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more (e.g., about 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more) mg of a type of chirally controlled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide is administered at a lower amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide is administered at a lower amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide due to improved efficacy. In some embodiments, a chirally controlled oligonucleotide is administered at a higher amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide. In some embodiments, a chirally controlled oligonucleotide is administered at a higher amount in a single dose, and/or in total dose, than a chirally uncontrolled oligonucleotide due to improved safety.

(1743) Pharmaceutical Compositions

(1744) When used as therapeutics, a provided oligonucleotide or oligonucleotide composition described herein is administered as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a provided oligonucleotides, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable inactive ingredient selected from pharmaceutically acceptable diluents, pharmaceutically acceptable excipients, and pharmaceutically acceptable carriers. In some embodiments, in provided compositions provided oligonucleotides may exist as salts, preferably pharmaceutically acceptable salts, e.g., sodium salts, ammonium salts, etc. In some embodiments, a salt of a provided oligonucleotide comprises two or more cations, for example, in some embodiments, up to the number of negatively charged acidic groups (e.g., phosphate, phosphorothioate, etc.) in an oligonucleotide. As appreciated by those skilled in the art, oligonucleotides described herein may be provided and/or utilized in a salt form, particularly a pharmaceutically acceptable salt form.

(1745) In some embodiments, the present disclosure provides salts of provided oligonucleotides, e.g., chirally controlled oligonucleotides, and pharmaceutical compositions thereof. In some embodiments, a salt is a pharmaceutically acceptable salt. In some embodiments, each hydrogen ion that may be donated to a base (e.g., under conditions of an aqueous solution, a pharmaceutical composition, etc.) is replaced by a non-H.sup.+ cation. For example, in some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is an all-metal ion salt, wherein each hydrogen ion (for example, of OH, SH, etc., acidic enough in water) of each internucleotidic linkage (e.g., a natural phosphate linkage, a phosphorothioate diester linkage, etc.) is replaced by a metal ion. In some embodiments, a provided salt is an all-sodium salt. In some embodiments, a provided pharmaceutically acceptable salt is an all-sodium salt. In some embodiments, a provided salt is an all-sodium salt, wherein each internucleotidic linkage which is a natural phosphate linkage (acid form OP(O)(OH)O), if any, exists as its sodium salt form (OP(O)(ONa)O), and each internucleotidic linkage which is a phosphorothioate diester linkage (phosphorothioate internucleotidic linkage; acid form OP(O)(SH)O), if any, exists as its sodium salt form (OP(O)(SNa)O).

(1746) In some embodiments, the pharmaceutical composition is formulated for intravenous injection, oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration or otic administration. In some embodiments, the pharmaceutical composition is a tablet, a pill, a capsule, a liquid, an inhalant, a nasal spray solution, a suppository, a suspension, a gel, a colloid, a dispersion, a suspension, a solution, an emulsion, an ointment, a lotion, an eye drop or an ear drop.

(1747) In some embodiments, the present disclosure provides a pharmaceutical composition comprising chirally controlled oligonucleotide, or composition thereof, in admixture with a pharmaceutically acceptable excipient. One of skill in the art will recognize that the pharmaceutical compositions include the pharmaceutically acceptable salts of the chirally controlled oligonucleotide, or composition thereof, described above.

(1748) A variety of supramolecular nanocarriers can be used to deliver nucleic acids. Example nanocarriers include, but are not limited to liposomes, cationic polymer complexes and various polymeric. Complexation of nucleic acids with various polycations is another approach for intracellular delivery; this includes use of PEGlyated polycations, polyethyleneamine (PEI) complexes, cationic block co-polymers, and dendrimers. Several cationic nanocarriers, including PEI and polyamidoamine dendrimers help to release contents from endosomes. Other approaches include use of polymeric nanoparticles, polymer micelles, quantum dots and lipoplexes. In some embodiments, an oligonucleotide is conjugated to another molecular.

(1749) Additional nucleic acid delivery strategies are known in addition to the example delivery strategies described herein.

(1750) In therapeutic and/or diagnostic applications, the compounds of the disclosure can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington, The Science and Practice of Pharmacy, (20th ed. 2000).

(1751) Provided oligonucleotides, and compositions thereof, are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from about 0.01 to about 1000 mg, from about 0.5 to about 100 mg, from about 1 to about 50 mg per day, and from about 5 to about 100 mg per day are examples of dosages that may be used. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

(1752) Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and may include, by way of example but not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts may be found in, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000). Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate, pamoate (embonate), phosphate, salicylate, succinate, sulfate, or tartrate.

(1753) As appreciated by a person having oridinary skill in the art, oligonucleotides may be formulated as a number of salts for, e.g., pharmaceutical uses. In some embodiments, a salt is a metal cation salt and/or ammonium salt. In some embodiments, a salt is a metal cation salt of an oligonucleotide. In some embodiments, a salt is an ammonium salt of an oligonucleotide. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a salt is a sodium salt of an oligonucleotide. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed with oligonucleotides. As appreciated by a person having oridinary skill in the art, a salt of an oligonucleotide may contain more than one cations, e.g., sodium ions, as there may be more than one anions within an oligonucleotide.

(1754) Depending on the specific conditions being treated, such agents may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained-low release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington, The Science and Practice of Pharmacy (20th ed. 2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articullar, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

(1755) For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

(1756) Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.

(1757) Compounds, e.g., oligonucleotides, can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject (e.g., patient) to be treated.

(1758) For nasal or inhalation delivery, the agents of the disclosure may also be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances such as, saline, preservatives, such as benzyl alcohol, absorption promoters, and fluorocarbons.

(1759) In certain embodiments, oligonucleotides and compositions are delivered to the CNS. In certain embodiments, oligonucleotides and compositions are delivered to the cerebrospinal fluid. In certain embodiments, oligonucleotides and compositions are administered to the brain parenchyma. In certain embodiments, oligonucleotides and compositions are delivered to an animal/subject by intrathecal administration, or intracerebroventricular administration. Broad distribution of oligonucleotides and compositions, described herein, within the central nervous system may be achieved with intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.

(1760) In certain embodiments, parenteral administration is by injection, by, e.g., a syringe, a pump, etc. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue, such as striatum, caudate, cortex, hippocampus and cerebellum.

(1761) In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decreases median effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or 50. In certain embodiments, the targeted tissue is brain tissue. In certain embodiments the targeted tissue is striatal tissue. In certain embodiments, decreasing EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.

(1762) In certain embodiments, an oligonucleotide is delivered by injection or infusion once every month, every two months, every 90 days, every 3 months, every 6 months, twice a year or once a year.

(1763) Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

(1764) In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of an active compound into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

(1765) Pharmaceutical preparations for oral use can be obtained by combining an active compound with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

(1766) Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

(1767) Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, an active compound may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

(1768) In some embodiments, any DMD oligonucleotide, or combination thereof, described herein, or any composition comprising a DMD oligonucleotide described herein, can be combined with any pharmaceutical preparation described herein or known in the art.

(1769) Certain Embodiments of Conjugates and Additional Chemical Moieties

(1770) In some embodiments, provided oligonucleotides comprise one or more additional chemical moieties (e.g., other than typical moieties of nucleobases, sugars and/or internucleotidic linkages, etc.), optionally through a linker. In some embodiments, a chemical moiety is a lipid moiety. In some embodiments, a chemical moiety is a carbohydrate moiety. In some embodiments, a chemical moiety is a targeting moiety. In some embodiments, a chemical moiety is a moiety of a ligand. In some embodiments, a chemical moiety can increase delivery of oligonucleotides to certain organelles, cells, tissues, organs, and/or organisms. In some embodiments, a chemical moiety enhances one or more of desired properties and/or activities. Certain example chemical moieties utilized in certain oligonucleotides are presented in the Tables (e.g., various Mod in Table A1). In some embodiments, a chemical moiety comprises one or more sugar moieties or derivatives thereof, e.g., glucose, mannose, etc. In some embodiments, a chemical moiety is or comprises a lipid moiety. In some embodiments, a chemical moiety is or comprises a vitamin E moiety. In some embodiments, a chemical moiety comprises one or more peptide moieties. In some embodiments, a peptide is a cell-penetrating peptide. In some embodiments, a peptide is a ligand of a protein, e.g., a cell surface receptor. In some embodiments, a peptide is a Tfr1 peptide. Certain example peptide moieties are utilized to prepare oligonucleotides described in the Tables, e.g., Table 1A. In some embodiments, a chemical moiety comprises one or more basic moieties. In some embodiments, a basic moiety is positively charged at, e.g. about pH 7.4. In some embodiments, a basic moiety is or comprises a guanidine moiety. In some embodiments, a basic moiety is or comprises N(R.sup.1).sub.2, wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, a basic moiety is or comprises N(R.sup.1).sub.3, wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, a basic moiety is or comprises NC(N(R.sup.1).sub.2).sub.2, wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, each R.sup.1 is independently R as described in the present disclosure. In some embodiments, each R.sup.1 is independently optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.1 is methyl. In some embodiments, one or two R.sup.1 are the same. In some embodiments, each R.sup.1 is the same. In some embodiments, at least one R.sup.1 is different from another R.sup.1. In some embodiments, a basic moiety is NC(N(CH.sub.3).sub.2).sub.2. In some embodiments, a chemical moiety comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sugar, peptide, lipid, and/or basic moieties. In some embodiments, the number is 1. In some embodiments, the number is 2. In some embodiments, the number is 3. In some embodiments, the number is 4. In some embodiments, the number is 5. In some embodiments, the number is 6. In some embodiments, a chemical moiety comprises a ligand moiety of a protein, e.g., a receptor protein of a target cell. In some embodiments, a ligand is a ligand for a vitamin E receptor. In some embodiments, a ligand is for Tfr1 receptor. Chemical moieties as described and demonstrated in the present disclosure include and can be utilized as carbohydrate moieties, lipid moieties, targeting moieties, etc., and can provide a variety of functions, e.g., improving delivery, one or more properties, activities, etc.

(1771) In some embodiments, the present disclosure provides oligonucleotides comprising additional chemistry moieties, optionally connected to the oligonucleotide moiety through a linker. In some embodiments, the present disclosure provides oligonucleotides comprising (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3- ,

(1772) wherein:

(1773) each R.sup.D is independently a chemical moiety; each of L.sup.M1, L.sup.M2, and L.sup.M3 is independently L; and
b is 1-1000.

(1774) In some embodiments, each of L.sup.M1, L.sup.M2, and L.sup.M3 is independently a covalent bond, or a bivalent or multivalent, optionally substituted, linear or branched group selected from a C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, C(R).sub.2, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]; and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L.

(1775) In some embodiments, L.sup.M1 comprises one or more N(R) and one or more C(O). In some embodiments, a linker (e.g., L, L.sup.M, etc.) or L.sup.M1 is or comprises

(1776) ##STR00751##
wherein n.sup.L is 1-8. In some embodiments, a linker or -L.sup.M1-L.sup.M2-L.sup.M3- is

(1777) ##STR00752##
or a salt form thereof, wherein n.sup.L is 1-8. In some embodiments, a linker or -L.sup.M1-L.sup.M2-L.sup.M3- is

(1778) ##STR00753##
or a salt form thereof, wherein: n.sup.L is 1-8. each amino group independently connects to a moiety; and the P atom connects to the 5-OH of the oligonucleotide.

(1779) In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(1780) ##STR00754##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(1781) ##STR00755##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(1782) ##STR00756##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(1783) ##STR00757##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(1784) ##STR00758##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(1785) ##STR00759##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(1786) ##STR00760##
In some embodiments, a linker, or L.sup.M1, is or comprises

(1787) ##STR00761##
In some embodiments, the moiety and linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises:

(1788) ##STR00762##
In some embodiments, the moiety and linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises:

(1789) ##STR00763##
In some embodiments, a linker is

(1790) ##STR00764##
In some embodiments, the moiety and linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises:

(1791) ##STR00765##
In some embodiments, the moiety and linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises:

(1792) ##STR00766##

(1793) In some embodiments, n.sup.L is 1-8. In some embodiments, n.sup.L is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n.sup.L is 1. In some embodiments, n.sup.L is 2. In some embodiments, n.sup.L is 3. In some embodiments, n.sup.L is 4. In some embodiments, n.sup.L is 5. In some embodiments, n.sup.L is 6. In some embodiments, n.sup.L is 7. In some embodiments, n.sup.L is 8.

(1794) In some embodiments, L.sup.M2 is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, C(R).sub.2, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O; and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L. In some embodiments, L.sup.M2 is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group having 1-5 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, C(R).sub.2, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), or P(O)(R). In some embodiments, L.sup.M2 is is a covalent bond, or a bivalent, optionally substituted, linear or branched C.sub.1-10 aliphatic wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, C(R).sub.2, O, S, N(R), or C(O). In some embodiments, L.sup.M2 is NH(CH.sub.2).sub.6, wherein NH is bonded to L.sup.M1.

(1795) In some embodiments, L.sup.M3 is is P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR), OP(O)(SR), OP(O)(R), OP(O)(NR), OP(S)(OR), OP(S)(SR), OP(S)(R), OP(S)(NR), OP(R), OP(OR), OP(SR), OP(NR), or OP(OR)[B(R).sub.3]. In some embodiments, L.sup.M3 is OP(O)(OR), or OP(O)(SR), wherein O is bonded to L.sup.M2. In some embodiments, the P atom is connected to a sugar unit, a nucleobase unit, or an internucleotidic linkage. In some embodiments, the P atom is connected to a OH group through formation of a PO bond. In some embodiments, the P atom is connected to the 5-OH group through formation of a PO bond.

(1796) In some embodiments, L.sup.M1 is a covalent bond. In some embodiments, L.sup.M2 is a covalent bond. In some embodiments, L.sup.M3 is a covalent bond. In some embodiments, L.sup.M1 is L.sup.M2 as described in the present disclosure. In some embodiments, L.sup.M1 is L.sup.M3 as described in the present disclosure. In some embodiments, L.sup.M2 is L.sup.M1 as described in the present disclosure. In some embodiments, L.sup.M2 is L.sup.M3 as described in the present disclosure. In some embodiments, L.sup.M3 is L.sup.M1 as described in the present disclosure. In some embodiments, L.sup.M3 is L.sup.M2 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M2 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M3 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1-L.sup.M2, wherein each of L.sup.M1 and L.sup.M2 is independently as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1-L.sup.M3, wherein each of L.sup.M1 and L.sup.M3 is independently as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M2-L.sup.M3, wherein each of L.sup.M2 and L.sup.M3 is independently as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1-L.sup.M2-L.sup.M3, wherein each of L.sup.M1, L.sup.M2 and L.sup.M3 is independently as described in the present disclosure.

(1797) In some embodiments, each R.sup.D is independently a chemical moiety as described in the present disclosure. In some embodiments, R.sup.D is an additional chemical moiety. In some embodiments, R.sup.D is targeting moiety. In some embodiments, R.sup.D is or comprises a carbohydrate moiety. In some embodiments, R.sup.D is or comprises a lipid moiety. In some embodiments, R.sup.D is or comprises a ligand moiety for, e.g., cell receptors such as a sigma receptor, an asialoglycoprotein receptor, etc. In some embodiments, a ligand moiety is or comprises an anisamide moiety, which may be a ligand moiety for a sigma receptor. In some embodiments, a ligand moiety is or comprises a lipid. In some embodiments, a ligand moiety is or comprises a GalNAc moiety, which may be a ligand moiety for an asialoglycoprotein receptor. In some embodiments, R.sup.D is selected from optionally substituted phenyl,

(1798) ##STR00767##
wherein n is 0 or 1, and each other variable is independently as described in the present disclosure. In some embodiments, R.sup.s is F. In some embodiments, R.sup.s is OMe. In some embodiments, R.sup.s is OH. In some embodiments, R.sup.s is NHAc. In some embodiments, R.sup.s is NHCOCF.sub.3. In some embodiments, R is H. In some embodiments, R is H. In some embodiments, R.sup.2s is NHAc, and R.sup.5s is OH. In some embodiments, R.sup.2s is p-anisoyl, and R.sup.5s is OH. In some embodiments, R.sup.2s is NHAc and R.sup.5s is p-anisoyl. In some embodiments, R.sup.2s is OH, and R.sup.5s is p-anisoyl. In some embodiments, R.sup.D is selected from

(1799) ##STR00768## ##STR00769## ##STR00770##
Further embodiments of R.sup.D includes additional chemical moiety embodiments, e.g., those described in the examples.

(1800) In some embodiments, n is 1. In some embodiments, n is 0.

(1801) In some embodiments, n is 1. In some embodiments, n is 2.

(1802) In some embodiments, a provided oligonucleotide, e.g., DMD oligonucleotide, is conjugated to an additional component (chemical moiety). In some embodiments, a composition comprises any DMD oligonucleotide, or combination thereof, described herein, can be conjugated to any chemical moiety described herein or known in the art.

(1803) In some embodiments, a composition comprising a provided oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component which is any of: Sulfonamide (Carbonic Anhydrases IV inhibitor); Cleavable lipid; Transferrin Receptor 1 (CD71, TfR) ligand; OCTN2 transporter targeting (L-Cartinine); Glut4 and Glut1 Receptor ligand; Mannose Receptor C1 (Mrc1) and Mannose 6P Receptor (M6Pr) ligand; Cleavable Lipid; Cholesterol; or a Peptide (including, but not limited to, a short delivery peptide or cell-penetrating peptide (CPP)).

(1804) Variously oligonucleotides have been designed and/or constructed which comprise an additional component which is, comprises or is derived from: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); and Mannose (tri- and hex-antennary, alpha and beta); and various synthesis schemes for these additional components and oligonucleotides comprising them or molecules derived from them have been devised.

(1805) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from

(1806) ##STR00771##
WV-DL-14 is also known as WV-DL-014. In some embodiments, gambogic acid or a derivative thereof binds to Transferrin receptor (CD71).

(1807) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from L-cartinine, which binds to the OCTN2 transporter. In some embodiments, a composition comprising a DMD oligonucleotide comprises an additional component which is derived from

(1808) ##STR00772##

(1809) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a sulfonamide or a derivative thereof.

(1810) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of:

(1811) ##STR00773## ##STR00774##

(1812) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is or comprises or comprises a derivative of:

(1813) ##STR00775##

(1814) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is or comprises or comprises a derivative of:

(1815) ##STR00776##

(1816) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of: WV-DL-001, WV-DL-002, WV-DL-003, WV-DL-006, WV-DL-007, WV-DL-008, WV-DL-009, WV-DL-010, WV-DL-011, WV-DL-012, or WV-D1-014, and other additional components, wherein the terminal-COOH is used to conjugate the additional component to a linker or to an oligonucleotide. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of: WV-DL-001, WV-DL-002, WV-DL-003, WV-DL-006, WV-DL-007, WV-DL-008, WV-DL-009, WV-DL-010, WV-DL-011, WV-DL-012, or WV-DI-014, and other additional components, wherein the terminal-COOH is used to conjugate the additional component to a linker, wherein the conjugation process converts the COOH to a C(O) which connects a linker. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of: WV-DL-001, WV-DL-002, WV-DL-003, WV-DL-006, WV-DL-007, WV-DL-008, WV-DL-009, WV-DL-010, WV-DL-011, WV-DL-012, or WV-DI-014, and other additional components, wherein the terminal COOH is used to conjugate the additional component to a linker, wherein the conjugation process replaces the COOH with C(O) which connects to NH of a linker (e.g., L001). A non-limiting example of a product of this process for conjugation, using an additional component derived from WV-DL-006 is shown here:

(1817) ##STR00777##
wherein WV-DL-005 indicates the additional component.

(1818) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a lipid. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a lipid, including but not limited to a lipid described herein.

(1819) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component, wherein the additional component is conjugated to the oligonucleotide via a cleavable linker. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component which is a lipid, wherein the lipid is conjugated to the oligonucleotide via a cleavable linker. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide, comprises an additional component which is a lipid, including but not limited to a lipid described herein, wherein the lipid is conjugated to the oligonucleotide via a cleavable linker.

(1820) In some embodiments a cleavable linker comprises an ester. In some embodiments, a cleavable linker is cleavable within a cell, allowing the oligonucleotide to be physically separated from the additional component.

(1821) In some embodiments a cleavable linker is or comprises:

(1822) ##STR00778##

(1823) Non-limiting examples of an oligonucleotide conjugated to a lipid(s) via a cleavable linker are shown here:

(1824) ##STR00779##

(1825) A non-limiting example of an oligonucleotide comprising an additional component which is stearic acid, linked to the oligonucleotide via a cleavable linker is shown here:

(1826) ##STR00780##
wherein stearic acid indicates the additional component.

(1827) A non-limiting reagent useful for conjugating stearic acid through a cleavable linker and its example preparation and use are shown below:

(1828) ##STR00781##

(1829) A non-limiting reagent useful for conjugating a cholesterol derivative through a cleavable linker, and its example preparation, are shown here:

(1830) ##STR00782##

(1831) In some embodiments, a composition comprising an oligonucleotide comprises an additional component derived from:

(1832) ##STR00783##

(1833) In some embodiments, a composition comprising an oligonucleotide comprises an additional component derived from either of:

(1834) ##STR00784##

(1835) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises a mannose receptor ligand. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises a mannose receptor ligand which is a mannose receptor inhibitor. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from any of:

(1836) ##STR00785## ##STR00786##
where the arrow indicates a COOH which can be used to conjugate the additional component to an oligonucleotide, optionally via a linker.

(1837) A non-limiting example of a procedure for preparing an additional component comprising a mannose receptor ligand is shown here:

(1838) ##STR00787## ##STR00788##

(1839) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a ligand (or derivative thereof) that binds to a glucose or Glut4 receptor. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a ligand (or derivative thereof) that binds to a glucose receptor. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is a ligand (or derivative thereof) that binds to and inhibits a glucose receptor. In some embodiments, a ligand (or derivative thereof) that binds to a glucose or Glut4 receptor is mono-, bi-, tri, or hex-antennary. In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component which is derived from:

(1840) ##STR00789##

(1841) A non-limiting example of a procedure for synthesis of a tri-antennary glucose receptor inhibitor is shown here:

(1842) ##STR00790## ##STR00791##

(1843) A non-limiting example of a procedure for synthesis of a hex-antennary glucose receptor inhibitor is shown here:

(1844) ##STR00792## ##STR00793##

(1845) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component increases internalization of the oligonucleotide via receptor -mediated endocytosis.

(1846) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer.

(1847) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer which is a peptide aptamer, a RNA apatamer, a DNA aptamer, or an aptamer which comprises a RNA nucleotide, a DNA nucleotide, a modified nucleotide, and/or an amino acid and/or peptide.

(1848) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer which binds to a receptor.

(1849) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer which binds to a receptor which is a mannose receptor, a mannose-6-phosphate receptor or transferrin receptor.

(1850) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer that increases internalization of the oligonucleotide.

(1851) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is an aptamer that increases internalization of the oligonucleotide via receptor -mediated endocytosis.

(1852) In some embodiments, an oligonucleotide, e.g., a DMD oligonucleotide comprises an additional component, wherein the additional component is or comprises a peptide. In some embodiments, a peptide is a cell-penetrating peptide (CPP). In some embodiments, a CPP is arginine-rich. In some embodiments, a CPP has or comprises the amino acid sequence of RRQPPRSISSHPC (SEQ ID NO: 3252) or RRQPPRSISSHP (SEQ ID NO: 3253).

(1853) A non-limiting example of a procedure for conjugating a peptide to a DMD oligonucleotide is shown here (SEQ ID NOS 3253 and 3253, respectively, in order of appearance):

(1854) ##STR00794##

(1855) In some embodiments, a peptide comprises the amino acid sequence of RC or RRC. In some embodiments, a peptide comprises a structure of either of:

(1856) ##STR00795##

(1857) Provided oligonucleotides, e.g., DMD oligonucleotides, may be conjugated as PMOs to cell-penetrating peptides. Yokota et al. 2012 Nucl. Acid Ther. 22: 306; Wu et al. 2009 Mol. Ther. 17: 864-871; Goyenvalle et al. 2010 Mol. Ther. 18, 198-205; Jearawiriyapaisarn et al. 2010 Cardiovasc. Res. 85, 444-453; Crisp et al. 2011 Hum. Mol. Genet. 20, 413-421; Widrick et al. 2011; Wu et al. 2011 PLOS One 6, e19906.

(1858) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises one or more peptide and/or peptide tag. In some embodiments, a peptide is or comprises a muscle-targeting heptapeptide (MSP). In some embodiments, the sequence of a muscle-targeting helptapeptide is or comprises the sequence of ASSLNIAXB (SEQ ID NO: 3254). In some embodiments, a peptide is or comprises a cell-penetrating peptide. In some embodiments, the sequence of a cell-penetrating peptide comprises multiple arginines. In some embodiments, the sequence of a cell-penetrating peptide is or comprises RXRRBRRXRRBRXB (SEQ ID NO: 3255).

(1859) In some embodiments, the sequence of a peptide is or comprises a sequence of: ASSLNIAXB (SEQ ID NO: 3254), RXRRBRRXRRBRXB (SEQ ID NO: 3255), RXRRXRRXRRXRXB (SEQ ID NO: 3256), ASSLNIAXB-RXRRBRRXRRBRXB (SEQ ID NO: 3257), RXRRBRRXRRBRXB-ASSLNIAXB (SEQ ID NO: 3258), or any sequence comprising both ASSLNIAXB and either RXRRBRRXRRBRXB (SEQ ID NO: 3255) or RXRRXRRXRRXRXB(SEQ ID NO: 3256), wherein R is L-arginine, X is 6-aminohexanoic acid, and B is beta-alanine.

(1860) A muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) may be conjugated to provided oligonucleotides in accordance with the present disclosure. Yin et al. 2009 Hum. Mol. Genet. 18: 4405-4414. Yokota et al. 2009 Arch. Neurol. 66: 32.

(1861) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises anisamide or a derivative thereof.

(1862) In some embodiments, a composition comprising an oligonucleotide, e.g., a DMD oligonucleotide comprises one or more guanidinium group. vPMOs are reportedly morpholino oligomers conjugated with delivery moiety containing eight terminal guanidinium groups on a dendrimer scaffold that enable entry into cells. Morcos et al. 2008 Biotechniques 45: 613-618; Yokota et al. 2012 Nucl. Acid Ther. 22: 306.

(1863) In some embodiments, an oligonucleotide, e.g., DMD oligonucleotide is delivered using a leash. A non-limiting example of a leash is reported in: Gebski et al. 2003 Hum. Mol. Gen. 12: 1801-1811.

(1864) In some embodiments, an additional chemical moiety is cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; Gambogic acid; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).

(1865) Certain chemical moieties, e.g., lipid moieties, carbohydrate moieties, targeting moieties, etc. and linker moieties for connecting such moieties to oligonucleotide chains (e.g., via sugars, nucleobases, internucleotidic linkages, etc.) are described in the Tables as example; some of such chemical and linker moieties and related technologies for their preparation, conjugation with oligonucleotide chains, and uses are described in e.g., WO 2017/062862, WO 2017/192679, WO 2017/210647, etc.

(1866) Lipids

(1867) In some embodiments, an additional chemical moiety/component is a lipid moiety. In some embodiments, the present disclosure provided oligonucleotide compositions further comprise one or more lipids. In some embodiments, incorporation of lipid moieties into oligonucleotides can provide unexpected, greatly improved properties (e.g., activities, toxicities, distribution, pharmacokinetics, etc.).

(1868) A composition can be obtained by combining an active compound with a lipid. In some embodiments, the lipid is conjugated to an active compound. In some embodiments, the lipid is not conjugated to an active compound. In some embodiments, a lipid comprises a C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C.sub.1-4 aliphatic group. In some embodiments, a lipid comprises a C.sub.10-C.sub.60 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.60 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C.sub.1-4 aliphatic group. In some embodiments, a lipid comprises a C.sub.10-C.sub.80 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.80 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C.sub.1-4 aliphatic group. In some embodiments, a lipid comprises a C.sub.10-C.sub.100 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.100 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C.sub.1-4 aliphatic group.

(1869) In some embodiments, a lipid comprises an optionally substituted, C.sub.10-C.sub.80 saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, a C.sub.1-C.sub.6 heteroaliphatic moiety, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), and C(O)O, wherein each variable is independently as defined and described herein. In some embodiments, a lipid comprises an optionally substituted C.sub.10-C.sub.80 saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an optionally substituted C.sub.10-C.sub.80 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.80 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C.sub.1-4 aliphatic group. In some embodiments, a lipid comprises an optionally substituted, C.sub.10-C.sub.60 saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, a C.sub.1-C.sub.6 heteroaliphatic moiety, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), and C(O)O, wherein each variable is independently as defined and described herein. In some embodiments, a lipid comprises an optionally substituted C.sub.10-C.sub.60 saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an optionally substituted C.sub.10-C.sub.60 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.60 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C.sub.1-4 aliphatic group. In some embodiments, a lipid comprises an optionally substituted, C.sub.10-C.sub.40 saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, a C.sub.1-C.sub.6 heteroaliphatic moiety, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2, SC(O), C(O)S, OC(O), and C(O)O, wherein each variable is independently as defined and described herein. In some embodiments, a lipid comprises an optionally substituted C.sub.10-C.sub.40 saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an optionally substituted C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain, optionally substituted with one or more C.sub.1-4 aliphatic group. In some embodiments, a lipid comprises an unsubstituted C.sub.10-C.sub.80 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises no more than one optionally substituted C.sub.10-C.sub.80 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises two or more optionally substituted C.sub.10-C.sub.80 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an unsubstituted C.sub.10-C.sub.60 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises no more than one optionally substituted C.sub.10-C.sub.60 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises two or more optionally substituted C.sub.10-C.sub.60 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises an unsubstituted C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises no more than one optionally substituted C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises two or more optionally substituted C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid comprises a C.sub.10-C.sub.40 linear, saturated or partially unsaturated, aliphatic chain. In some embodiments, a lipid is selected from the group consisting of: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl. In some embodiments, a lipid is not conjugated to an oligonucleotide chain (whether through one or more linker moieties or not). In some embodiments, a lipid is conjugated to an oligonucleotide chain, optionally through one or more linker moieties.

(1870) In some embodiments, a lipid is selected from the group consisting of: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl. In some embodiments, a lipid has a structure of any of:

(1871) ##STR00796##
In some embodiments, an active compound is an oligonucleotide described herein. In some embodiments, an active compound is an oligonucleotide capable of mediating skipping of an exon in dystrophin. In some embodiments, an active compound is an oligonucleotide capable of mediating skipping of exon 51 in dystrophin. In some embodiments, an active compound is a nucleic acid of a sequence comprising or consisting of any sequence of any nucleic acid described herein. In some embodiments, an active compound is a nucleic acid of a sequence comprising or consisting of any sequence of any oligonucleotide listed in Table A1. In some embodiments, a composition comprises a lipid and an an active compound, and further comprises another component selected from: another lipid, and a targeting compound or moiety. In some embodiments, a lipid includes, without limitation: an amino lipid; an amphipathic lipid; an anionic lipid; an apolipoprotein; a cationic lipid; a low molecular weight cationic lipid; a cationic lipid such as CLinDMA and DLinDMA; an ionizable cationic lipid; a cloaking component; a helper lipid; a lipopeptide; a neutral lipid; a neutral zwitterionic lipid; a hydrophobic small molecule; a hydrophobic vitamin; a PEG-lipid; an uncharged lipid modified with one or more hydrophilic polymers; phospholipid; a phospholipid such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; a stealth lipid; a sterol; a cholesterol; and a targeting lipid; and any other lipid described herein or reported in the art. In some embodiments, a composition comprises a lipid and a portion of another lipid capable of mediating at least one function of another lipid. In some embodiments, a targeting compound or moiety is capable of targeting a compound (e.g., a composition comprising a lipid and a active compound) to a particular cell or tissue or subset of cells or tissues. In some embodiments, a targeting moiety is designed to take advantage of cell- or tissue-specific expression of particular targets, receptors, proteins, or other subcellular components; In some embodiments, a targeting moiety is a ligand (e.g., a small molecule, antibody, peptide, protein, carbohydrate, aptamer, etc.) that targets a composition to a cell or tissue, and/or binds to a target, receptor, protein, or other subcellular component.

(1872) In some embodiments, incorporation of a lipid moiety for delivery of an active compound allow (e.g., do not prevent or interfere with) the function of an active compound. Non-limiting example lipids include: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl.

(1873) In some embodiments, lipid conjugation, such as conjugation with fatty acids, may improve one or more properties of oligonucleotides. In some embodiments, lipid conjugation improves delivery.

(1874) In some embodiments, as supported by experimental data, conjugation with lipids can increase skipping efficiency.

(1875) In some embodiments, a composition for delivery of an active compound is capable of targeting an active compound to particular cells or tissues, as desired. In some embodiments, a composition for delivery of an active compound is capable of targeting an active compound to a muscle cell or tissue. In some embodiments, the present disclosure pertains to compositions and methods related to delivery of active compounds, wherein the compositions comprise an active compound a lipid. In some embodiments to a muscle cell or tissue, the lipid is selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl. Example compositions were prepared comprising an active compound (WV-942) and a lipid, and these compositions were capable of delivering an active compound to target cells and tissues, e.g., muscle cells and tissues. The example lipids used include stearic acid, oleic acid, alpha-linolenic acid, gamma-linolenic acids, cis-DHA, turbinaric acid and dilinoleyl acid.

(1876) Various compositions comprising an active compound and any of: stearic acid, oleic acid, alpha-linolenic acid, gamma-linolenic acid, cis-DHA or turbinaric acid, were able to deliver an active compound to various tissues, including gastrocnemius muscle tissue, heart muscle tissue, quadriceps muscle tissue, gastrocnemius muscle tissue, and diaphragm muscle tissue.

(1877) In some embodiments, a composition comprising a lipid, selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl, and an active compound is capable of delivering an active compound to extra-hepatic cells and tissues, e.g., muscle cells and tissues.

(1878) In some embodiments, a lipid has the structure of R.sup.LDOH, wherein wherein R.sup.LD is an optionally substituted, C.sub.10-C.sub.80 saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, CC, a C.sub.1-C.sub.6 heteroaliphatic moiety, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O), N(R)C(O)O, OC(O)N(R), S(O), S(O).sub.2, S(O).sub.2N(R), N(R)S(O).sub.2 SC(O), C(O)S, OC(O), and C(O)O. In some embodiments, a lipid has the structure of R.sup.LDC(O)OH. In some embodiments, R.sup.LD is

(1879) ##STR00797##
Example oligonucleotides comprising such R.sup.LD groups are described herein and in WO 2017/062862, the description of R.sup.LD is incorporated herein by reference.

(1880) In some embodiments, a lipid is conjugated to an active compound optionally through a linker moiety. In some embodiments, a linker is L.sup.M. In some embodiments, a linker is L. In some embodiments, -L-comprises a bivalent aliphatic chain. In some embodiments, -L-comprises a phosphate group. In some embodiments, -L-comprises a phosphorothioate group. In some embodiments, -L- has the structure of C(O)NH(CH.sub.2).sub.6OP(O)(S.sup.). In some embodiments, -L- has the structure of C(O)NH(CH.sub.2).sub.6OP(O)(O.sup.).

(1881) Lipids, optionally through linkers, can be conjugated to oligonucleotides at various suitable locations. In some embodiments, lipids are conjugated through the 5-OH group. In some embodiments, lipids are conjugated through the 3-OH group. In some embodiments, lipids are conjugated through one or more sugar moieties. In some embodiments, lipids are conjugated through one or more bases. In some embodiments, lipids are incorporated through one or more internucleotidic linkages. In some embodiments, an oligonucleotide may contain multiple conjugated lipids which are independently conjugated through its 5-OH, 3-OH, sugar moieties, base moieties and/or internucleotidic linkages.

(1882) In some embodiments, a composition comprises an oligonucleotide, e.g., DMD oligonucleotide and a lipid selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid, arachidonic acid, and dilinoleyl, wherein the lipid is directly conjugated to the biologically active agent (without a linker interposed between the lipid and the biologically active agent). In some embodiments, a composition comprises an oligonucleotide and a lipid selected from: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), turbinaric acid and dilinoleyl, wherein the lipid is directly conjugated to the biologically active agent (without a linker interposed between the lipid and the biologically active agent).

(1883) In some embodiments, a composition comprises a DMD oligonucleotide and any lipid known in the art, wherein the lipid is conjugated or not conjugated to the oligonucleotide.

(1884) Non-limiting examples of lipids, and methods of making them and conjugating them are provided in, for example, WO 2017/062862, the lipids and related methods of which are incorporated herein by reference.

(1885) Targeting Moieties

(1886) In some embodiments, an additional chemical moiety/component is a targeting moiety. In some embodiments, a provided composition further comprises a targeting moiety. In some embodiments, a targeting moiety is conjugated to an oligonucleotide chain. In some embodiments, a biologically active agent is conjugated to both a lipid and an oligonucleotide chain. Various targeting moieties can be used in accordance with the present disclosure, e.g., lipids, antibodies, peptides, carbohydrates, etc.

(1887) Targeting moieties can be incorporated into provided technologies through many types of methods in accordance with the present disclosure. In some embodiments, targeting moieties are chemically conjugated with oligonucleotides.

(1888) In some embodiments, provided compositions comprise two or more targeting moieties. In some embodiments, provided oligonucleotides comprise two or more conjugated targeting moieties. In some embodiments, the two or more conjugated targeting moieties are the same. In some embodiments, the two or more conjugated targeting moieties are different. In some embodiments, provided oligonucleotides comprise no more than one targeting moiety. In some embodiments, oligonucleotides of a provided composition comprise different types of conjugated targeting moieties. In some embodiments, oligonucleotides of a provided composition comprise the same type of targeting moieties.

(1889) Targeting moieties can be conjugated to oligonucleotides optionally through linkers. Various types of linkers in the art can be utilized in accordance of the present disclosure. In some embodiments, a linker comprises a phosphate group, which can, for example, be used for conjugating targeting moieties through chemistry similar to those employed in oligonucleotide synthesis. In some embodiments, a linker comprises an amide, ester, or ether group. In some embodiments, a linker is L.sup.M. In some embodiments, a linker has the structure of -L-. Targeting moieties can be conjugated through either the same or different linkers compared to lipids.

(1890) Targeting moieties, optionally through linkers, can be conjugated to oligonucleotides at various suitable locations. In some embodiments, targeting moieties are conjugated through the 5-OH group. In some embodiments, targeting moieties are conjugated through the 3-OH group. In some embodiments, targeting moieties are conjugated through one or more sugar moieties. In some embodiments, targeting moieties are conjugated through one or more bases. In some embodiments, targeting moieties are incorporated through one or more internucleotidic linkages. In some embodiments, an oligonucleotide may contain multiple conjugated targeting moieties which are independently conjugated through its 5-OH, 3-OH, sugar moieties, base moieties and/or internucleotidic linkages. Targeting moieties and lipids can be conjugated either at the same, neighboring and/or separated locations. In some embodiments, a targeting moiety is conjugated at one end of an oligonucleotide, and a lipid is conjugated at the other end.

(1891) In some embodiments, a targeting moiety interacts with a protein on the surface of targeted cells. In some embodiments, such interaction facilitates internalization into targeted cells. In some embodiments, a targeting moiety comprises a sugar moiety. In some embodiments, a targeting moiety comprises a polypeptide moiety. In some embodiments, a targeting moiety comprises an antibody. In some embodiments, a targeting moiety is an antibody. In some embodiments, a targeting moiety comprises an inhibitor. In some embodiments, a targeting moiety is a moiety from a small molecule inhibitor. In some embodiments, an inhibitor is an inhibitor of a protein on the surface of targeted cells. In some embodiments, an inhibitor is a carbonic anhydrase inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase inhibitor expressed on the surface of target cells. In some embodiments, a carbonic anhydrase is I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV or XVI. In some embodiments, a carbonic anhydrase is membrane bound. In some embodiments, a carbonic anhydrase is IV, IX, XII or XIV. In some embodiments, an inhibitor is for IV, IX, XII and/or XIV. In some embodiments, an inhibitor is a carbonic anhydrase III inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase IV inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase IX inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase XII inhibitor. In some embodiments, an inhibitor is a carbonic anhydrase XIV inhibitor. In some embodiments, an inhibitor comprises or is a sulfonamide (e.g., those described in Supuran, CT. Nature Rev Drug Discover 2008, 7, 168-181, which sulfonamides are incorporated herein by reference). In some embodiments, an inhibitor is a sulfonamide. In some embodiments, targeted cells are muscle cells.

(1892) In some embodiments, a targeting moiety is R.sup.LD or R.sup.CD or R.sup.TD as defined and described in the present disclosure. In some embodiments, R.sup.CD comprises or is

(1893) ##STR00798##
In some embodiments, R.sup.CD comprises or is

(1894) ##STR00799##
In some embodiments, R.sup.CD comprises or is

(1895) ##STR00800##
In some embodiments, R.sup.TD is a sulfonamide moiety as described in the present disclosure. In some embodiments, R.sup.TD comprises or is

(1896) ##STR00801##
In some embodiments, R.sup.TD or R.sup.CD comprises or is

(1897) ##STR00802##
In some embodiments, R.sup.TD or R.sup.CD comprises or is

(1898) ##STR00803##
In some embodiments, R.sup.TD comprises or is

(1899) ##STR00804##
In some embodiments, R.sup.TD or R.sup.CD comprises or is

(1900) ##STR00805##
In some embodiments, R.sup.TD or R.sup.CD comprises or is

(1901) ##STR00806##
In some embodiments, R.sup.TD comprises or is

(1902) ##STR00807##
In some embodiments, R.sup.TD comprises or is

(1903) ##STR00808##
In some embodiments, R.sup.TD or R.sup.CD comprises or is

(1904) ##STR00809##
In some embodiments, R.sup.TD or R.sup.CD comprises or is

(1905) ##STR00810##
In some embodiments, R.sup.TD comprises or is

(1906) ##STR00811##
In some embodiments, R.sup.TD comprises or is

(1907) ##STR00812##
In some embodiments, R.sup.LD is a targeting moiety that comprises or is a lipid moiety. In some embodiments, X is O. In some embodiments, X is S.

(1908) In some embodiments, the present disclosure provides technologies (e.g., reagents, methods, etc.) for conjugating various moieties to oligonucleotide chains. In some embodiments, the present disclosure provides technologies for conjugating targeting moiety to oligonucleotide chains. In some embodiments, the present disclosure provides acids comprising targeting moieties for conjugation, e.g., R.sup.LDCOOH. In some embodiments, the present disclosure provides linkers for conjugation, e.g., L.sup.LD. A person having ordinary skill in the art understands that many known and widely practiced technologies can be utilized for conjugation with oligonucleotide chains in accordance with the present disclosure. In some embodiments, a provided acid is

(1909) ##STR00813##
In some embodiments, a provided acid is

(1910) ##STR00814##
In some embodiments, a provided acid is

(1911) ##STR00815##

(1912) In some embodiments, a provided acid is

(1913) ##STR00816##
In some embodiments, a provided acid is a fatty acid, which can provide a lipid moiety as a targeting moiety. In some embodiments, the present disclosure provides methods and reagents for preparing such acids.

(1914) In some embodiments, an additional chemical moiety, e.g., one comprising a guanidine moiety, may be incorporated into an oligonucleotide to improve one or more properties and/or activities. In some embodiments, such an additional chemical moiety is useful for improving delivery. In some embodiments, an additional chemical moiety comprises one or more group having the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein. In some embodiments, an additional chemical moiety comprises one or more group having the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein. In some embodiments, such a chemical moiety has the structure of formula R.sup.1-[-L-L.sup.P]n-, wherein each L.sup.P independently has the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2 as described herein, and each other variable is independently as described herein. In some embodiments, R.sup.1 is OH. In some embodiments, R.sup.1 is H. In some embodiments, each L is independently optionally substituted bivalent C.sub.1-10 aliphatic. In some embodiments, each L is independently (CH.sub.2).sub.3 alkylene. In some embodiments, each L is independently C.sub.1-6 alkylene. In some embodiments, each L.sup.P is independently n001

(1915) ##STR00817##
In some embodiments, an additional chemical moiety is

(1916) ##STR00818##
In some embodiments, an additional chemical moiety is bonded to 5-end carbon of an oligonucleotide chain. In some embodiments, it may be incorporated, e.g., using reagents including those illustrated below:

(1917) ##STR00819##
In some embodiments, an additional chemical moiety may be linked to an oligonucleotide chain through a cleavable group, e.g., a phosphate group, to an oligonucleotide chain (e.g., at the 5-end carbon):

(1918) ##STR00820##
In some embodiments, L is a sugar moiety as described herein. For example, in some embodiments, L is

(1919) ##STR00821##
In some embodiments, an additional chemical moiety is

(1920) ##STR00822##
In some embodiments, it is bonded to 5-end carbon of an oligonucleotide chain. In some embodiments, it may be incorporated, e.g., using reagents including those illustrated below:

(1921) ##STR00823##
In some embodiments, additional chemical moieties described herein may comprise one or more alkyl chain. In some embodiments, additional chemical moieties described herein may comprise one or more lipid moieties. Those skilled in the art appreciates that many other embodiments of L.sup.P, including neutral internucleotidic linkage moieties, may be utilized in additional chemical moieties, e.g., n009. In some embodiments, an additional chemical moiety is

(1922) ##STR00824##
In some embodiments, an additional chemical moiety is

(1923) ##STR00825##
As described herein, in some embodiments, an additional chemical moiety may be bonded to the 5-end carbon of an oligonucleotide chain. In some embodiments, an additional chemical moiety may be incorporated, e.g., using reagents including those illustrated below:

(1924) ##STR00826## ##STR00827##
Those skilled in the art will appreciate that many other technologies, including synthetic chemical technologies, can be utilized in accordance with the present disclosure to provide compounds, e.g., oligonucleotides, reagents for incorporating additional chemical moieties, etc.

(1925) In some embodiments, provided compounds, e.g., reagents, products (e.g., oligonucleotides, amidites, etc.) etc. are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97% or 99% pure. In some embodiments, the purity is at least 50%. In some embodiments, the purity is at least 75%. In some embodiments, the purity is at least 80%. In some embodiments, the purity is at least 85%. In some embodiments, the purity is at least 90%. In some embodiments, the purity is at least 95%. In some embodiments, the purity is at least 96%. In some embodiments, the purity is at least 97%. In some embodiments, the purity is at least 98%. In some embodiments, the purity is at least 99%.

(1926) Combination Therapy

(1927) In some embodiments, a subject is administered an additional treatment (including, but not limited to, a therapeutic agent or method) in additional to provided oligonucleotide or oligonucleotide composition, e.g., a composition comprising a DMD oligonucleotide. In some embodiments, a composition comprising a DMD oligonucleotide(s) (or two or more compositions, each comprising a DMD oligonucleotide) is administered to a patient along with an additional treatment.

(1928) In some embodiments, the present disclosure pertains to a method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising (a) administering to a subject susceptible thereto or suffering therefrom a composition comprising a provided oligonucleotide, and (b) administering to the subject an additional treatment which is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy. In some embodiments, an additional treatment is a composition comprising a second oligonucleotide.

(1929) In some embodiments, an additional treatment is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy by itself. In some embodiments, an additional treatment is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy when administered with a provided oligonucleotide.

(1930) In some embodiments, an additional treatment is administered to the subject prior to, after or simultaneously with a composition comprising a provided oligonucleotide, e.g., a provided DMD oligonucleotide. In some embodiments, a composition comprises both a DMD oligonucleotide(s) and an additional treatment. In some embodiments, a DMD oligonucleotide(s) and an additional treatment(s) are in separate compositions. In some embodiments, the present disclosure provides technologies (e.g., compositions, methods, etc.) for combination therapy, for example, with other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides and/or compositions may be used together with one or more other therapeutic agents. In some embodiments, provided compositions comprise provided oligonucleotides, and one or more other therapeutic agents. In some embodiments, the one or more other therapeutic agents may have one or more different targets, and/or one or more different mechanisms toward targets, when compared to provided oligonucleotides in the composition. In some embodiments, a therapeutic agent is an oligonucleotide. In some embodiments, a therapeutic agent is a small molecule drug. In some embodiments, a therapeutic agent is a protein. In some embodiments, a therapeutic agent is an antibody. A number of therapeutic agents may be utilized in accordance with the present disclosure. For example, oligonucleotides for DMD may be used together with one or more therapeutic agents that modulate utrophin production (utrophin modulators). In some embodiments, a utrophin modulator promotes production of utrophin. In some embodiments, a utrophin modulator is ezutromid. In some embodiments, a utrophin modulator is

(1931) ##STR00828##
or a pharmaceutically acceptable salt thereof. In some embodiments, provided oligonucleotides or compositions thereof are administered prior to, concurrently with, or subsequent to one or more other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides or compositions thereof are administered concurrently with one or more other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides or compositions thereof are administered prior to one or more other therapeutic agents and/or medical procedures. In some embodiments, provided oligonucleotides or compositions thereof are administered subsequent to one or more other therapeutic agents and/or medical procedures. In some embodiments, provide compositions comprise one or more other therapeutic agents.

(1932) In some embodiments, a composition comprising a DMD oligonucleotide is co-administered with an additional agent in order to improve skipping of a DMD exon of interest. In some embodiments, an additional agent is an antibody, oligonucleotide, protein or small molecule. In some embodiments, an additional agent interferes with a protein involved in splicing. In some embodiments, an additional agent interferes with a protein involved in splicing, wherein the protein is a SR protein.

(1933) In some embodiments, an additional agent interferes with a protein involved in splicing, wherein the protein is a SR protein, which contains a protein domain with one or more long repeats of serine (S) and arginine (R) amino acid residues. SR proteins are reportedly heavily phosphorylated in cells and are involved in constitutive and alternative splicing. Long et al. 2009 Biochem. J. 417: 15-27; Shepard et al. 2009 Genome Biol. 10: 242. In some embodiments, an additional agent is a chemical compound that inhibits or decreases a SR protein kinase. In some embodiments, a chemical compound that inhibits or decreases a SR protein kinase is SRPIN340. SRPIN340 is reported in, for example, Fukuhura et al. 2006 Proc. Natl. Acad. Sci. USA 103: 11329-11333. In some embodiments, a chemical compound is a kinase inhibitor specific for Cdc-like kinases (Clks) that are also able to phosphorylate SR proteins. In some embodiments, a kinase inhibitor specific for Cdc-like kinases (Clks) that are also able to phosphorylate SR proteins is TG003. TG003 reportedly affected splicing both in vitro and in vivo. Nowak et al. 2010 J. Biol. Chem. 285: 5532-5540; Muraki et al. 2004 J. Biol. Chem. 279: 24246-24254; Yomoda et al. 2008 Genes Cells 13: 233-244; and Nishida et al. 2011 Nat Commun. 2: 308.

(1934) In some embodiments, in a patient afflicted with muscular dystrophy, muscle tissue is replaced by fat and connective tissue, and affected muscles may look larger due to increased fat content, a condition known as pseudohypertrophy. In some embodiments, a composition comprising a DMD oligonucleotide(s) is administered along with a treatment which reduces or prevents development of fat or fibrous or connective tissue, or replacement of muscle tissue by fat or fibrous or connective tissue.

(1935) In some embodiments, a composition comprising a DMD oligonucleotide(s) is administered along with a treatment which reduces or prevents development of fat or fibrous or connective tissue, or replacement of muscle tissue by fat or fibrous or connective tissue, wherein the treatment is an antibody to connective tissue growth factor (CTGF), a central mediator of fibrosis (e.g., FG-3019). In some embodiments, a composition comprising a DMD oligonucleotide(s) is administered along with an agent which reduces the fat content of the human body.

(1936) Additional treatments incude: slowing the progression of the disease by immune modulators (eg, steroids and transforming growth factor -beta inhibitors), inducing or introducing proteins that may compensate for dystrophin deficiency in the myofiber (eg, utrophin, biglycan, and laminin), or bolstering the muscle's regenerative response (eg, myostatin and activin 2B).

(1937) In some embodiments, an additional treatment is a small molecule capable of restoring normal balance of calcium within muscle cells.

(1938) In some embodiments, an additional treatment is a small molecule capable of restoring normal balance of calcium within muscle cells by correcting the activity of a type of channel called the ryanodine receptor calcium channel complex (RyR). In some embodiments, such a small molecule is Rycal ARM210 (ARMGO Pharma, Tarry Town, NY).

(1939) In some embodiments, an additional treatment is a flavonoid.

(1940) In some embodiments, an additional treatment is a flavonoid such as Epicatechin. Epicatechin is a flavonoid found in dark chocolate harvested from the cacao tree which has been reported in animals and humans to increase the production of new mitochondria in heart and muscle (e.g., mitochondrial biogenesis) while concurrently stimulating the regeneration of muscle tissue.

(1941) In some embodiments, an additional treatment is follistatin gene therapy.

(1942) In some embodiments, an additional treatment is adeno-associated virus delivery of follistatin 344 to increase muscle strength and prevent muscle wasting and fibrosis.

(1943) In some embodiments, an additional treatment is glucocorticoid.

(1944) In some embodiments, an additional treatment is prednisone.

(1945) In some embodiments, an additional treatment is deflazacort.

(1946) In some embodiments, an additional treatment is vamorolone (VBP15).

(1947) In some embodiments, an additional treatment is delivery of an exogenous Dystrophin gene or synthetic version or portion thereof, such as a microdystrophin gene.

(1948) In some embodiments, an additional treatment is delivery of an exogenous Dystrophin gene or portion thereof, such as a microdystrophin gene, such as SGT-001, an adeno-associated viral (AAV) vector -mediated gene transfer system for delivery of a synthetic dystrophin gene or microdystrophin (Solid BioSciences, Cambridge, Mass.).

(1949) In some embodiments, an additional treatment is stem cell treatment.

(1950) In some embodiments, an additional treatment is a steroid.

(1951) In some embodiments, an additional treatment is a corticosteroid.

(1952) In some embodiments, an additional treatment is prednisone.

(1953) In some embodiments, an additional treatment is a beta-2 agonist.

(1954) In some embodiments, an additional treatment is an ion channel inhibitor.

(1955) In some embodiments, an additional treatment is a calcium channel inhibitor which is a xanthin. In some embodiments, an additional treatment is a calcium channel inhibitor which is methylxanthine. In some embodiments, an additional treatment is a calcium channel inhibitor which is pentoxifylline. In some embodiments, an additional treatment is a calcium channel inhibitor which is a methylxanthine derivative selected from: pentoxifylline, furafylline, lisofylline, propentofylline, pentifylline, theophylline, torbafylline, albifylline, enprofylline and derivatives thereof.

(1956) In some embodiments, an additional treatment is a treatment for heart disease or cardiovascular disease.

(1957) In some embodiments, an additional treatment is a calcium channel inhibitor.

(1958) In some embodiments, an additional treatment is a blood pressure medicine.

(1959) In some embodiments, an additional treatment is surgery.

(1960) In some embodiments, an additional treatment is surgery to fix shortened muscles, straighten the spine, or treat a heart or lung problem.

(1961) In some embodiments, an additional treatment is a brace, walker, standing walker, or other mechanical aid for walking.

(1962) In some embodiments, an additional treatment is exercise and/or physical therapy.

(1963) In some embodiments, an additional treatment is assisted ventilation.

(1964) In some embodiments, an additional treatment is anticonvulsant, immunosuppressant or treatment for constipation.

(1965) In some embodiments, an additional treatment is an inhibitor of NF-B.

(1966) In some embodiments, an additional treatment comprises salicylic acid and/or docosahexaenoic acid (DHA).

(1967) In some embodiments, an additional treatment is edasalonexent (CAT-1004, Catabasis), a conjugate of salicylic acid and docosahexaenoic acid (DHA).

(1968) In some embodiments, an additional treatment is a cell-based therapeutic.

(1969) In some embodiments, an additional treatment is comprises allogeneic cardiosphere-derived cells.

(1970) In some embodiments, an additional treatment is CAP-1002 (Capricor).

(1971) Certain Embodiments of Variables

(1972) Embodiments of variables are extensive described in the present disclosure. Those skilled in the art appreciate that an embodiment described for one variable may be optionally and independently combined with embodiments for other variables, and such combinations, wherever and whenever appropriate, are within the scope of the present disclosure. Embodiments of a variable (e.g. R) given when describing one variable that can be such variable (e.g., R.sup.1, which can be R) are generally applicable to other variables that can be the same variable (e.g., R.sup.s, which can be R). Various embodiments of many variables are also described in other sections of the present disclosure.

(1973) In some embodiments, P.sup.L is P(W). In some embodiments, P.sup.L is P. In some embodiments, P.sup.L is a chiral P(P*). In some embodiments, P.sup.L is P.fwdarw.B(R).sub.3.

(1974) In some embodiments, W is O. In some embodiments, W is S. In some embodiments, W is Se. In some embodiments, W is N(-L-R.sup.5).

(1975) In some embodiments, X is O. In some embodiments, X is S. In some embodiments, X is N(-L-R.sup.5). In some embodiments, -L-R.sup.5 is R, which is taken together with a R group of -L-R.sup.1 (e.g., a C(R) in L) to form a double bond or a ring as described in the present disclosure. In some embodiments, X is L.

(1976) In some embodiments, Y is O. In some embodiments, Y is S. In some embodiments, Z is O. In some embodiments, Z is S. In some embodiments, Y is O and Z is O.

(1977) In some embodiments, W is O, Y is O and Z is O. In some embodiments, W is S, Y is O and Z is O.

(1978) In some embodiments, R.sup.1 is H. In some embodiments, R.sup.1 is -L-R. In some embodiments, R.sup.1 is halogen. In some embodiments, R.sup.1 is CN. In some embodiments, R.sup.1 is NO.sub.2. In some embodiments, R.sup.1 is -L-Si(R).sub.3. In some embodiments, R.sup.1 is OR. In some embodiments, R.sup.1 is SR. In some embodiments, R.sup.1 is N(R).sub.2.

(1979) In some embodiments, R.sup.1 is R as described in the present disclosure.

(1980) In some embodiments, X-L-R.sup.1 comprises or is an optionally substituted moiety of a chiral auxiliary (e.g., HX-L-R.sup.1 is an optionally substituted (e.g., capped) chiral auxiliary), e.g., as used in chirally controlled oligonucleotide synthesis, such as those described in US20150211006, US 20150211006, WO 2017015555, WO 2017015575, WO 2017062862, or WO 2017160741, chiral auxiliaries of each of which are incorporated herein by reference.

(1981) In some embodiments, X-L-R.sup.1 is OR. In some embodiments, X-L-R.sup.1 is OH. In some embodiments, X-L-R.sup.1 is SR. In some embodiments, X-L-R.sup.1 is SH.

(1982) In some embodiments, X-L-R.sup.1 is R. In some embodiments, R is CH.sub.3. In some embodiments, R is CH.sub.2CH.sub.3. In some embodiments, R is CH.sub.2CH.sub.2CH.sub.3. In some embodiments, R is CH.sub.2OCH.sub.3. In some embodiments, R is CH.sub.3CH.sub.2OCH.sub.2. In some embodiments, R is PhCH.sub.2OCH.sub.2. In some embodiments, R is HCCCH.sub.2. In some embodiments, R is H.sub.3CCCCH.sub.2. In some embodiments, R is CH.sub.2CHCH.sub.2. In some embodiments, R is CH.sub.3SCH.sub.2. In some embodiments, R is CH.sub.2COOCH.sub.3. In some embodiments, R is CH.sub.2COOCH.sub.2CH.sub.3. In some embodiments, R is CH.sub.2CONHCH.sub.3.

(1983) In some embodiments, X-L-R.sup.1 is comprises a guanidine moiety. In some embodiments, X-L-R.sup.1 is or comprises

(1984) ##STR00829##
In some embodiments, X-L-R.sup.1 is -L-W.sup.z, wherein W.sup.z is selected from

(1985) ##STR00830##
wherein R is R and n is 0-15. In some embodiments, R and R are independently

(1986) ##STR00831##
In some embodiments, L is OCH.sub.2CH.sub.2. In some embodiments, n is 0-3. In some embodiments, each R.sup.s is independently H, OCH.sub.3, F, CN, CH.sub.3, NO.sub.2, CF.sub.3, or OCF.sub.3. In some embodiments, R and R are the same. In some embodiments, R and R are different.

(1987) In some embodiments, In some embodiments, X-L-R.sup.1 is

(1988) ##STR00832##
wherein each R is independently as described in the present disclosure. In some embodiments, two R on two different nitrogen atoms are taken together to form an optionally substituted ring as described in the present disclosure. In some embodiments, a ring is saturated. In some embodiments, a ring is monocyclic. In some embodiments, a ring is 3-10 membered. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring has no additional ring heteroatoms in addition to the two nitrogen atoms.

(1989) In some embodiments, R.sup.5 is R.sup.1 as described in the present disclosure. In some embodiments, R.sup.5 is H. In some embodiments, R.sup.5 is R as described in the present disclosure.

(1990) In some embodiments, L is a bivalent optionally substituted methylene group. In some embodiments, L is CH.sub.2. In some embodiments, each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L.

(1991) In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched C.sub.1-30 aliphatic group, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, or C(O)O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C.sub.1-6 alkylene, C.sub.1-6 alkenylene, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, and C(O)O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, and C(O)O.

(1992) In some embodiments, L is a covalent bond. In some embodiments, L is optionally substituted bivalent C.sub.1-30 aliphatic. In some embodiments, L is optionally substituted bivalent C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from boron, oxygen, nitrogen, sulfur, phosphorus and silicon.

(1993) In some embodiments, aliphatic moieties, e.g. those of L, L.sup.s, L.sup.M, R, etc., either monovalent or bivalent or multivalent, and can contain any number of carbon atoms (before any optional substitution) within its range, e.g., C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25, C.sub.26, C.sub.27, C.sub.28, C.sub.29, C.sub.30, etc. In some embodiments, heteroaliphatic moieties, e.g. those of L, R, etc., either monovalent or bivalent or multivalent, and can contain any number of carbon atoms (before any optional substitution) within its range, e.g., C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25, C.sub.26, C.sub.27, C.sub.28, C.sub.29, C.sub.30, etc.

(1994) In some embodiments, a methylene unit of a linker, e.g., L, L.sup.s, L.sup.M, etc., is replaced with Cy, wherein Cy is as described in the present disclosure. In some embodiments, one or more methylene unit is optionally and independently substituted with O, S, N(R), C(O), S(O), S(O).sub.2, P(O)(OR), P(O)(SR), P(S)(OR), or P(S)(OR). In some embodiments, a methylene unit is replaced with O. In some embodiments, a methylene unit is replaced with S. In some embodiments, a methylene unit is replaced with N(R). In some embodiments, a methylene unit is replaced with C(O). In some embodiments, a methylene unit is replaced with S(O). In some embodiments, a methylene unit is replaced with S(O).sub.2. In some embodiments, a methylene unit is replaced with P(O)(OR). In some embodiments, a methylene unit is replaced with P(O)(SR). In some embodiments, a methylene unit is replaced with P(O)(R). In some embodiments, a methylene unit is replaced with P(O)(NR). In some embodiments, a methylene unit is replaced with P(S)(OR). In some embodiments, a methylene unit is replaced with P(S)(SR). In some embodiments, a methylene unit is replaced with P(S)(R). In some embodiments, a methylene unit is replaced with P(S)(NR). In some embodiments, a methylene unit is replaced with P(R). In some embodiments, a methylene unit is replaced with P(OR). In some embodiments, a methylene unit is replaced with P(SR). In some embodiments, a methylene unit is replaced with P(NR). In some embodiments, a methylene unit is replaced with P(OR)[B(R).sub.3]. In some embodiments, one or more methylene unit is optionally and independently substituted with O, S, N(R), C(O), S(O), S(O).sub.2, P(O)(OR), P(O)(SR), P(S)(OR), or P(S)(OR). In some embodiments, a methylene unit is replaced with OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, each of which may independently be an internucleotidic linkage.

(1995) In some embodiments, L or L.sup.s (e.g., when L.sup.s is L), e.g., when connected to R.sup.s or a sugar ring, is CH.sub.2. In some embodiments, L is C(R).sub.2, wherein at least one R is not hydrogen. In some embodiments, L is CHR. In some embodiments, R is hydrogen. In some embodiments, L is CHR, wherein R is not hydrogen. In some embodiments, C of CHR is chiral. In some embodiments, L is (R)CHR, wherein C of CHR is chiral. In some embodiments, L is (S)CHR, wherein C of CHR is chiral. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is optionally substituted C.sub.1-5 aliphatic. In some embodiments, R is optionally substituted C.sub.1-5 alkyl. In some embodiments, R is optionally substituted C.sub.1-4 aliphatic. In some embodiments, R is optionally substituted C.sub.1-4 alkyl. In some embodiments, R is optionally substituted C.sub.1-3 aliphatic. In some embodiments, R is optionally substituted C.sub.1-3 alkyl. In some embodiments, R is optionally substituted C.sub.2 aliphatic. In some embodiments, R is optionally substituted methyl. In some embodiments, R is C.sub.1-6 aliphatic. In some embodiments, R is C.sub.1-6 alkyl. In some embodiments, R is C.sub.1-5 aliphatic. In some embodiments, R is C.sub.1-5 alkyl. In some embodiments, R is C.sub.1-4 aliphatic. In some embodiments, R is C.sub.1-4 alkyl. In some embodiments, R is C.sub.1-3 aliphatic. In some embodiments, R is C.sub.1-3 alkyl. In some embodiments, R is C.sub.2 aliphatic. In some embodiments, R is methyl. In some embodiments, R is C.sub.1-6 haloaliphatic. In some embodiments, R is C.sub.1-6 haloalkyl. In some embodiments, R is C.sub.1-5 haloaliphatic. In some embodiments, R is C.sub.1-5 haloalkyl. In some embodiments, R is C.sub.1-4 haloaliphatic. In some embodiments, R is C.sub.1-4 haloalkyl. In some embodiments, R is C.sub.1-3 haloaliphatic. In some embodiments, R is C.sub.1-3 haloalkyl. In some embodiments, R is C.sub.2 haloaliphatic. In some embodiments, R is methyl substituted with one or more halogen. In some embodiments, R is CF.sub.3. In some embodiments, L is optionally substituted CHCH. In some embodiments, L is optionally substituted (E)-CHCH. In some embodiments, L is optionally substituted (Z)CHCH. In some embodiments, L is CC.

(1996) In some embodiments, L comprises at least one phosphorus atom. In some embodiments, at least one methylene unit of L is replaced with P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O.

(1997) In some embodiments, L is bonded to a phosphorus of an linkage (e.g., when X is a covalent bond), e.g., the phosphorus of a linkage having formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, such an linkage is an internucleotidic linkage. In some embodiments, such an linkage is a chirally controlled internucleotidic linkage.

(1998) In some embodiments, L is Cy. In some embodiments, L is CC.

(1999) In some embodiments, Lis a bivalent, optionally substituted, linear or branched C.sub.1-30 aliphatic group wherein one or more methylene units are optionally and independently replaced as described in the present disclosure. In some embodiments, Lis a bivalent, optionally substituted, linear or branched C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms wherein one or more methylene units are optionally and independently replaced as described in the present disclosure.

(2000) In some embodiments, a heteroaliphatic group in the present disclosure, e.g., of L, R (including any variable that can be R), etc., comprises a

(2001) ##STR00833##
moiety. In some embodiments, N is directly bonded to a phosphorus atom. In some embodiments, a heteroaliphatic group comprises a

(2002) ##STR00834##
moiety. In some embodiments, a heteroaliphatic group comprises

(2003) ##STR00835##
a moiety. In some embodiments, such a moiety is directly bonded to a phosphorus atom. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is isopropyl.

(2004) In some embodiments, Cy is optionally substituted bivalent monocyclic, bicyclic or polycyclic C.sub.3-20 cycloaliphatic. In some embodiments, Cy is optionally substituted bivalent monocyclic, bicyclic or polycyclic C.sub.6-20 aryl. In some embodiments, Cy is optionally substituted monocyclic, bicyclic or polycyclic 3-20 membered heterocyclyl ring having 1-5 heteroatoms. In some embodiments, Cy is optionally substituted monocyclic, bicyclic or polycyclic 5-20 membered heterocyclyl ring having 1-5 heteroatoms, wherein at least one heteroatom is oxygen. In some embodiments, Cy is 3-10 membered. In some embodiments, Cy is 3-membered. In some embodiments, Cy is 4-membered. In some embodiments, Cy- is 5-membered. In some embodiments, Cy is 6-membered. In some embodiments, Cy- is 7-membered. In some embodiments, Cy is 8-membered. In some embodiments, Cy is 9-membered. In some embodiments, Cy is 10-membered. In some embodiments, Cy is optionally substituted bivalent tetrahydrofuran ring. In some embodiments, Cy is an optionally substituted furanose moiety. In some embodiments, Cy is an optionally substituted bivalent 5-membered heteroaryl ring having 1-4 heteroatoms. In some embodiments, at least one heteroatom is nitrogen. In some embodiments, each heteroatom is nitrogen. In some embodiments, Cy is an optionally substituted bivalent triazole ring. In some embodiments, In some embodiments, Cy is optionally substituted

(2005) ##STR00836##
In some embodiments, Cy is

(2006) ##STR00837##
In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is isopropyl.

(2007) In some embodiments, Cy.sup.L is an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon. In some embodiments, Cy.sup.L is trivalent. In some embodiments, Cy.sup.L is tetravalent. In some embodiments, one or more CH in a moiety, e.g., L, L.sup.s, L.sup.M, etc. are independently substituted with a trivalent Cy.sup.L group. In some embodiments, one or more carbon atoms in a moiety, e.g., L, L.sup.s, L.sup.M, etc. are independently substituted with a tetravalent Cy.sup.L group. In some embodiments, one or more CH in a moiety, e.g., L, L.sup.s, L.sup.M, etc. are independently substituted with a trivalent Cy.sup.L group, and one or more carbon atoms in a moiety, e.g., L, L.sup.s, L.sup.M, etc. are independently substituted with a tetravalent Cy.sup.L group.

(2008) In some embodiments, Cy.sup.L is monocyclic. In some embodiments, Cy.sup.L is bicyclic. In some embodiments, Cy.sup.L is polycyclic.

(2009) In some embodiments, Cy.sup.L is saturated. In some embodiments, Cy.sup.L is partially unsaturated. In some embodiments, Cy.sup.L is aromatic. In some embodiments, Cy.sup.L is or comprises a saturated ring moiety. In some embodiments, Cy.sup.L is or comprises a partially unsaturated ring moiety. In some embodiments, Cy.sup.L is or comprises an aromatic ring moiety.

(2010) In some embodiments, Cy.sup.L is an optionally substituted C.sub.3-20 cycloaliphatic ring as described in the present disclosure (for example, those described for R but tetravalent). In some embodiments, a ring is an optionally substituted saturated C.sub.3-20 cycloaliphatic ring. In some embodiments, a ring is an optionally substituted partially unsaturated C.sub.3-20 cycloaliphatic ring. A cycloaliphatic ring can be of various sizes as described in the present disclosure. In some embodiments, a ring is 3, 4, 5, 6, 7, 8, 9, or 10-membered. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring is 8-membered. In some embodiments, a ring is 9-membered. In some embodiments, a ring is 10-membered. In some embodiments, a ring is an optionally substituted cyclopropyl moiety. In some embodiments, a ring is an optionally substituted cyclobutyl moiety. In some embodiments, a ring is an optionally substituted cyclopentyl moiety. In some embodiments, a ring is an optionally substituted cyclohexyl moiety. In some embodiments, a ring is an optionally substituted cycloheptyl moiety. In some embodiments, a ring is an optionally substituted cyclooctanyl moiety. In some embodiments, a cycloaliphatic ring is a cycloalkyl ring. In some embodiments, a cycloaliphatic ring is monocyclic. In some embodiments, a cycloaliphatic ring is bicyclic. In some embodiments, a cycloaliphatic ring is polycyclic. In some embodiments, a ring is a cycloaliphatic moiety as described in the present disclosure for R with more valences.

(2011) In some embodiments, Cy.sup.L is an optionally substituted 6-20 membered aryl ring. In some embodiments, a ring is an optionally substituted trivalent or tetravalent phenyl moiety. In some embodiments, a ring is a tetravalent phenyl moiety. In some embodiments, a ring is an optionally substituted naphthalene moiety. A ring can be of different size as described in the present disclosure. In some embodiments, an aryl ring is 6-membered. In some embodiments, an aryl ring is 10-membered. In some embodiments, an aryl ring is 14-membered. In some embodiments, an aryl ring is monocyclic. In some embodiments, an aryl ring is bicyclic. In some embodiments, an aryl ring is polycyclic. In some embodiments, a ring is an aryl moiety as described in the present disclosure for R with more valences.

(2012) In some embodiments, Cy.sup.L is an optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms, e.g., independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Cy.sup.L is an optionally substituted 5-20 membered heteroaryl ring having 1-10 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, Cy.sup.L is an optionally substituted 5-6 membered heteroaryl ring having 1-4 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, Cy.sup.L is an optionally substituted 5-membered heteroaryl ring having 1-4 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, Cy.sup.L is an optionally substituted 6-membered heteroaryl ring having 1-4 heteroatoms, e.g., independently selected from oxygen, nitrogen, and sulfur. In some embodiments, as described in the present disclosure, heteroaryl rings can be of various sizes and contain various numbers and/or types of heteroatoms. In some embodiments, a heteroaryl ring contains no more than one heteroatom. In some embodiments, a heteroaryl ring contains more than one heteroatom. In some embodiments, a heteroaryl ring contains no more than one type of heteroatom. In some embodiments, a heteroaryl ring contains more than one type of heteroatoms. In some embodiments, a heteroaryl ring is 5-membered. In some embodiments, a heteroaryl ring is 6-membered. In some embodiments, a heteroaryl ring is 8-membered. In some embodiments, a heteroaryl ring is 9-membered. In some embodiments, a heteroaryl ring is 10-membered. In some embodiments, a heteroaryl ring is monocyclic. In some embodiments, a heteroaryl ring is bicyclic. In some embodiments, a heteroaryl ring is polycyclic. In some embodiments, a heteroaryl ring is a nucleobase moiety, e.g., A, T, C, G, U, etc. In some embodiments, a ring is a heteroaryl moiety as described in the present disclosure for R with more valences. In some embodiments, as in linkers described in the present disclosure, Cy.sup.L is

(2013) In some embodiments, Cy.sup.L is a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Cy.sup.L is a 3-20 membered heterocyclyl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, a heterocyclyl ring is saturated. In some embodiments, a heterocyclyl ring is partially unsaturated. A heterocyclyl ring can be of various sizes as described in the present disclosure. In some embodiments, a ring is 3, 4, 5, 6, 7, 8, 9, or 10-membered. In some embodiments, a ring is 3-membered. In some embodiments, a ring is 4-membered. In some embodiments, a ring is 5-membered. In some embodiments, a ring is 6-membered. In some embodiments, a ring is 7-membered. In some embodiments, a ring is 8-membered. In some embodiments, a ring is 9-membered. In some embodiments, a ring is 10-membered. Heterocyclyl rings can contain various numbers and/or types of heteroatoms. In some embodiments, a heterocyclyl ring contains no more than one heteroatom. In some embodiments, a heterocyclyl ring contains more than one heteroatom. In some embodiments, a heterocyclyl ring contains no more than one type of heteroatom. In some embodiments, a heterocyclyl ring contains more than one type of heteroatoms. In some embodiments, a heterocyclyl ring is monocyclic. In some embodiments, a heterocyclyl ring is bicyclic. In some embodiments, a heterocyclyl ring is polycyclic. In some embodiments, a ring is a heterocyclyl moiety as described in the present disclosure for R with more valences.

(2014) As readily appreciated by a person having ordinary skill in the art, many suitable ring moieties are extensively described in and can be used in accordance with the present disclosure, for example, those described for R (which may have more valences for Cy.sup.L).

(2015) In some embodiments, Cy.sup.L is a sugar moiety in a nucleic acid. In some embodiments, Cy.sup.L is an optionally substituted furanose moiety. In some embodiments, Cy.sup.L is a pyranose moiety. In some embodiments, Cy.sup.L is an optionally substituted furanose moiety found in DNA. In some embodiments, Cy.sup.L is an optionally substituted furanose moiety found in RNA. In some embodiments, Cy.sup.L is an optionally substituted 2-deoxyribofuranose moiety. In some embodiments, Cy.sup.L is an optionally substituted ribofuranose moiety. In some embodiments, substitutions provide sugar modifications as described in the present disclosure. In some embodiments, an optionally substituted 2-deoxyribofuranose moiety and/or an optionally substituted ribofuranose moiety comprise substitution at a 2-position. In some embodiments, a 2-position is a 2-modification as described in the present disclosure. In some embodiments, a 2-modification is F. In some embodiments, a 2-modification is OR, wherein R is as described in the present disclosure. In some embodiments, R is not hydrogen. In some embodiments, Cy.sup.L is a modified sugar moiety, such as a sugar moiety in LNA, alpha-L-LNA or GNA. In some embodiments, Cy.sup.L is a modified sugar moiety, such as a sugar moiety in ENA. In some embodiments, Cy.sup.L is a terminal sugar moiety of an oligonucleotide, connecting an internucleotidic linkage and a nucleobase. In some embodiments, Cy.sup.L is a terminal sugar moiety of an oligonucleotide, for example, when that terminus is connected to a solid support optionally through a linker. In some embodiments, Cy.sup.L is a sugar moiety connecting two internucleotidic linkages and a nucleobase. Example sugars and sugar moieties are extensively described in the present disclosure.

(2016) In some embodiments, Cy.sup.L is a nucleobase moiety. In some embodiments, a nucleobase is a natural nucleobase, such as A, T, C, G, U, etc. In some embodiments, a nucleobase is a modified nucleobase. In some embodiments, Cy.sup.L is optionally substituted nucleobase moiety selected from A, T, C, G, U, and 5mC. Example nucleobases and nucleobase moieties are extensively described in the present disclosure.

(2017) In some embodiments, two Cy.sup.L moieties are bonded to each other, wherein one Cy.sup.L is a sugar moiety and the other is a nucleobase moiety. In some embodiments, such a sugar moiety and nucleobase moiety forms a nucleoside moiety. In some embodiments, a nucleoside moiety is natural. In some embodiments, a nucleoside moiety is modified. In some embodiments, Cy.sup.L is an optionally substituted natural nucleoside moiety selected from adenosine, 5-methyluridine, cytidine, guanosine, uridine, 5-methylcytidine, 2-deoxyadenosine, thymidine, 2-deoxycytidine, 2-deoxyguanosine, 2-deoxyuridine, and 5-methyl-2-deoxycytidine. Example nucleosides and nucleosides moieties are extensive described in the present disclosure.

(2018) Ring A.sup.L can be either be monovalent, bivalent or polyvalent. In some embodiments, Ring A.sup.L is monovalent (e.g., when g is 0 and no substitution). In some embodiments, Ring A.sup.L is bivalent. In some embodiments, Ring A.sup.L is polyvalent. In some embodiments, Ring A.sup.L is bivalent and is Cy. In some embodiments, Ring A.sup.L is an optionally substituted bivalent triazole ring. In some embodiments, Ring A.sup.L is trivalent and is Cy.sup.L. In some embodiments, Ring A.sup.L is tetravalent and is Cy.sup.L. In some embodiments, Ring A.sup.L is optionally substituted

(2019) ##STR00838##

(2020) In some embodiments, X-L-R.sup.1 is optionally substituted alkynyl. In some embodiments, X-L-R.sup.1 is CCH. In some embodiments, an alkynyl group, e.g., CCH, can react with a number of reagents through various reactions to provide further modifications. For example, in some embodiments, an alkynyl group can react with azides through click chemistry. In some embodiments, an azide has the structure of R.sup.1N.sub.3.

(2021) In some embodiments, each R.sup.s is independently H, halogen, CN, N.sub.3, NO, NO.sub.2, -L-R, -L-Si(R).sub.3, -L-OR, -L-SR, -L-N(R).sub.2, O-L-R, O-L-Si(R).sub.3, O-L-OR, O-L.sup.sSR, or O-L.sup.sN(R).sub.2 as described in the present disclosure.

(2022) In some embodiments, R.sup.s is R, wherein R is as described in the present disclosure. In some embodiments, R.sup.s is R, wherein R is as described in the present disclosure. In some embodiments, R.sup.s is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.s is methyl. In some embodiments, R.sup.s is optionally substituted C.sub.1-30 heteroaliphatic. In some embodiments, R.sup.s comprises one or more silicon atoms. In some embodiments, R.sup.s is CH.sub.2Si(Ph).sub.2CH.sub.3.

(2023) In some embodiments, R.sup.s is -L-R. In some embodiments, R.sup.s is -L-R wherein -L- is a bivalent, optionally substituted C.sub.1-30 heteroaliphatic group. In some embodiments, R.sup.s is CH.sub.2Si(Ph).sub.2CH.sub.3.

(2024) In some embodiments, R.sup.s is F. In some embodiments, R.sup.s is Cl. In some embodiments, R.sup.s is Br. In some embodiments, R.sup.s is I. In some embodiments, R.sup.s is CN. In some embodiments, R.sup.s is N.sub.3. In some embodiments, R.sup.s is NO. In some embodiments, R.sup.s is NO.sub.2. In some embodiments, R.sup.s is -L-Si(R).sub.3. In some embodiments, R.sup.s is Si(R).sub.3. In some embodiments, R.sup.s is -L-R. In some embodiments, R.sup.s is R. In some embodiments, R.sup.s is -L-OR. In some embodiments, R.sup.s is OR. In some embodiments, R.sup.s is -L-SR. In some embodiments, R.sup.s is SR. In some embodiments, R.sup.s is -L-N(R).sub.2. In some embodiments, R.sup.s is N(R).sub.2. In some embodiments, R.sup.s is O-L-R. In some embodiments, R.sup.s is O-L-Si(R).sub.3. In some embodiments, R.sup.s is O-L-OR. In some embodiments, R.sup.s is O-L-SR. In some embodiments, R.sup.s is O-L-N(R).sub.2. In some embodiments, R.sup.s is a 2-modification as described in the present disclosure. In some embodiments, R.sup.s is OR, wherein R is as described in the present disclosure. In some embodiments, R.sup.s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.s is OMe. In some embodiments, R.sup.s is OCH.sub.2CH.sub.2OMe. In some embodiments, R.sup.s is R.sup.1s, R.sup.2s, R.sup.3s, R.sup.4s, or R.sup.5s as described in the present disclosure.

(2025) In some embodiments, g is 0-20. In some embodiments, g is 1-20. In some embodiments, g is 1-5. In some embodiments, g is 1. In some embodiments, g is 2. In some embodiments, g is 3. In some embodiments, g is 4. In some embodiments, g is 5. In some embodiments, g is 6. In some embodiments, g is 7. In some embodiments, g is 8. In some embodiments, g is 9. In some embodiments, g is 10. In some embodiments, g is 11. In some embodiments, g is 12. In some embodiments, g is 13. In some embodiments, g is 14. In some embodiments, g is 15. In some embodiments, g is 16. In some embodiments, g is 17. In some embodiments, g is 18. In some embodiments, g is 19. In some embodiments, g is 20.

(2026) In some embodiments,

(2027) ##STR00839##
is

(2028) ##STR00840##
In some embodiments,

(2029) ##STR00841##
is

(2030) ##STR00842##
In some embodiments,

(2031) ##STR00843##
is

(2032) ##STR00844##

(2033) In some embodiments, each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms, e.g., independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, Ring A is an optionally substituted ring, which ring is as described in the present disclosure. In some embodiments, Ring A comprises an oxygen ring atom. In some embodiments, Ring A is or comprises a ring of a sugar moiety. In some embodiments, a ring is

(2034) ##STR00845##
In some embodiments, a ring is

(2035) ##STR00846##
In some embodiments, a ring is

(2036) ##STR00847##
In some embodiments, a ring is a bicyclic ring, e.g., found in a sugar moiety of LNA.

(2037) In some embodiments, a sugar unit is of the structure

(2038) ##STR00848##
wherein each variable is independently as described in the present disclosure. In some embodiments, a nucleoside unit is of the structure

(2039) ##STR00849##
wherein each variable is independently as described in the present disclosure.

(2040) In some embodiments, L.sup.s is C(R.sup.5s).sub.2 and

(2041) ##STR00850##
is as described in the present disclosure. In some embodiments, L.sup.s is CHR.sup.5s and

(2042) ##STR00851##
is as described in the present disclosure. In some embodiments, L.sup.s is C(R).sub.2 and

(2043) ##STR00852##
is as described in the present disclosure. In some embodiments, L.sup.s is CHR and

(2044) ##STR00853##
is as described in the present disclosure.

(2045) In some embodiments,

(2046) ##STR00854##
is

(2047) ##STR00855##
BA is connected at Cl, and each of R.sup.1s, R.sup.2s, R.sup.3s, R.sup.4s and R.sup.5s is independently as described in the present disclosure. In some embodiments,

(2048) ##STR00856##
is

(2049) ##STR00857##
wherein R.sup.2s is as described in the present disclosure. In some embodiments,

(2050) ##STR00858##
is

(2051) ##STR00859##
wherein R.sup.2s is not-OH. In some embodiments,

(2052) ##STR00860##
is

(2053) ##STR00861##
wherein R.sup.2s and R.sup.4s are R, and the two R groups are taken together with their intervening atoms to form an optionally substituted ring. In some embodiments,

(2054) ##STR00862##
or Ring A, is optionally substituted

(2055) ##STR00863##
In some embodiments,

(2056) ##STR00864##
or Ring A, is

(2057) ##STR00865##
In some embodiments,

(2058) ##STR00866##
or Ring A, is

(2059) ##STR00867##

(2060) In some embodiments, each of R.sup.1s, R.sup.2s, R.sup.3s, R.sup.4s, and R.sup.5s is independently R.sup.s, wherein R.sup.s is as described in the present disclosure.

(2061) In some embodiments, R.sup.1s is R.sup.s wherein R.sup.s is as described in the present disclosure. In some embodiments, R.sup.1s is at 1-position (BA is at 1-position). In some embodiments, R.sup.1s is H. In some embodiments, R.sup.1s is F. In some embodiments, R.sup.1s is Cl. In some embodiments, R.sup.1s is Br. In some embodiments, R.sup.1s is I. In some embodiments, R.sup.1s is CN. In some embodiments, R.sup.1s is N.sub.3. In some embodiments, R.sup.1s is NO. In some embodiments, R.sup.1s is NO.sub.2. In some embodiments, R.sup.1s is -L-R. In some embodiments, R.sup.1s is R. In some embodiments, R.sup.1s is -L-OR. In some embodiments, R.sup.1s is OR. In some embodiments, R.sup.1s is -L-SR. In some embodiments, R.sup.1s is SR. In some embodiments, R.sup.1s is L-L-N(R).sub.2. In some embodiments, R.sup.1s is N(R).sub.2. In some embodiments, R.sup.1s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.1s is OMe. In some embodiments, R.sup.1s is -MOE. In some embodiments, R.sup.1s is hydrogen. In some embodiments, R.sup.s at one 1-position is hydrogen, and R.sup.s at the other 1-position is not hydrogen as described herein. In some embodiments, R.sup.s at both 1-positions are hydrogen. In some embodiments, R.sup.s at one 1-position is hydrogen, and the other 1-position is connected to an internucleotidic linkage. In some embodiments, R.sup.1s is F. In some embodiments, R.sup.1s is Cl. In some embodiments, R.sup.1s is Br. In some embodiments, R.sup.1s is I. In some embodiments, R.sup.1s is CN. In some embodiments, R.sup.1s is N.sub.3. In some embodiments, R.sup.1s is NO. In some embodiments, R.sup.1s is NO.sub.2. In some embodiments, R.sup.1s is -L-R. In some embodiments, R.sup.1s is R. In some embodiments, R.sup.1s is -L-OR. In some embodiments, R.sup.1s is OR. In some embodiments, R.sup.1s is -L-SR. In some embodiments, R.sup.1s is SR. In some embodiments, R.sup.1s is -L-N(R).sub.2. In some embodiments, R.sup.1s is N(R).sub.2. In some embodiments, R.sup.1s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.1s is OH. In some embodiments, R.sup.1s is OMe. In some embodiments, R.sup.1s is -MOE. In some embodiments, R.sup.1s is hydrogen. In some embodiments, one R.sup.1s at a 1-position is hydrogen, and the other R.sup.1s at the other 1-position is not hydrogen as described herein. In some embodiments, R.sup.1s at both 1-positions are hydrogen. In some embodiments, R.sup.1s is O-L-OR. In some embodiments, R.sup.1s is O-L-OR, wherein L is optionally substituted C.sub.1-6 alkylene, and R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1s is O-(optionally substituted C.sub.1-6 alkylene)-OR. In some embodiments, R.sup.15 is O-(optionally substituted C.sub.1-6 alkylene)-OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.1s is OCH.sub.2CH.sub.2OMe.

(2062) In some embodiments, R.sup.2s is R.sup.s wherein R.sup.s is as described in the present disclosure. In some embodiments, if there are two R.sup.2s at the 2-position, one R.sup.2s is H and the other is not. In some embodiments, R.sup.2s is at 2-position (BA is at 1-position). In some embodiments, R.sup.2s is H. In some embodiments, R.sup.2s is F. In some embodiments, R.sup.2s is Cl. In some embodiments, R.sup.2s is Br. In some embodiments, R.sup.2s is I. In some embodiments, R.sup.2s is CN. In some embodiments, R.sup.2s is N.sub.3. In some embodiments, R.sup.2s is NO. In some embodiments, R.sup.2s is NO.sub.2. In some embodiments, R.sup.2s is -L-R. In some embodiments, R.sup.2s is R. In some embodiments, R.sup.2s is -L-OR. In some embodiments, R.sup.2s is OR. In some embodiments, R.sup.2s is -L-SR. In some embodiments, R.sup.2s is SR. In some embodiments, R.sup.2s is L-L-N(R).sub.2. In some embodiments, R.sup.2s is N(R).sub.2. In some embodiments, R.sup.2s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.2s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.2s is OMe. In some embodiments, R.sup.2s is -MOE. In some embodiments, R.sup.2s is hydrogen. In some embodiments, R.sup.s at one 2-position is hydrogen, and R.sup.s at the other 2-position is not hydrogen as described herein. In some embodiments, R.sup.s at both 2-positions are hydrogen. In some embodiments, R.sup.s at one 2-position is hydrogen, and the other 2-position is connected to an internucleotidic linkage. In some embodiments, R.sup.2s is F. In some embodiments, R.sup.2s is Cl. In some embodiments, R.sup.2s is Br. In some embodiments, R.sup.2s is I. In some embodiments, R.sup.2s is CN. In some embodiments, R.sup.2s is N.sub.3. In some embodiments, R.sup.2s is NO. In some embodiments, R.sup.2s is NO.sub.2. In some embodiments, R.sup.2s is -L-R. In some embodiments, R.sup.2s is R. In some embodiments, R.sup.2s is -L-OR. In some embodiments, R.sup.2s is OR. In some embodiments, R.sup.2s is -L-SR. In some embodiments, R.sup.2s is SR. In some embodiments, R.sup.2s is -L-N(R).sub.2. In some embodiments, R.sup.2s is N(R).sub.2. In some embodiments, R.sup.2s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.2s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.2s is OH. In some embodiments, R.sup.2s is OMe. In some embodiments, R.sup.2s is -MOE. In some embodiments, R.sup.2s is hydrogen. In some embodiments, one R.sup.2s at a 2-position is hydrogen, and the other R.sup.2s at the other 2-position is not hydrogen as described herein. In some embodiments, R.sup.2s at both 2-positions are hydrogen. In some embodiments, R.sup.2s is O-L-OR. In some embodiments, R.sup.2s is O-L-OR, wherein L is optionally substituted C.sub.1-6 alkylene, and R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.2s is O-(optionally substituted C.sub.1-6 alkylene)-OR. In some embodiments, R.sup.2s is O-(optionally substituted C.sub.1-6 alkylene)-OR, wherein R is optionally substituted C.sub.16 alkyl. In some embodiments, R.sup.2s is OCH.sub.2CH.sub.2OMe.

(2063) In some embodiments, R.sup.2s comprises a guanidine moiety. In some embodiments, R.sup.2s comprises

(2064) ##STR00868##
In some embodiments, R.sup.2s is -L-W.sup.z, wherein W.sup.z is selected from

(2065) ##STR00869##
wherein R is R and n is 0-15. In some embodiments, R and R are independently

(2066) ##STR00870##
In some embodiments, L is OCH.sub.2CH.sub.2. In some embodiments, n is 0-3. In some embodiments, each R.sup.s is independently H, OCH.sub.3, F, CN, CH.sub.3, NO.sub.2, CF.sub.3, or OCF.sub.3. In some embodiments, R and R are the same. In some embodiments, R and R are different.

(2067) In some embodiments, R.sup.3s is R.sup.s wherein R.sup.s is as described in the present disclosure. In some embodiments, R.sup.3s is at 3-position (BA is at 1-position). In some embodiments, R.sup.3s is H. In some embodiments, R.sup.3s is F. In some embodiments, R.sup.3s is Cl. In some embodiments, R.sup.3s is Br. In some embodiments, R.sup.3s is I. In some embodiments, R.sup.3s is CN. In some embodiments, R.sup.3s is N.sub.3. In some embodiments, R.sup.3s is NO. In some embodiments, R.sup.3s is NO.sub.2. In some embodiments, R.sup.3s is -L-R. In some embodiments, R.sup.3s is R. In some embodiments, R.sup.3s is -L-OR. In some embodiments, R.sup.3s is OR. In some embodiments, R.sup.3s is -L-SR. In some embodiments, R.sup.3s is SR. In some embodiments, R.sup.3s is -L-N(R).sub.2. In some embodiments, R.sup.3s is N(R).sub.2. In some embodiments, R.sup.3s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.3s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.3s is OMe. In some embodiments, R.sup.3s is -MOE. In some embodiments, R.sup.3s is hydrogen. In some embodiments, R.sup.s at one 3-position is hydrogen, and R.sup.s at the other 3-position is not hydrogen as described herein. In some embodiments, R.sup.s at both 3-positions are hydrogen. In some embodiments, R.sup.s at one 3-position is hydrogen, and the other 3-position is connected to an internucleotidic linkage. In some embodiments, R.sup.3s is F. In some embodiments, R.sup.3s is Cl. In some embodiments, R.sup.3s is Br. In some embodiments, R.sup.3s is I. In some embodiments, R.sup.3s is CN. In some embodiments, R.sup.3s is N.sub.3. In some embodiments, R.sup.3s is NO. In some embodiments, R.sup.3s is NO.sub.2. In some embodiments, R.sup.3s is -L-R. In some embodiments, R.sup.3s is R. In some embodiments, R.sup.3s is -L-OR. In some embodiments, R.sup.3s is OR. In some embodiments, R.sup.3s is -L-SR. In some embodiments, R.sup.3s is SR. In some embodiments, R.sup.3s is L-L-N(R).sub.2. In some embodiments, R.sup.3s is N(R).sub.2. In some embodiments, R.sup.3s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.3s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.3s is OH. In some embodiments, R.sup.3s is OMe. In some embodiments, R.sup.3s is -MOE. In some embodiments, R.sup.3s is hydrogen.

(2068) In some embodiments, R.sup.4s is R.sup.s wherein R.sup.s is as described in the present disclosure. In some embodiments, R.sup.4s is at 4-position (BA is at 1-position). In some embodiments, R.sup.4s is H. In some embodiments, R.sup.4s is F. In some embodiments, R.sup.4s is Cl. In some embodiments, R.sup.4s is Br. In some embodiments, R.sup.4s is I. In some embodiments, R.sup.4s is CN. In some embodiments, R.sup.4s is N.sub.3. In some embodiments, R.sup.4s is NO. In some embodiments, R.sup.4s is NO.sub.2. In some embodiments, R.sup.4s is -L-R. In some embodiments, R.sup.4s is R. In some embodiments, R.sup.4s is -L-OR. In some embodiments, R.sup.4s is OR. In some embodiments, R.sup.4s is -L-SR. In some embodiments, R.sup.4s is SR. In some embodiments, R.sup.4s is -L-N(R).sub.2. In some embodiments, R.sup.4s is N(R).sub.2. In some embodiments, R.sup.4s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.4s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.4s is OMe. In some embodiments, R.sup.4s is -MOE. In some embodiments, R.sup.4s is hydrogen. In some embodiments, R.sup.s at one 4-position is hydrogen, and R.sup.s at the other 4-position is not hydrogen as described herein. In some embodiments, R.sup.s at both 4-positions are hydrogen. In some embodiments, R.sup.s at one 4-position is hydrogen, and the other 4-position is connected to an internucleotidic linkage. In some embodiments, R.sup.4s is F. In some embodiments, R.sup.4s is Cl. In some embodiments, R.sup.4s is Br. In some embodiments, R.sup.4s is I. In some embodiments, R.sup.4s is CN. In some embodiments, R.sup.4s is N.sub.3. In some embodiments, R.sup.4s is NO. In some embodiments, R.sup.4s is NO.sub.2. In some embodiments, R.sup.4s is -L-R. In some embodiments, R.sup.4s is R. In some embodiments, R.sup.4s is -L-OR. In some embodiments, R.sup.4s is OR. In some embodiments, R.sup.4s is -L-SR. In some embodiments, R.sup.4s is SR. In some embodiments, R.sup.4s is L-L-N(R).sub.2. In some embodiments, R.sup.4s is N(R).sub.2. In some embodiments, R.sup.4s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.4s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.4s is OH. In some embodiments, R.sup.4s is OMe. In some embodiments, R.sup.4s is -MOE. In some embodiments, R.sup.4s is hydrogen.

(2069) In some embodiments, R.sup.5s is R.sup.s wherein R.sup.s is as described in the present disclosure. In some embodiments, R.sup.5s is R wherein R is as described in the present disclosure. In some embodiments, R.sup.5s is H. In some embodiments, two or more R.sup.5s are connected to the same carbon atom, and at least one is not H. In some embodiments, R.sup.5s is not H. In some embodiments, R.sup.5s is F. In some embodiments, R.sup.5s is Cl. In some embodiments, R.sup.5s is Br. In some embodiments, R.sup.5s is I. In some embodiments, R.sup.5s is CN. In some embodiments, R.sup.5s is N.sub.3. In some embodiments, R.sup.5s is NO. In some embodiments, R.sup.5s is NO.sub.2. In some embodiments, R.sup.5s is -L-R. In some embodiments, R.sup.5s is R. In some embodiments, R.sup.5s is -L-OR. In some embodiments, R.sup.5s is OR. In some embodiments, R.sup.5s is -L-SR. In some embodiments, R.sup.5s is SR. In some embodiments, R.sup.5s is L-L-N(R).sub.2. In some embodiments, R.sup.5s is N(R).sub.2. In some embodiments, R.sup.5s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.5s is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.5s is OH. In some embodiments, R.sup.5s is OMe. In some embodiments, R.sup.5s is -MOE. In some embodiments, R.sup.5s is hydrogen.

(2070) In some embodiments, R.sup.5s is optionally substituted C.sub.1-6 aliphatic as described in the present disclosure, e.g., C.sub.1-6 aliphatic embodiments described for R or other variables. In some embodiments, R.sup.5s is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.5s is optionally substituted methyl, wherein each substituent, if any, independently comprises no more than one carbon atoms. In some embodiments, R.sup.5s is optionally substituted methyl, wherein each substituent, if any, independently is halogen. In some embodiments, R.sup.5s is methyl. In some embodiments, R.sup.5s is ethyl.

(2071) In some embodiments, R.sup.5s is a protected hydroxyl group suitable for oligonucleotide synthesis. In some embodiments, R.sup.5s is OR, wherein R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.5s is DMTrO. Example protecting groups are widely known for use in accordance with the present disclosure. For additional examples, see Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991, and U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, and/or WO 2017/210647, protecting groups of each of which are hereby incorporated by reference.

(2072) In some embodiments, two or more of R.sup.1s, R.sup.2s, R.sup.3s, R.sup.4s, and R.sup.5s are R and can be taken together with intervening atom(s) to form a ring as described in the present disclosure. In some embodiments, R.sup.2s and R.sup.4s are R taken together to form a ring, and a sugar moiety can be a bicyclic sugar moiety, e.g., a LNA sugar moiety.

(2073) In some embodiments, L.sup.s is L as described in the present disclosure.

(2074) In some embodiments, L.sup.s is C(R.sup.5s).sub.2, wherein each R.sup.5s is independently as described in the present disclosure. In some embodiments, one of R.sup.5s is H and the other is not H. In some embodiments, none of R.sup.5s is H. In some embodiments, L is CHR.sup.5s, wherein each R.sup.5s is independently as described in the present disclosure. In some embodiments, the carbon atom of C(R.sup.5s).sub.2 is stereorandom. In some embodiments, it is of R configuration. In some embodiments, it is of S configuration. In some embodiments, C(R.sup.5s).sub.2 is 5-C, optionally substituted, of a sugar moiety. In some embodiments, the C of C(R.sup.5s).sub.2 is of R configuration. In some embodiments, the C of C(R.sup.5s).sub.2 is of S configuration. As described in the present disclosure, in some embodiments, R.sup.5s is optionally substituted C.sub.1-6 aliphatic; in some embodiments, R.sup.5s is methyl.

(2075) In some embodiments, provided compounds comprise one or more bivalent or multivalent optionally substituted rings, e.g., Ring A, Cy.sup.L, those formed by two or more R groups (R and (combinations of) variables that can be R) taken together, etc. In some embodiments, a ring is a cycloaliphatic, aryl, heteroaryl, or heterocyclyl group as described for R but bivalent or multivalent. As appreciated by those skilled in the art, ring moieties described for one variable, e.g., Ring A, can also be applicable to other variables, e.g., Cy.sup.L, if requirements of the other variables, e.g., number of heteroatoms, valence, etc., are satisfied. Example rings are extensively described in the present disclosure.

(2076) In some embodiments, a ring, e.g., in Ring A, R, etc. which is optionally substituted, is a 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2077) In some embodiments, a ring can be of any size within its range, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered.

(2078) In some embodiments, a ring is monocyclic. In some embodiments, a ring is saturated and monocyclic. In some embodiments, a ring is monocyclic and partially saturated. In some embodiments, a ring is monocyclic and aromatic.

(2079) In some embodiments, a ring is bicyclic. In some embodiments, a ring is polycyclic. In some embodiments, a bicyclic or polycyclic ring comprises two or more monocyclic ring moieties, each of which can be saturated, partially saturated, or aromatic, and each which can contain no or 1-10 heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a saturated monocyclic ring. In some embodiments, a bicyclic or polycyclic ring comprises a saturated monocyclic ring containing no heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a saturated monocyclic ring comprising one or more heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a partially saturated monocyclic ring. In some embodiments, a bicyclic or polycyclic ring comprises a partially saturated monocyclic ring containing no heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a partially saturated monocyclic ring comprising one or more heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises an aromatic monocyclic ring. In some embodiments, a bicyclic or polycyclic ring comprises an aromatic monocyclic ring containing no heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises an aromatic monocyclic ring comprising one or more heteroatoms. In some embodiments, a bicyclic or polycyclic ring comprises a saturated ring and a partially saturated ring, each of which independently contains one or more heteroatoms. In some embodiments, a bicyclic ring comprises a saturated ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a bicyclic ring comprises an aromatic ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises a saturated ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises an aromatic ring and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises an aromatic ring and a saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a polycyclic ring comprises an aromatic ring, a saturated ring, and a partially saturated ring, each of which independently comprises no, or one or more heteroatoms. In some embodiments, a ring comprises at least one heteroatom. In some embodiments, a ring comprises at least one nitrogen atom. In some embodiments, a ring comprises at least one oxygen atom. In some embodiments, a ring comprises at least one sulfur atom.

(2080) As appreciated by those skilled in the art in accordance with the present disclosure, a ring is typically optionally substituted. In some embodiments, a ring is unsubstituted. In some embodiments, a ring is substituted. In some embodiments, a ring is substituted on one or more of its carbon atoms. In some embodiments, a ring is substituted on one or more of its heteroatoms. In some embodiments, a ring is substituted on one or more of its carbon atoms, and one or more of its heteroatoms. In some embodiments, two or more substituents can be located on the same ring atom. In some embodiments, all available ring atoms are substituted. In some embodiments, not all available ring atoms are substituted. In some embodiments, in provided structures where rings are indicated to be connected to other structures

(2081) ##STR00871##
optionally substituted is to mean that, besides those structures already connected, remaining substitutable ring positions, if any, are optionally substituted.

(2082) In some embodiments, a ring is a bivalent or multivalent C.sub.3-30 cycloaliphatic ring. In some embodiments, a ring is a bivalent or multivalent C.sub.3-20 cycloaliphatic ring. In some embodiments, a ring is a bivalent or multivalent C.sub.3-10 cycloaliphatic ring. In some embodiments, a ring is a bivalent or multivalent 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 3-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 4-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 5-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 6-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent 7-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, a ring is a bivalent or multivalent cyclohexyl ring. In some embodiments, a ring is a bivalent or multivalent cyclopentyl ring. In some embodiments, a ring is a bivalent or multivalent cyclobutyl ring. In some embodiments, a ring is a bivalent or multivalent cyclopropyl ring.

(2083) In some embodiments, a ring is a bivalent or multivalent C.sub.6-30 aryl ring. In some embodiments, a ring is a bivalent or multivalent phenyl ring.

(2084) In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic saturated, partially unsaturated or aryl ring. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic saturated ring. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic partially unsaturated ring. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic aryl ring. In some embodiments, a ring is a bivalent or multivalent naphthyl ring.

(2085) In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring is a bivalent or multivalent 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

(2086) In some embodiments, a ring is a bivalent or multivalent 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen.

(2087) In some embodiments, a ring is a bivalent or multivalent 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, a ring is a bivalent or multivalent 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2088) In certain embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 6,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2089) In some embodiments, a ring is a bivalent or multivalent 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring is a bivalent or multivalent 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 3-membered heterocyclic ring having one heteroatom selected from nitrogen, oxygen or sulfur. In some embodiments, a ring is a bivalent or multivalent 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2090) In some embodiments, a ring is a bivalent or multivalent 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a bivalent or multivalent 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2091) In some embodiments, a ring is a bivalent or multivalent 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a bivalent or multivalent 6,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2092) In some embodiments, a ring formed by two or more groups taken together, which is typically optionally substituted, is a monocyclic saturated 5-7 membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a monocyclic saturated 5-membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a monocyclic saturated 6-membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a monocyclic saturated 7-membered ring having no additional heteroatoms in addition to intervening heteroatoms, if any.

(2093) In some embodiments, a ring formed by two or more groups taken together is a bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring formed by two or more groups taken together is a bicyclic, saturated, partially unsaturated, or aryl 5-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 8-10 membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 8-membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 9-membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is a bicyclic and saturated 10-membered bicyclic ring having no additional heteroatoms in addition to intervening heteroatoms, if any. In some embodiments, a ring formed by two or more groups taken together is bicyclic and comprises a 5-membered ring fused to a 5-membered ring. In some embodiments, a ring formed by two or more groups taken together is bicyclic and comprises a 5-membered ring fused to a 6-membered ring. In some embodiments, the 5-membered ring comprises one or more intervening nitrogen, phosphorus and oxygen atoms as ring atoms. In some embodiments, a ring formed by two or more groups taken together comprises a ring system having the backbone structure of

(2094) ##STR00872##

(2095) In some embodiments, a ring formed by two or more groups taken together is a polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, a ring formed by two or more groups taken together is a polycyclic, saturated, partially unsaturated, or aryl 3-30 membered ring having, in addition to the intervening heteroatoms, if any, 0-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

(2096) In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-10 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-9 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-8 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-7 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-6 membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms.

(2097) In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 6-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 7-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 8-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 9-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 10-membered monocyclic ring whose ring atoms comprise one or more intervening nitrogen, phosphorus and/or oxygen atoms.

(2098) In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 5-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 6-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 7-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 8-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 9-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring formed by two or more groups taken together is monocyclic, bicyclic or polycyclic and comprises a 10-membered ring whose ring atoms consist of carbon atoms and the intervening nitrogen, phosphorus and oxygen atoms.

(2099) In some embodiments, rings described herein are unsubstituted. In some embodiments, rings described herein are substituted. In some embodiments, substituents are selected from those described in example compounds provided in the present disclosure.

(2100) In some embodiments, each BA is independently an optionally substituted group selected from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon; each Ring A is independently an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form there, wherein each variable is independently as described in the present disclosure.

(2101) In some embodiments, each BA is independently an optionally substituted C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein the heteroaryl comprises one or more heteroatoms selected from oxygen and nitrogen; each Ring A is independently an optionally substituted 5-10 membered monocyclic or bicyclic saturated ring having 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, wherein the ring comprises at least one oxygen atom; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or salt form thereof, wherein each variable is independently as described in the present disclosure.

(2102) In some embodiments, each BA is independently an optionally substituted A, T, C, G, or U, or an optionally substituted tautomer of A, T, C, G, or U; each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or salt form thereof, wherein each variable is independently as described in the present disclosure.

(2103) In some embodiments, each BA is independently an optionally substituted or protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil; each Ring A is independently an optionally substituted 5-7 membered monocyclic or bicyclic saturated ring having one or more oxygen atoms; and each L.sup.P independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or salt form thereof, wherein each variable is independently as described in the present disclosure.

(2104) In some embodiments, R.sup.5s-L.sup.s- is CH.sub.2OH. In some embodiments, R.sup.5s-L.sup.s- is CH(R.sup.5s)OH, wherein R.sup.5s is as described in the present disclosure.

(2105) In some embodiments, BA is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is an optionally substituted group selected from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a natural nucleobase moiety, and a modified nucleobase moiety. In some embodiments, BA is optionally substituted C.sub.5-30 heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, BA is optionally substituted natural nucleobases and tautomers thereof. In some embodiments, BA is protected natural nucleobases and tautomers thereof. Various nucleobase protecting groups for oligonucleotide synthesis are known and can be utilized in accordance with the present disclosure. In some embodiments, BA is an optionally substituted nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof. In some embodiments, BA is an optionally protected nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, and tautomers thereof.

(2106) In some embodiments, BA is optionally substituted C.sub.3-30 cycloaliphatic. In some embodiments, BA is optionally substituted C.sub.6-30 aryl. In some embodiments, BA is optionally substituted C.sub.3-30 heterocyclyl. In some embodiments, BA is optionally substituted C.sub.5-30 heteroaryl. In some embodiments, BA is an optionally substituted natural base moiety. In some embodiments, BA is an optionally substituted modified base moiety. BA is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.3-30 heterocyclyl, and C.sub.5-30 heteroaryl. In some embodiments, BA is an optionally substituted group selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, C.sub.3-30 heterocyclyl, C.sub.5-30 heteroaryl, and a natural nucleobase moiety.

(2107) In some embodiments, BA is connected through an aromatic ring. In some embodiments, BA is connected through a heteroatom. In some embodiments, BA is connected through a ring heteroatom of an aromatic ring. In some embodiments, BA is connected through a ring nitrogen atom of an aromatic ring.

(2108) In some embodiments, BA is a natural nucleobase moiety. In some embodiments, BA is an optionally substituted natural nucleobase moiety. In some embodiments, BA is a substituted natural nucleobase moiety. In some embodiments, BA is optionally substituted, or an optionally substituted tautomer of, A, T, C, U, or G. In some embodiments, BA is natural nucleobase A, T, C, U, or G. In some embodiments, BA is an optionally substituted group selected from natural nucleobases A, T, C, U, and G.

(2109) In some embodiments, BA is an optionally substituted purine base residue. In some embodiments, BA is a protected purine base residue. In some embodiments, BA is an optionally substituted adenine residue. In some embodiments, BA is a protected adenine residue. In some embodiments, BA is an optionally substituted guanine residue. In some embodiments, BA is a protected guanine residue. In some embodiments, BA is an optionally substituted cytosine residue. In some embodiments, BA is a protected cytosine residue. In some embodiments, BA is an optionally substituted thymine residue. In some embodiments, BA is a protected thymine residue. In some embodiments, BA is an optionally substituted uracil residue. In some embodiments, BA is a protected uracil residue. In some embodiments, BA is an optionally substituted 5-methylcytosine residue. In some embodiments, BA is a protected 5-methylcytosine residue.

(2110) In some embodiments, s is 0-20. In some embodiments, s is 1-20. In some embodiments, s is 1-5. In some embodiments, s is 1. In some embodiments, s is 2. In some embodiments, s is 3. In some embodiments, s is 4. In some embodiments, s is 5. In some embodiments, s is 6. In some embodiments, s is 7. In some embodiments, s is 8. In some embodiments, s is 9. In some embodiments, s is 10. In some embodiments, s is 11. In some embodiments, s is 12. In some embodiments, s is 13. In some embodiments, s is 14. In some embodiments, s is 15. In some embodiments, s is 16. In some embodiments, s is 17. In some embodiments, s is 18. In some embodiments, s is 19. In some embodiments, s is 20.

(2111) In some embodiments, L.sup.P is an internucleotidic linkage. In some embodiments, L.sup.P is an internucleotidic linkage of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. In some embodiments, L.sup.P is a natural phosphate linkage. In some embodiments, L.sup.P is a non-negatively charged internucleotidic linkage. In some embodiments, L.sup.P is a neutral internucleotidic linkage. In some embodiments, L.sup.P is a negatively-charged internucleotidic linkage. In some embodiments, L.sup.P is a phosphorothioate internucleotidic linkage. In some embodiments, L.sup.P is a chirally controlled internucleotidic linkage.

(2112) In some embodiments, z is 1-1000. In some embodiments, z+1 is an oligonucleotide length as described in the present disclosure. In some embodiments, z is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000. In some embodiments, z is 10-100. In some embodiments, z is 10-50. In some embodiments, z is 15-100. In some embodiments, z is 20-50. In some embodiments, z is no less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, z is no less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, z is no more than 50, 60, 70, 80, 90, 100, 150, or 200. In some embodiments, z is 5-50, 10-50, 14-50, 14-45, 14-40, 14-35, 14-30, 14-25, 14-100, 14-150, 14-200, 14-250, 14-300, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-100, 15-150, 15-200, 15-250, 15-300, 16-50, 16-45, 16-40, 16-35, 16-30, 16-25, 16-100, 16-150, 16-200, 16-250, 16-300, 17-50, 17-45, 17-40, 17-35, 17-30, 17-25, 17-100, 17-150, 17-200, 17-250, 17-300, 18-50, 18-45, 18-40, 18-35, 18-30, 18-25, 18-100, 18-150, 18-200, 18-250, 18-300, 19-50, 19-45, 19-40, 19-35, 19-30, 19-25, 19-100, 19-150, 19-200, 19-250, or 19-300. In some embodiments, z is 10. In some embodiments, z is 11. In some embodiments, z is 12. In some embodiments, z is 13. In some embodiments, z is 14. In some embodiments, z is 15. In some embodiments, z is 16. In some embodiments, z is 17. In some embodiments, z is 18. In some embodiments, z is 19. In some embodiments, z is 20. In some embodiments, z is 21. In some embodiments, z is 22. In some embodiments, z is 23. In some embodiments, z is 24. In some embodiments, z is 25. In some embodiments, z is 26. In some embodiments, z is 27. In some embodiments, z is 28. In some embodiments, z is 29. In some embodiments, z is 30. In some embodiments, z is 31. In some embodiments, z is 32. In some embodiments, z is 33. In some embodiments, z is 34.

(2113) In some embodiments, L.sup.3E is -L- or -L-L-. In some embodiments, L.sup.3E is -L-. In some embodiments, L.sup.3E is -L-L-. In some embodiments, L.sup.3E is a covalent bond. In some embodiments, L.sup.3E is a linker used in oligonucleotide synthesis. In some embodiments, L.sup.3E is a linker used in solid phase oligonucleotide synthesis. Various types of linkers are known and can be utilized in accordance with the present disclosure. In some embodiments, a linker is a succinate linker (OC(O)CH.sub.2CH.sub.2C(O)). In some embodiments, a linker is an oxalyl linker (OC(O)C(O)). In some embodiments, L.sup.3E is a succinyl-piperidine linker (SP) linker. In some embodiments, L.sup.3E is a succinyl linker. In some embodiments, L.sup.3E is a Q-linker. In some embodiments, L.sup.3E is O.

(2114) In some embodiments, R.sup.3E is R, -L-R, OR, or a solid support. In some 23E is R as embodiments, R.sup.3E is R as described in the present disclosure. In some embodiments, R.sup.3E described in the present disclosure. In some embodiments, R.sup.3E is hydrogen. In some embodiments, R.sup.3E is -L-R. In some embodiments, R.sup.3E is OR. In some embodiments, R.sup.3E is a support for oligonucleotide synthesis. In some embodiments, R.sup.3E is a solid support. In some embodiments, a solid support is a CPG support. In some embodiments, a solid support is a polystyrene support. In some embodiments, R.sup.3E is H. In some embodiments, -L.sup.3-R.sup.3E is H. In some embodiments, R.sup.3E is OH. In some embodiments, -L.sup.3-R.sup.3E is OH. In some embodiments, R.sup.3E is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.3E is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.3E is OR. In some embodiments, R.sup.3E is OH. In some embodiments, R.sup.3E is OR, wherein R is not hydrogen. In some embodiments, R.sup.3E is OR, wherein R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R.sup.3E is a 3-end cap (e.g., those used in RNAi technologies).

(2115) In some embodiments, R.sup.3E is a solid support. In some embodiments, R.sup.3E is a solid support for oligonucleotide synthesis. Various types of solid support are known and can be utilized in accordance with the present disclosure. In some embodiments, a solid support is HCP. In some embodiments, a solid support is CPG.

(2116) In some embodiments, R is R, C(O)R, C(O)OR, or S(O).sub.2R, wherein R is as described in the present disclosure. In some embodiments, R is R, wherein R is as described in the present disclosure. In some embodiments, R is C(O)R, wherein R is as described in the present disclosure. In some embodiments, R is C(O)OR, wherein R is as described in the present disclosure. In some embodiments, R is S(O).sub.2R, wherein R is as described in the present disclosure. In some embodiments, R is hydrogen. In some embodiments, R is not hydrogen. In some embodiments, R is R, wherein R is optionally substituted C.sub.1-20 aliphatic as described in the present disclosure. In some embodiments, R is R, wherein R is optionally substituted C.sub.1-20 heteroaliphatic as described in the present disclosure. In some embodiments, R is R, wherein R is optionally substituted C.sub.6-20 aryl as described in the present disclosure. In some embodiments, R is R, wherein R is optionally substituted C.sub.6-20 arylaliphatic as described in the present disclosure. In some embodiments, R is R, wherein R is optionally substituted C.sub.6-20 arylheteroaliphatic as described in the present disclosure. In some embodiments, R is R, wherein R is optionally substituted 5-20 membered heteroaryl as described in the present disclosure. In some embodiments, R is R, wherein R is optionally substituted 3-20 membered heterocyclyl as described in the present disclosure. In some embodiments, two or more R are R, and are optionally and independently taken together to form an optionally substituted ring as described in the present disclosure.

(2117) In some embodiments, each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2118) In some embodiments, each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2119) In some embodiments, each R is independently H, or an optionally substituted group selected from C.sub.1-20 aliphatic, C.sub.1-20 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-20 aryl, C.sub.6-20 arylaliphatic, C.sub.6-20 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-20 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or two R groups are optionally and independently taken together to form a covalent bond, or: two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2120) In some embodiments, each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2121) In some embodiments, each R is independently H, or an optionally substituted group selected from C.sub.1-20 aliphatic, C.sub.1-20 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-20 aryl, C.sub.6-20 arylaliphatic, C.sub.6-20 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-20 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2122) In some embodiments, R is hydrogen. In some embodiments, R is not hydrogen. In some embodiments, R is an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl, a 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and a 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2123) In some embodiments, R is hydrogen or an optionally substituted group selected from C.sub.1-20 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, an 8-10 membered bicyclic saturated, partially unsaturated or aryl ring, a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 4-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, a 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2124) In some embodiments, R is optionally substituted C.sub.1-30 aliphatic. In some embodiments, R is optionally substituted C.sub.1-20 aliphatic. In some embodiments, R is optionally substituted C.sub.1-15 aliphatic. In some embodiments, R is optionally substituted C.sub.1-10 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 aliphatic. In some embodiments, R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R is optionally substituted hexyl, pentyl, butyl, propyl, ethyl or methyl. In some embodiments, R is optionally substituted hexyl. In some embodiments, R is optionally substituted pentyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is optionally substituted propyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is hexyl. In some embodiments, R is pentyl. In some embodiments, R is butyl. In some embodiments, R is propyl. In some embodiments, R is ethyl. In some embodiments, R is methyl. In some embodiments, R is isopropyl. In some embodiments, R is n-propyl. In some embodiments, R is tert-butyl. In some embodiments, R is sec-butyl. In some embodiments, R is n-butyl. In some embodiments, R is (CH.sub.2).sub.2CN.

(2125) In some embodiments, R is optionally substituted C.sub.3-30 cycloaliphatic. In some embodiments, R is optionally substituted C.sub.3-20 cycloaliphatic. In some embodiments, R is optionally substituted C.sub.3-10 cycloaliphatic. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is cyclopropyl.

(2126) In some embodiments, R is an optionally substituted 3-30 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 3-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is an optionally substituted 7-membered saturated or partially unsaturated carbocyclic ring. In some embodiments, R is optionally substituted cycloheptyl. In some embodiments, R is cycloheptyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is cyclohexyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is cyclopentyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is cyclobutyl. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is cyclopropyl.

(2127) In some embodiments, when R is or comprises a ring structure, e.g., cycloaliphatic, cycloheteroaliphatic, aryl, heteroaryl, etc., the ring structure can be monocyclic, bicyclic or polycyclic. In some embodiments, R is or comprises a monocyclic structure. In some embodiments, R is or comprises a bicyclic structure. In some embodiments, R is or comprises a polycyclic structure.

(2128) In some embodiments, R is optionally substituted C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C.sub.1-20 heteroaliphatic having 1-10 heteroatoms. In some embodiments, R is optionally substituted C.sub.1-20 heteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus or silicon, optionally including one or more oxidized forms of nitrogen, sulfur, phosphorus or selenium. In some embodiments, R is optionally substituted C.sub.1-30 heteroaliphatic comprising 1-10 groups independently selected from

(2129) ##STR00873##

(2130) In some embodiments, R is optionally substituted C.sub.6-30 aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is substituted phenyl.

(2131) In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated, partially unsaturated or aryl ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic saturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic partially unsaturated ring. In some embodiments, R is an optionally substituted 8-10 membered bicyclic aryl ring. In some embodiments, R is optionally substituted naphthyl.

(2132) In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

(2133) In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen. In some embodiments, R is a substituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6 membered monocyclic heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, sulfur, and oxygen.

(2134) In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. In some embodiments, R is an optionally substituted 6-membered monocyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2135) In some embodiments, R is an optionally substituted 5-membered monocyclic heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is selected from optionally substituted pyrrolyl, furanyl, or thienyl.

(2136) In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered heteroaryl ring having one nitrogen atom, and an additional heteroatom selected from sulfur or oxygen. Example R groups include but are not limited to optionally substituted pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or isoxazolyl.

(2137) In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. Example R groups include but are not limited to optionally substituted triazolyl, oxadiazolyl or thiadiazolyl.

(2138) In some embodiments, R is an optionally substituted 5-membered heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. Example R groups include but are not limited to optionally substituted tetrazolyl, oxatriazolyl and thiatriazolyl.

(2139) In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-4 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-3 nitrogen atoms. In other embodiments, R is an optionally substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having four nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having three nitrogen atoms. In some embodiments, R is an optionally substituted 6-membered heteroaryl ring having two nitrogen atoms. In certain embodiments, R is an optionally substituted 6-membered heteroaryl ring having one nitrogen atom. Example R groups include but are not limited to optionally substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or tetrazinyl.

(2140) In certain embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted indolyl. In some embodiments, R is an optionally substituted azabicyclo[3.2.1]octanyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted azaindolyl. In some embodiments, R is an optionally substituted benzimidazolyl. In some embodiments, R is an optionally substituted benzothiazolyl. In some embodiments, R is an optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2141) In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2142) In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is optionally substituted benzofuranyl. In some embodiments, R is optionally substituted benzo[b]thienyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted oxazolopyridiyl, thiazolopyridinyl or imidazopyridinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2143) In some embodiments, R is optionally substituted 1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo [3,2-b]pyrrolyl, 4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl, thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl, pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some embodiments, R is optionally substituted dihydropyrroloimidazolyl, 1H-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl or imidazo[5,1-b]thiazolyl.

(2144) In certain embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1 heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted quinolinyl. In some embodiments, R is an optionally substituted isoquinolinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinazoline or a quinoxaline.

(2145) In some embodiments, R is 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is 3-30 membered heterocyclic ring having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is 3-30 membered heterocyclic ring having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

(2146) In some embodiments, R is an optionally substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is a substituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-7 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 6-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 7-membered partially unsaturated monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 3-membered heterocyclic ring having one heteroatom selected from nitrogen, oxygen or sulfur. In some embodiments, R is optionally substituted 4-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 5-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 6-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 7-membered heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2147) In some embodiments, R is an optionally substituted 3-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 7-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2148) In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms. In some embodiments, R is an optionally substituted 4-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 4-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.

(2149) In some embodiments, R is an optionally substituted 5-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 5-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.

(2150) In some embodiments, R is an optionally substituted 6-membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is nitrogen. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is oxygen. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having no more than 1 heteroatom, wherein the heteroatom is sulfur. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 oxygen atoms. In some embodiments, R is an optionally substituted 6-membered partially unsaturated heterocyclic ring having 2 nitrogen atoms.

(2151) In certain embodiments, R is a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is optionally substituted oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl, azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl, imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl, dioxolanedionyl, oxathiolanedionyl, piperazinedionyl, morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl.

(2152) In certain embodiments, R is an optionally substituted 5-6 membered partially unsaturated monocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or oxazolinyl group.

(2153) In some embodiments, R is an optionally substituted 7-10 membered bicyclic saturated or partially unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolinyl. In some embodiments, R is optionally substituted isoindolinyl. In some embodiments, R is optionally substituted 1, 2, 3, 4-tetrahydroquinolinyl. In some embodiments, R is optionally substituted 1, 2, 3, 4-tetrahydroisoquinolinyl. In some embodiments, R is an optionally substituted azabicyclo[3.2.1]octanyl.

(2154) In some embodiments, R is an optionally substituted 8-10 membered bicyclic heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2155) In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted 1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl, 4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl, thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl, pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted dihydropyrroloimidazolyl, 1H-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl or imidazo[5,1-b]thiazolyl. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2156) In some embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having one heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted indolyl. In some embodiments, R is optionally substituted benzofuranyl. In some embodiments, R is optionally substituted benzo[b]thienyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted azaindolyl. In some embodiments, R is optionally substituted benzimidazolyl. In some embodiments, R is optionally substituted benzothiazolyl. In some embodiments, R is optionally substituted benzoxazolyl. In some embodiments, R is an optionally substituted indazolyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted oxazolopyridiyl, thiazolopyridinyl or imidazopyridinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted purinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl, thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl, thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments, R is an optionally substituted 5,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2157) In certain embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In other embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinolinyl. In some embodiments, R is optionally substituted isoquinolinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having two heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted quinazolinyl, phthalazinyl, quinoxalinyl or naphthyridinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having three heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted pyridopyrimidinyl, pyridopyridazinyl, pyridopyrazinyl, or benzotriazinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having four heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, R is optionally substituted pyridotriazinyl, pteridinyl, pyrazinopyrazinyl, pyrazinopyridazinyl, pyridazinopyridazinyl, pyrimidopyridazinyl or pyrimidopyrimidinyl. In some embodiments, R is an optionally substituted 6,6-fused heteroaryl ring having five heteroatoms independently selected from nitrogen, oxygen, and sulfur.

(2158) In some embodiments, R is optionally substituted C.sub.6-30 arylaliphatic. In some embodiments, R is optionally substituted C.sub.6-20 arylaliphatic. In some embodiments, R is optionally substituted C.sub.6-10 arylaliphatic. In some embodiments, an aryl moiety of the arylaliphatic has 6, 10, or 14 aryl carbon atoms. In some embodiments, an aryl moiety of the arylaliphatic has 6 aryl carbon atoms. In some embodiments, an aryl moiety of the arylaliphatic has 10 aryl carbon atoms. In some embodiments, an aryl moiety of the arylaliphatic has 14 aryl carbon atoms. In some embodiments, an aryl moiety is optionally substituted phenyl.

(2159) In some embodiments, R is optionally substituted C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is optionally substituted C.sub.6-20 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C.sub.6-20 arylheteroaliphatic having 1-10 heteroatoms independently selected from oxygen, nitrogen, and sulfur. In some embodiments, R is optionally substituted C.sub.6-10 arylheteroaliphatic having 1-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is optionally substituted C.sub.6-10 arylheteroaliphatic having 1-5 heteroatoms independently selected from oxygen, nitrogen, and sulfur.

(2160) In some embodiments, two R groups are optionally and independently taken together to form a covalent bond. In some embodiments, CO is formed. In some embodiments, CC is formed. In some embodiments, CC is formed.

(2161) In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-10 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-6 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-5 membered monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2162) In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-20 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-10 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-10 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-5 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-6 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In some embodiments, two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-5 membered monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-3 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

(2163) In some embodiments, heteroatoms in R groups, or in the structures formed by two or more R groups taken together, are selected from oxygen, nitrogen, and sulfur. In some embodiments, a formed ring is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-membered. In some embodiments, a formed ring is saturated. In some embodiments, a formed ring is partially saturated. In some embodiments, a formed ring is aromatic. In some embodiments, a formed ring comprises a saturated, partially saturated, or aromatic ring moiety. In some embodiments, a formed ring comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aromatic ring atoms. In some embodiments, a formed contains no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aromatic ring atoms. In some embodiments, aromatic ring atoms are selected from carbon, nitrogen, oxygen and sulfur.

(2164) In some embodiments, a ring formed by two or more R groups (or two or more groups selected from R and variables that can be R) taken together is a C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl, 5-30 membered heteroaryl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or 3-30 membered heterocyclyl having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, ring as described for R, but bivalent or multivalent.

(2165) As appreciated by those skilled in the art, embodiments of R described in the present disclosure can also independently be embodiments for variables that can be R.

(2166) In some embodiments, a is 1-100. In some embodiments, a is 1-50. In some embodiments, a is 1-40. In some embodiments, a is 1-30. In some embodiments, a is 1-20. In some embodiments, a is 1-15. In some embodiments, a is 1-10. In some embodiments, a is 1-9. In some embodiments, a is 1-8. In some embodiments, a is 1-7. In some embodiments, a is 1-6. In some embodiments, a is 1-5. In some embodiments, a is 1-4. In some embodiments, a is 1-3. In some embodiments, a is 1-2. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6. In some embodiments, a is 7. In some embodiments, a is 8. In some embodiments, a is 9. In some embodiments, a is 10. In some embodiments, a is more than 10.

(2167) In some embodiments, b is 1-100. In some embodiments, b is 1-50. In some embodiments, b is 1-40. In some embodiments, b is 1-30. In some embodiments, b is 1-20. In some embodiments, b is 1-15. In some embodiments, b is 1-10. In some embodiments, b is 1-9. In some embodiments, b is 1-8. In some embodiments, b is 1-7. In some embodiments, b is 1-6. In some embodiments, b is 1-5. In some embodiments, b is 1-4. In some embodiments, b is 1-3. In some embodiments, b is 1-2. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5. In some embodiments, b is 6. In some embodiments, b is 7. In some embodiments, b is 8. In some embodiments, b is 9. In some embodiments, b is 10. In some embodiments, b is 1. In some embodiments, b is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more.

(2168) In some embodiments, LED is L. In some embodiments, L.sup.LD is bivalent L.sup.M

(2169) In some embodiments, L.sup.M is -L.sup.M1-L.sup.M2-L.sup.M3- as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M2 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M3 as described in the present disclosure. In some embodiments, L.sup.M is L as described in the present disclosure.

(2170) In some embodiments, L.sup.M1 is L. In some embodiments, L.sup.M2 is L. In some embodiments, L.sup.M3 is L. In some embodiments, L.sup.M1 is a covalent bond. In some embodiments, L.sup.M2 is a covalent bond. In some embodiments, L.sup.M3 is a covalent bond. In some embodiments, L.sup.M1 is L.sup.M2 as described in the present disclosure. In some embodiments, L.sup.M1 is L.sup.M3 as described in the present disclosure. In some embodiments, L.sup.M2 is L.sup.M1 as described in the present disclosure. In some embodiments, L.sup.M2 is L.sup.M3 as described in the present disclosure. In some embodiments, L.sup.M3 is L.sup.M1 as described in the present disclosure. In some embodiments, L.sup.M3 is L.sup.M2 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M2 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M3 as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1-L.sup.M2, wherein each of L.sup.M1 and L.sup.M2 is independently as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1-L.sup.M3, wherein each of L.sup.M1 and L.sup.M3 is independently as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M2-L.sup.M3, wherein each of L.sup.M2 and L.sup.M3 is independently as described in the present disclosure. In some embodiments, L.sup.M is L.sup.M1-L.sup.M2-L.sup.M3, wherein each of L.sup.M1, L.sup.M2 and L.sup.M3 is independently as described in the present disclosure.

(2171) In some embodiments, L.sup.M1 comprises one or more N(R) and one or more C(O). In some embodiments, a linker or L.sup.M1 is or comprises

(2172) ##STR00874##
wherein n.sup.L is 1-8. In some embodiments, a linker or -L.sup.M1-L.sup.M2-L.sup.M3- is

(2173) ##STR00875##
or a salt form thereof, wherein n.sup.L is 1-8. In some embodiments, a linker or -L.sup.M1-L.sup.M2-L.sup.M3- is

(2174) ##STR00876##
or a salt form thereof, wherein: n.sup.L is 1-8. each amino group independently connects to a moiety; and the P atom connects to the 5-OH of the oligonucleotide.

(2175) In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(2176) ##STR00877##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(2177) ##STR00878##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(2178) ##STR00879##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(2179) ##STR00880##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(2180) ##STR00881##
In some embodiments, the moiety and the linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises

(2181) ##STR00882##
In some embodiments the moiety and the linker or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3- is or comprises

(2182) ##STR00883##
In some embodiments, the linker, or L.sup.M1, is or comprises

(2183) ##STR00884##
In some embodiments, the moiety and linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises:

(2184) ##STR00885##
In some embodiments, the moiety and linker, or (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises:

(2185) ##STR00886##

(2186) In some embodiments, n.sup.L is 1-8. In some embodiments, n.sup.L is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n.sup.L is 1. In some embodiments, n.sup.L is 2. In some embodiments, n.sup.L is 3. In some embodiments, n.sup.L is 4. In some embodiments, n.sup.L is 5. In some embodiments, n.sup.L is 6. In some embodiments, n.sup.L is 7. In some embodiments, n.sup.L is 8.

(2187) In some embodiments, at least one L.sup.M is directly bound to a sugar unit of a provided oligonucleotide. In some embodiments, a L.sup.M directly binds to a sugar unit incorporates a lipid moiety into an oligonucleotide. In some embodiments, a L.sup.M directly binds to a sugar unit incorporates a carbohydrate moiety into an oligonucleotide. In some embodiments, a L.sup.M directly binds to a sugar unit incorporates a R.sup.LD group into an oligonucleotide. In some embodiments, a L.sup.M directly binds to a sugar unit incorporates a R.sup.CD group into an oligonucleotide. In some embodiments, L.sup.M is directed bound through 5-OH of an oligonucleotide chain. In some embodiments, L.sup.M is directed bound through 3-OH of an oligonucleotide chain.

(2188) In some embodiments, at least one L.sup.M is directly bound to an internucleotidic linkage unit of a provided oligonucleotide. In some embodiments, a L.sup.M directly binds to an internucleotidic linkage unit incorporates a lipid moiety into an oligonucleotide. In some embodiments, a L.sup.M directly binds to an internucleotidic linkage unit incorporates a carbohydrate moiety into an oligonucleotide. In some embodiments, a L.sup.M directly binds to an internucleotidic linkage unit incorporates a R.sup.LD group into an oligonucleotide. In some embodiments, a L.sup.M directly binds to an internucleotidic linkage unit incorporates a R.sup.CD group into an oligonucleotide.

(2189) In some embodiments, at least one L.sup.M is directly bound to a nucleobase unit of a provided oligonucleotide. In some embodiments, a L.sup.M directly binds to a nucleobase unit incorporates a lipid moiety into an oligonucleotide. In some embodiments, a L.sup.M directly binds to a nucleobase unit incorporates a carbohydrate moiety into an oligonucleotide. In some embodiments, a L.sup.M directly binds to a nucleobase unit incorporates a R.sup.LD group into an oligonucleotide. In some embodiments, a L.sup.M directly binds to a nucleobase unit incorporates a R.sup.CD group into an oligonucleotide.

(2190) In some embodiments, L.sup.M is bivalent. In some embodiments, L.sup.M is multivalent. In some embodiments, L.sup.M is

(2191) ##STR00887##
wherein L.sup.M is directly bond to a nucleobase, for example, as in:

(2192) ##STR00888##
In some embodiments, L.sup.M is

(2193) ##STR00889##
In some embodiments, L.sup.M is

(2194) ##STR00890##
In some embodiments, L.sup.M is

(2195) ##STR00891##
In some embodiments, L.sup.M is

(2196) ##STR00892##
In some embodiments, a linker moiety, e.g., L.sup.M, L.sup.M1, L.sup.M2, L.sup.M3, L, L.sup.s, etc., is or comprises

(2197) ##STR00893##
In some embodiments, a linker moiety, e.g., L.sup.M, L.sup.M1, L.sup.M2, L.sup.M3, L, L.sup.s, etc., is or comprises

(2198) ##STR00894##

(2199) In some embodiments, R.sup.D is a lipid moiety. In some embodiments, R.sup.D is a targeting moiety. In some embodiments, R.sup.D is a carbohydrate moiety. In some embodiments, R.sup.D is a sulfonamide moiety. In some embodiments, R.sup.D is an antibody or a fragment thereof. In some embodiments, R.sup.D is R.sup.LD as described in the present disclosure. In some embodiments, R.sup.D is R.sup.CD as described in the present disclosure. In some embodiments, R.sup.D is R.sup.TD as described in the present disclosure.

(2200) In some embodiments, a lipid moiety has the structure of R.sup.LD. In some embodiments, R.sup.LD is optionally substituted C.sub.10, C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, or C.sub.25 to C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25, C.sub.26, C.sub.27, C.sub.28, C.sub.29, C.sub.30, C.sub.35, C.sub.40, C.sub.45, C.sub.50, C.sub.60, C.sub.70, or C.sub.80 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.10-80 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.20-80 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.10-70 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.20-70 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.10-60 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.20-60 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.10-50 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.20-50 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.10-40 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.20-40 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.10-30 aliphatic. In some embodiments, R.sup.LD is optionally substituted C.sub.20-30 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.10, C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, or C.sub.25 to C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25, C.sub.26, C.sub.27, C.sub.28, C.sub.29, C.sub.30, C.sub.35, C.sub.40, C.sub.45, C.sub.50, C.sub.60, C.sub.70, or C.sub.80 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.10-80 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.20-80 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.10-70 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.20-70 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.10-60 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.20-60 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.10-50 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.20-50 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.10-40 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.20-40 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.10-30 aliphatic. In some embodiments, R.sup.LD is unsubstituted C.sub.20-30 aliphatic.

(2201) In some embodiments, R.sup.LD is not hydrogen. In some embodiments, R.sup.LD is a lipid moiety. In some embodiments, R.sup.LD is a targeting moiety. In some embodiments, R.sup.LD is a targeting moiety comprising a carbohydrate moiety. In some embodiments, R.sup.LD is a GalNAc moiety.

(2202) In some embodiments, R.sup.TD is R.sup.LD, wherein R.sup.LD is independently as described in the present disclosure. In some embodiments, R.sup.TD is R.sup.CD, wherein R.sup.CD is independently as described in the present disclosure. In some embodiments, R.sup.TD comprises a sulfonamide moiety. In some embodiments, a R.sup.TD comprises a carbohydrate moiety. In some embodiments, a R.sup.TD comprises a GalNAc moiety.

(2203) In some embodiments, R.sup.CD is an optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, C(R).sub.2, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O; and one or more carbon atoms are optionally and independently replaced with Cy.sup.L. In some embodiments, R.sup.CD is an optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, C(R).sub.2, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O; and one or more carbon atoms are independently replaced with a monosaccharide, disaccharide or polysaccharide moiety. In some embodiments, R.sup.CD is an optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, C(R).sub.2, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O; and one or more carbon atoms are independently replaced with a GalNac moiety.

(2204) In some embodiments, each R.sup.D is independently a chemical moiety as described in the present disclosure. In some embodiments, R.sup.D is an additional chemical moiety. In some embodiments, R.sup.D is targeting moiety. In some embodiments, R.sup.D is or comprises a carbohydrate moiety. In some embodiments, R.sup.D is or comprises a lipid moiety. In some embodiments, R.sup.D is or comprises a ligand moiety for, e.g., cell receptors such as a sigma receptor, an asialoglycoprotein receptor, etc. In some embodiments, a ligand moiety is or comprises an anisamide moiety, which may be a ligand moiety for a sigma receptor. In some embodiments, a ligand moiety is or comprises a lipid. In some embodiments, a ligand moiety is or comprises a GalNAc moiety, which may be a ligand moiety for an asialoglycoprotein receptor. In some embodiments, R.sup.D is selected from optionally substituted phenyl,

(2205) ##STR00895##
wherein n.sup.L is 0 or 1, and each other variable is independently as described in the present disclosure. In some embodiments, R.sup.s is F. In some embodiments, R.sup.s is OMe. In some embodiments, R.sup.s is OH. In some embodiments, R.sup.s is NHAc. In some embodiments, R.sup.s is NHCOCF.sub.3. In some embodiments, R is H. In some embodiments, R is H. In some embodiments, R.sup.2s is NHAc, and R.sup.5s is OH. In some embodiments, R.sup.2s is p-anisoyl, and R.sup.5s is OH. In some embodiments, R.sup.2s is NHAc and R.sup.5s is p-anisoyl. In some embodiments, R.sup.2s is OH, and R.sup.5s is p-anisoyl. In some embodiments, R.sup.D is selected from

(2206) ##STR00896## ##STR00897## ##STR00898##
Further embodiments of R.sup.D includes additional chemical moiety embodiments, e.g., those described in the examples.

(2207) In some embodiments, R.sup.D, R.sup.LD or R.sup.TD is or comprises

(2208) ##STR00899##
In some embodiments, R.sup.D, R.sup.LD or R.sup.TD is or comprises

(2209) ##STR00900##
In some embodiments, R.sup.D, R.sup.LD or R.sup.TD is or comprises

(2210) ##STR00901##
In some embodiments, R.sup.D, R.sup.LD or R.sup.TD is or comprises

(2211) ##STR00902##
In some embodiments, R.sup.D, R.sup.LD, R.sup.CD or R.sup.TD is or comprises

(2212) ##STR00903##
In some embodiments, R.sup.D, R.sup.LD, or R.sup.TD is or comprises

(2213) ##STR00904##
In some embodiments, R.sup.D, R.sup.LD, R.sup.CD or R.sup.TD is or comprises N(R.sup.1).sub.2, wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, R.sup.D, R.sup.LD, R.sup.CD or R.sup.TD is or comprises N(R.sup.1).sub.3, wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, R.sup.D, R.sup.LD, R.sup.CD or R.sup.TD is or comprises one or more guanidine moieties. In some embodiments, R.sup.D, R.sup.LD, R.sup.CD or R.sup.TD is or comprises NC(N(R.sup.1).sub.2), wherein each R.sup.1 is independently as described in the present disclosure. In some embodiments, R.sup.D or R.sup.TD is or comprises

(2214) ##STR00905##
In some embodiments, R.sup.D, R.sup.LD or R.sup.TD is or comprises

(2215) ##STR00906##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2216) ##STR00907##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2217) ##STR00908##
In some embodiments, R.sup.D, R.sup.CD, or R.sup.TD is or comprises

(2218) ##STR00909##
In some embodiments, R.sup.D, R.sup.LD, or R.sup.TD is or comprises

(2219) ##STR00910##
In some embodiments, R.sup.D, R.sup.CD, or R.sup.TD is or comprises

(2220) ##STR00911##
In some embodiments, R.sup.D, R.sup.LD, or R.sup.TD is or comprises

(2221) ##STR00912##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2222) ##STR00913##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2223) ##STR00914##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2224) ##STR00915##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2225) ##STR00916##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2226) ##STR00917##
In some embodiments, R.sup.D or R.sup.TD is or comprises

(2227) ##STR00918##
In some embodiments, R.sup.D, R.sup.CD, or R.sup.TD is or comprises

(2228) ##STR00919##
In some embodiments, R.sup.D, R.sup.CD, or R.sup.TD is or comprises

(2229) ##STR00920##
In some embodiments, R.sup.D, R.sup.CD, or R.sup.TD is or comprises

(2230) ##STR00921##
In some embodiments, R.sup.D, R.sup.LD, R.sup.CD or R.sup.TD comprises

(2231) ##STR00922##
In some embodiments, R.sup.D, R.sup.LD, R.sup.CD or R.sup.TD comprises

(2232) ##STR00923##

(2233) In some embodiments, n is 1. In some embodiments, n is 0.

(2234) In some embodiments, n is 1. In some embodiments, n is 2.

(2235) In some embodiments, a moiety of the present disclosure, e.g., a heteroaliphatic, heteroaryl, heterocyclyl, a ring, etc., may contain one or more heteroatoms. In some embodiments, a heteroatom is any atom that is not carbon and is not hydrogen. In some embodiments, each heteroatom is independently selected from boron, nitrogen, oxygen, sulfur, silicon and phosphorus. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus. In some embodiments, each heteroatom is independently selected from boron, nitrogen, oxygen, sulfur and phosphorus. In some embodiments, each heteroatom is independently selected from boron, nitrogen, oxygen, sulfur and silicon. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In some embodiments, at least one heteroatom is nitrogen. In some embodiments, at least one heteroatom is oxygen. In some embodiments, at least one heteroatom is sulfur.

(2236) In some embodiments, y, t, n and m, e.g., in a stereochemistry pattern, each are independently 1-20 as described in the present disclosure. In some embodiments, y is 1. In some embodiments, y is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10.

(2237) In some embodiments, n is 1. In some embodiments, n is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, n is 1-10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 3, 4, 5, 6, 7 or 8. In some embodiments, n is 4, 5, 6, 7 or 8. In some embodiments, n is 5, 6, 7 or 8. In some embodiments, n is 6, 7 or 8. In some embodiments, n is 7 or 8. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

(2238) In some embodiments, m is 0-50. In some embodiments, m is 1-50. In some embodiments, m is 1. In some embodiments, m is 2-50. In some embodiments, m is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, m is 3, 4, 5, 6, 7 or 8. In some embodiments, m is 4, 5, 6, 7 or 8. In some embodiments, m is 5, 6, 7 or 8. In some embodiments, m is 6, 7 or 8. In some embodiments, m is 7 or 8. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15. In some embodiments, m is 16. In some embodiments, m is 17. In some embodiments, m is 18. In some embodiments, m is 19. In some embodiments, m is 20. In some embodiments, m is 21. In some embodiments, m is 22. In some embodiments, m is 23. In some embodiments, m is 24. In some embodiments, m is 25. In some embodiments, m is at least 2. In some embodiments, m is at least 3. In some embodiments, m is at least 4. In some embodiments, m is at least 5. In some embodiments, m is at least 6. In some embodiments, m is at least 7. In some embodiments, m is at least 8. In some embodiments, m is at least 9. In some embodiments, m is at least 10. In some embodiments, m is at least 11. In some embodiments, m is at least 12. In some embodiments, m is at least 13. In some embodiments, m is at least 14. In some embodiments, m is at least 15. In some embodiments, m is at least 16. In some embodiments, m is at least 17. In some embodiments, m is at least 18. In some embodiments, m is at least 19. In some embodiments, m is at least 20. In some embodiments, m is at least 21. In some embodiments, m is at least 22. In some embodiments, m is at least 23. In some embodiments, m is at least 24. In some embodiments, m is at least 25. In some embodiments, m is at least greater than 25.

(2239) In some embodiments, t is 1-20. In some embodiments, t is 1. In some embodiments, t is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is 1-5. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9. In some embodiments, t is 10. In some embodiments, t is 11. In some embodiments, t is 12. In some embodiments, t is 13. In some embodiments, t is 14. In some embodiments, t is 15. In some embodiments, t is 16. In some embodiments, t is 17. In some embodiments, t is 18. In some embodiments, t is 19. In some embodiments, t is 20.

(2240) In some embodiments, each of t and m is independently at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, each of t and m is independently at least 3. In some embodiments, each of t and m is independently at least 4. In some embodiments, each of t and m is independently at least 5. In some embodiments, each of t and m is independently at least 6. In some embodiments, each of t and m is independently at least 7. In some embodiments, each of t and m is independently at least 8. In some embodiments, each of t and m is independently at least 9. In some embodiments, each of t and m is independently at least 10.

(2241) As used in the present disclosure, in some embodiments, one or more is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, one or more is one. In some embodiments, one or more is two. In some embodiments, one or more is three. In some embodiments, one or more is four. In some embodiments, one or more is five. In some embodiments, one or more is six. In some embodiments, one or more is seven. In some embodiments, one or more is eight. In some embodiments, one or more is nine. In some embodiments, one or more is ten. In some embodiments, one or more is at least one. In some embodiments, one or more is at least two. In some embodiments, one or more is at least three. In some embodiments, one or more is at least four. In some embodiments, one or more is at least five. In some embodiments, one or more is at least six. In some embodiments, one or more is at least seven. In some embodiments, one or more is at least eight. In some embodiments, one or more is at least nine. In some embodiments, one or more is at least ten. As used in the present disclosure, in some embodiments, at least one is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, at least one is one. In some embodiments, at least one is two. In some embodiments, at least one is three. In some embodiments, at least one is four. In some embodiments, at least one is five. In some embodiments, at least one is six. In some embodiments, at least one is seven. In some embodiments, at least one is eight. In some embodiments, at least one is nine. In some embodiments, at least one is ten.

(2242) In some embodiments, the present disclosure provides the following embodiments:

(2243) 1. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by:

(2244) 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages.
2. The oligonucleotide composition of embodiment 1, wherein the oligonucleotide composition being characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
3. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and the oligonucleotide composition being characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
4. The composition of any one of the preceding embodiments, wherein each chiral internucleotidic linkage of the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.
5. The composition of any one of the preceding embodiments, wherein each chiral modified internucleotidic linkage independently has a stereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus.
6. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications, which composition is chirally controlled and it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, pattern of backbone linkages and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type, wherein: the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of inclusion of a nucleic acid sequence is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
7. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Sp.
8. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Rp.
9. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; and 3) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages; the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of inclusion of a nucleic acid sequence is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
10. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently an internucleotidic linkage at least 50% of which exists in its non-negatively charged form at pH 7.4.
11. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently a neutral internucleotidic linkage, wherein at least 50% of the internucleotidic linkage exists in its neutral form at pH 7.4.
12. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.
13. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage, when the units which it connects are replaced with CH.sub.3, independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.
14. The composition of any one of the preceding embodiments, wherein the reference condition is absence of the composition.
15. The composition of any one of the preceding embodiments, wherein the reference condition is presence of a reference composition.
16. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no chirally controlled internucleotidic linkages.
17. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no non-negatively charged internucleotidic linkages.
18. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises one or more backbone linkages selected from phosphodiester, phosphorothioate and phosphodithioate linkages.
19. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise one or more sugar modifications.
20. The composition of any one of the preceding embodiments, wherein the sugar modifications comprise one or more modifications selected from: 2-O-methyl, 2-MOE, 2-F, morpholino and bicyclic sugar moieties.
21. The composition of any one of the preceding embodiments, wherein one or more sugar modifications are 2-F modifications.
22. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 5-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety.
23. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 3-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety.
24. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a middle region between the 5-end region and the 3-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.
25. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; and 3) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise: 1) a 5-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety; 2) a 3-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety; and 3) a middle region between the 5-end region and the 3-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.
26. The composition of embodiment 25, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of inclusion of a nucleic acid sequence is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
27. The composition of any one of the preceding embodiments, wherein the 5-end region comprises 1 or more nucleoside units not comprising a 2-F modified sugar moiety.
28. The composition of any one of the preceding embodiments, wherein the 3-end region comprises 1 or more nucleoside units not comprising a 2-F modified sugar moiety.
29. The composition of any one of the preceding embodiments, wherein the middle region comprises 1 or more nucleotidic units comprising no phosphodiester linkage.
30. The composition of any one of the preceding embodiments, wherein the first of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety and a modified internucleotidic linkage of the 5-end is the first, second, third, fourth or fifth nucleoside unit of the oligonucleotide from the 5-end, and the last of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety and a modified internucleotidic linkage of the 3-end is the last, second last, third last, fourth last, or fifth last nucleoside unit of the oligonucleotide.
31. The composition of any one of the preceding embodiments, wherein the 5-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
32. The composition of any one of the preceding embodiments, wherein the 5-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
33. The composition of any one of the preceding embodiments, wherein the 3-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
34. The composition of any one of the preceding embodiments, wherein the 3-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
35. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2-F modified sugar moiety in the 5-end region is independently a modified internucleotidic linkage.
36. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2-F modified sugar moiety in the 3-end region is independently a modified internucleotidic linkage.
37. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is independently a chiral internucleotidic linkage.
38. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.
39. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.
40. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage.
41. The composition of embodiment 35 or 36, wherein each modified internucleotidic linkage is a Sp chirally controlled phosphorothioate internucleotidic linkage.
42. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages.
43. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages each independently between a nucleoside unit comprising a 2-OR.sup.1 modified sugar moiety and a nucleoside unit comprising a 2-F modified sugar moiety, or between two nucleoside units each independently comprising a 2-OR.sup.1 modified sugar moiety, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl.
44. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
45. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages each independently between a nucleoside unit comprising a 2-OR.sup.1 modified sugar moiety and a nucleoside unit comprising a 2-F modified sugar moiety, or between two nucleoside units each independently comprising a 2-OR.sup.1 modified sugar moiety, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl.
46. The composition of embodiment 43 or 45, wherein 2-OR.sup.1 is 2-OCH.sub.3.
47. The composition of embodiment 43 or 45, wherein 2-OR.sup.1 is 2-OCH.sub.2CH.sub.2OCH.sub.3.
48. The composition of any one of the preceding embodiments, wherein the 5-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.
49. The composition of any one of the preceding embodiments, wherein the 5-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.
50. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 5-end region is a chiral modified internucleotidic linkage.
51. The composition of any one of the preceding embodiments, wherein the 3-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.
52. The composition of any one of the preceding embodiments, wherein the 3-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.
53. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 3-end region is a chiral modified internucleotidic linkage.
54. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.
55. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.
56. The composition of any one of embodiments 48-55, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.
57. The composition of any one of embodiments 48-55, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage wherein its chirally controlled linkage phosphorus has a Sp configuration.
58. The composition of any one of embodiments 48-57, wherein each chiral modified internucleotidic linkage is independently a chirally controlled phosphorothioate internucleotidic linkage.
59. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-negatively charged internucleotidic linkages.
60. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 neutral internucleotidic linkages.
61. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chiral internucleotidic linkage.
62. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chirally controlled internucleotidic linkage independently of Rp or Sp at its linkage phosphorus.
63. The composition of any one of the preceding embodiments, wherein the base sequence comprises a sequence having no more than 5 mismatches from a 20 base long portion of the dystrophin gene or its complement.
64. The composition of any one of the preceding embodiments, wherein the length of the base sequence of the oligonucleotides of the plurality is no more than 50 bases.
65. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled centers independently of Rp or Sp.
66. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 5 chirally controlled centers independently of Rp or Sp.
67. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 6 chirally controlled centers independently of Rp or Sp.
68. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 10 chirally controlled centers independently of Rp or Sp.
69. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular oligonucleotide type are capable of mediating skipping of one or more exons of the dystrophin gene.
70. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45, 51 or 53 of the dystrophin gene.
71. The composition of embodiment 70, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45 of the dystrophin gene.
72. The composition of embodiment 70, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 51 of the dystrophin gene.
73. The composition of embodiment 70, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 53 of the dystrophin gene.
74. The composition of any one of preceding embodiments, wherein the composition provides exon skipping of two or more exons.
75. The composition of embodiment 71, wherein the base sequence comprises a sequence having no more than 5 mismatches from a sequence of Table A1.
76. The composition of embodiment 71, wherein the base sequence comprises or is a sequence of Table A1.
77. The composition of embodiment 71, wherein the base sequence is a sequence of Table A1.
78. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality are oligonucleotides of an oligonucleotide selected from Table A1.
79. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
80. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.
81. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.
82. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.
83. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure.
84. The composition of any one of the preceding embodiments, wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
85. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.
86. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.
87. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.
88. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise or consist of a wing-core-wing structure, and wherein only one wing comprise one or more non-negatively charged internucleotidic linkages.
89. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
90. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.
91. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.
92. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.
93. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage.
94. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage or a natural phosphate internucleotidic linkage.
95. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage.
96. The composition of any one of embodiments 93-95, wherein the percentage is 50% or more.
97. The composition of any one of embodiments 93-95, wherein the percentage is 60% or more.
98. The composition of any one of embodiments 93-95, wherein the percentage is 75% or more.
99. The composition of any one of embodiments 93-95, wherein the percentage is 80% or more.
100. The composition of any one of embodiments 93-95, wherein the percentage is 90% or more.
101. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.
102. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.
103. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.
104. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.
105. The composition of any one of the preceding embodiments, wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
106. The composition of any one of the preceding embodiments, wherein all non-negatively charged internucleotidic linkages of the same oligonucleotide have the same constitution.
107. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.
108. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.
109. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.
110. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular type are structurally identical.
111. The composition of any one of the preceding claims, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a carbohydrate moiety, a peptide moiety, a receptor ligand moiety, or a moiety having the structure of N(R.sup.1).sub.2, N(R.sup.1).sub.3, or NC(N(R.sup.1).sub.2).sub.2.
112. The composition of any one of the preceding claims, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a guanidine moiety.
113. The composition of any one of the preceding claims, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises NC(N(CH.sub.3).sub.2).sub.2.
114. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the same constitution as oligonucleotides of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.
115. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.
116. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.
117. The composition of any one of embodiments 114-116, wherein the percentage is at least 10%.
118. The composition of any one of embodiments 114-116, wherein the percentage is at least 50%.
119. The composition of any one of embodiments 114-116, wherein the percentage is at least 80%.
120 The composition of any one of embodiments 114-116, wherein the percentage is at least 90%.
121. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage is a phosphoramidate linkage.
122. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage comprises a guanidine moiety.
123. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I:

(2245) ##STR00924##
or a salt form thereof, wherein: P.sup.L is P(W), P, or P.fwdarw.B(R).sub.3; W is O, N(-L-R.sup.5), S or Se; each of R.sup.1 and R.sup.5 is independently H, -L-R, halogen, CN, NO.sub.2, -L-Si(R).sub.3, OR, SR, or N(R).sub.2; each of X, Y and Z is independently O, S, N(-L-R.sup.5), or L; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
124. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I or a salt form thereof.
125. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-1 or a salt form thereof:

(2246) ##STR00925##
126. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-1 or a salt form thereof.
127. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-2 or a salt form thereof:

(2247) ##STR00926##
128. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof:

(2248) ##STR00927##
129. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof.
130. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
131. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
132. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.
133. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.
134. The composition of any one of embodiments 128-131, wherein the ring formed is a saturated ring.
135. The composition of any one of embodiments 128-131, wherein the ring formed is a partially unsaturated ring.
136. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II:

(2249) ##STR00928##
or a salt form thereof, wherein: P.sup.L is P(W), P, or P.fwdarw.B(R).sub.3; W is O, N(-L-R.sup.5), S or Se;
each of X, Y and Z is independently-O, S, N(-L-R.sup.5), or L; R.sup.s is H, -L-R, halogen, CN, NO.sub.2, -L-Si(R).sub.3, OR, SR, or N(R).sub.2; Ring A.sup.L is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R.sup.s is independently H, halogen, CN, N.sub.3, NO, NO.sub.2, -L-R, -L-Si(R).sub.3, -L-OR, -L-SR, -L-N(R).sub.2, O-L-R, O-L-Si(R).sub.3, O-L-OR, O-L-SR, or O-L-N(R).sub.2; g is 0-20; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or, two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
137. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II, or a salt form thereof.
138. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-1:

(2250) ##STR00929##
or a salt form thereof.
139. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-a-1, or a salt form thereof.
140. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-2:

(2251) ##STR00930##
or a salt form thereof.
141. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-a-2, or a salt form thereof.
142. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-b-1:

(2252) ##STR00931##
or a salt form thereof.
143. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-b-1, or a salt form thereof.
144. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-b-2:

(2253) ##STR00932##
or a salt form thereof.
145. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-b-2, or a salt form thereof.
146. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-c-1:

(2254) ##STR00933##
or a salt form thereof.
147. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-c-1, or a salt form thereof.
148. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-c-2:

(2255) ##STR00934##
or a salt form thereof.
149. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-c-2, or a salt form thereof.
150. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-d-1:

(2256) ##STR00935##
or a salt form thereof.
151. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-d-1, or a salt form thereof.
152. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-d-2:

(2257) ##STR00936##
or a salt form thereof.
153. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-d-2, or a salt form thereof.
154. The composition of any one of embodiments 136-153, wherein each non-negatively charged internucleotidic linkage has the same structure.
155. The composition of any one of the preceding embodiments, wherein, if applicable, each internucleotidic linkage in the oligonucleotides of the plurality that is not a non-negatively charged internucleotidic linkage independently has the structure of formula I.
156. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the oligonucleotides of the plurality independently has the structure of formula I.
157. The composition of any one of the preceding embodiments, wherein one or more P.sup.L is P(W).
158. The composition of any one of the preceding embodiments, wherein each P.sup.L is independently P(W).
159. The composition of any one of the preceding embodiments, wherein one or more W is O.
160. The composition of any one of the preceding embodiments, wherein each W is O.
161. The composition of any one of the preceding embodiments, wherein one or more Y is O.
162. The composition of any one of the preceding embodiments, wherein each Y is O.
163. The composition of any one of the preceding embodiments, wherein one or more Z is O.
164. The composition of any one of the preceding embodiments, wherein each Z is O.
165. The composition of any one of the preceding embodiments, wherein one or more X is O.
166. The composition of any one of the preceding embodiments, wherein one or more X is S.
167. The composition of any one of the preceding embodiments, wherein a non-negatively charged

(2258) ##STR00937##
internucleotidic linkage has the structure of
168. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

(2259) ##STR00938##
169. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

(2260) ##STR00939##
170. The composition of any one of the preceding embodiments, wherein for each internucleotidic linkage of formula I or a salt fore thereof that is not a non-negatively charged internucleotidic linkage, X is independently O or S, and -L.sup.s-R.sup.5 is H (natural phosphate linkage or phosphorothioate linkage, respectively).
171. The composition of any one of the preceding embodiments, wherein each phosphorothioate linkage, if any, in the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.
172. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled oligonucleotide composition.
173. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled oligonucleotide composition.
174. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a targeting moiety wherein the targeting moiety is independently connected to an oligonucleotide backbone through a linker.
175. The composition of embodiment 174, wherein the targeting moiety is a carbohydrate moiety.
176. The composition of embodiment 174 or 175, wherein the targeting moiety comprises or is a GalNAc moiety.
177. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a lipid moiety wherein the lipid moiety is independently connected to an oligonucleotide backbone through a linker.
178. The composition of any one of the preceding embodiments, wherein the oligonucleotide of the plurality comprise a pattern of backbone chiral centers of (Np/Op)t[(Rp)n(Sp)m]y, (Np/Op)t[(Op)n(Sp)m]y, (Np/Op)t[(Op/Rp)n(Sp)m]y, (Sp)t[(Rp)n(Sp)m]y, (Sp)t[(Op)n(Sp)m]y, (Sp)t[(Op/Rp)n(Sp)m]y, [(Rp)n(Sp)m]y, [(Op)n(Sp)m]y, [(Op/Rp)n(Sp)m]y, (Rp)t(Np)n(Rp)m, (Rp)t(Sp)n(Rp)m, (Rp)t[(Np/Op)n]y(Rp)m, (Rp)t[(Sp/Np)n]y(Rp)m, (Rp)t[(Sp/Op)n]y(Rp)m, (Np/Op)t(Np)n(Np/Op)m, (Np/Op)t(Sp)n(Np/Op)m, (Np/Op)t[(Np/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Np/Op)t[(Sp/Op)n]y(Np/Op)m, (Rp/Op)t(Np)n(Rp/Op)m, (Rp/Op)t(Sp)n(Rp/Op)m, (Rp/Op)t[(Np/Op)n]y(Rp/Op)m, (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m, or (Rp/Op)t[(Sp/Op)n]y(Rp/Op)m.
179. The composition of any one of the preceding embodiments, wherein the oligonucleotide of the plurality comprise a pattern of backbone chiral centers of (Sp)t[(Rp)n(Sp)m]y.
180. The composition of any one of the preceding embodiments, wherein y is 1.
181. The composition of any one of the preceding embodiments, wherein n is 1.
182. The composition of any one of the preceding embodiments, wherein t is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
183. The composition of any one of the preceding embodiments, wherein t is 4, 5, 6, 7, 8, 9 or 10.
184. The composition of any one of the preceding embodiments, wherein m is 2, 3, 4, 5, 6, 7, 8, 9 or 10.
185. The composition of any one of the preceding embodiments, wherein m is 4, 5, 6, 7, 8, 9 or 10.
186. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality has the structure of formula O-I or a salt thereof.
187. The composition of any one of the preceding embodiments, wherein L.sup.P in formula O-I independently has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.
188. The composition of any one of the preceding embodiments, wherein a

(2261) ##STR00940##
is

(2262) ##STR00941##
189. The composition of any one of the preceding embodiments, wherein a

(2263) ##STR00942##
is

(2264) ##STR00943##
190. The composition of any one of the preceding embodiments, wherein a

(2265) ##STR00944##
is

(2266) ##STR00945##
191. The composition of any one of the preceding embodiments, wherein a

(2267) ##STR00946##
is optionally substituted

(2268) ##STR00947##
192. The composition of any one of the preceding embodiments, wherein L.sup.s in formula O-I between L.sup.P and Ring A is C(R.sup.5s).sub.2.
193. The composition of any one of the preceding embodiments, wherein L.sup.s in formula O-I between L.sup.P and Ring A is CH(R.sup.5s).
194. The composition of any one of the preceding embodiments, wherein -L.sup.3E-R.sup.3E in formula O-I IS OH.
195. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality has the structure of A.sup.c-[-L.sup.LD-(R.sup.LD).sub.a].sub.b, A.sup.c-[-L.sup.M (R.sup.D).sub.a].sub.b, [(A.sup.c).sub.a-L.sup.M].sub.b-R.sup.D, (A.sup.c).sub.a-L.sup.M-(A.sup.c).sub.b, or (A.sup.c).sub.a-L.sup.M-(R.sup.D).sub.b, or a salt thereof.
196. The composition of embodiment 195, wherein H-A.sup.c, [H].sub.a-A.sup.c or [H].sub.b-A.sup.c is an oligonucleotide of any one of embodiments 186-194.
197. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more non-neutral internucleotidic linkages at the condition of the composition independently exist as a salt form.
198. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a salt form.
199. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a metal salt.
200. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as a metal salt.
201. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as sodium salt.
202. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage is independently a natural phosphate linkage (the neutral form of which is OP(O)(OH)O) or phosphorothioate internucleotidic linkage (the neutral form of which is OP(O)(SH)O).
203. The composition of any one of the preceding embodiments, wherein each heteroatom in heteroaliphatic, heteroalkyl, heterocyclyl, or heteroaryl is independently boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.
204. The composition of any one of the preceding embodiments, wherein each heteroatom in heteroaliphatic, heteroalkyl, heterocyclyl, or heteroaryl is independently nitrogen, oxygen, silicon, sulfur, or phosphorus.
205. The composition of any one of the preceding embodiments, wherein each heteroatom in heteroaliphatic, heteroalkyl, heterocyclyl, or heteroaryl is independently nitrogen, oxygen, or sulfur.
206. A pharmaceutical composition comprising an oligonucleotide composition of any one of the preceding embodiments and a pharmaceutically acceptable carrier.
207. A method for altering splicing of a target transcript, comprising administering an oligonucleotide composition of any one of the preceding embodiments.
208. The method of embodiment 207, wherein the splicing of the target transcript is altered relative to absence of the composition.
209. The method of any one of the preceding embodiments, wherein the alteration is that one or more exon is skipped at an increased level relative to absence of the composition.
210. The method of any one of the preceding embodiments, wherein the target transcript is pre-mRNA of dystrophin.
211. The method of any one of the preceding embodiments, wherein exon 51 of dystrophin is skipped at an increased level relative to absence of the composition.
212. The method of any one of embodiments 207-210, wherein exon 53 of dystrophin is skipped at an increased level relative to absence of the composition.
213. The method of any one of embodiments 207-210, wherein exon 45 of dystrophin is skipped at an increased level relative to absence of the composition.
214. The method of any one of the preceding embodiments, wherein two or more exons of dystrophin is skipped at an increased level relative to absence of the composition
215. The method of any one of the preceding embodiments, wherein a protein encoded by the mRNA with the exon skipped provides one or more functions better than a protein encoded by the corresponding mRNA without the exon skipping.
216. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition of any one of the preceding embodiments.
217. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising (a) administering to a subject susceptible thereto or suffering therefrom a composition of any one of the preceding embodiments, and (b) administering to the subject additional treatment.
218. The method of embodiment 217, wherein the additional treatment is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD).
219. The method of any one of the preceding embodiments, wherein the additional treatment comprises administering a composition of any one of the preceding embodiments, wherein oligonucleotides of the composition have a different base sequence.
220. The method of any one of the preceding embodiments, wherein the additional treatment comprises administering a composition of any one of the preceding embodiments, wherein oligonucleotides of the composition have a different base sequence and target a different exon.
221. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast or myotubule.
222. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell.
223. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell, which is contacted with the composition after 0, 4 or 7 days of pre-differentiation.
224. A composition comprising a combination comprising: (a) a first composition of any of the preceding embodiments; (b) a second composition of any of the preceding embodiments; and, optionally (c) a third composition of any of the preceding embodiments, wherein the first, second and third compositions are different.

EXEMPLIFICATION

(2269) The foregoing has been a description of certain non-limiting embodiments of the disclosure. Accordingly, it is to be understood that embodiments of the disclosure herein described are merely illustrative of applications of principles of the disclosure. Reference herein to details of illustrated embodiments is not intended to limit the scope of any claims.

(2270) Various methods for preparing, and for assessing properties and/or activities of, oligonucleotides and oligonucleotide compositions are widely known in the art and may be utilized in accordance with the present disclosure, including but not limited to those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and WO 2019/055951, the methods and reagents of each of which are incorporated herein by reference. In some embodiments, the present disclosure provides technologies for preparing oligonucleotides and compositions thereof, particularly chirally controlled oligonucleotides which comprise neutral backbones (e.g., n001, n002, n003, n004, n005, n006, n007, n008, n009, n010, etc.) and chirally controlled oligonucleotide compositions thereof, and technologies for assessing and using various oligonucleotides and compositions thereof. Among other things, Applicant describes herein example technologies for preparing, assessing and using provided oligonucleotides and oligonucleotide compositions.

(2271) Functions and advantage of certain embodiments of the present disclosure may be more fully understood from the examples described below. The following examples are intended to illustrate certain benefits of such embodiments.

Example 1. Example Synthesis of Oligonucleotide Compositions

(2272) Technologies for preparing oligonucleotide and compositions thereof are widely known in the art. In some embodiments, oligonucleotides and oligonucleotide compositions of the present disclosure were prepared using technologies, e.g., reagents (e.g., solid supports, coupling reagents, cleavage reagents, phosphoramidites, etc.), chiral auxiliaries, solvents (e.g., for reactions, washing, etc.), cycles, reaction conditions (e.g., time, temperature, etc.), etc., described in one or more of U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, and WO 2019/055951.

Example 2. Example Synthesis of Oligonucleotides Comprising an Internucleotidic Linkage Comprising a Triazole Moiety or an Alkyne Moiety

(2273) Various types of internucleotidic linkages can be prepared in accordance with the present disclosure. Described in this example is preparation of oligonucleotides comprising internucleotidic linkages comprising triazole moieties. As those skilled in the art appreciates, technology described herein can be readily utilized to conjugate various desirable moieties, e.g., those derived from GalNAc, lipids, peptides, ligands, etc. Among other things, such conjugation can be useful for delivery of oligonucleotides to various target systems (e.g., CNS, muscles, eye, etc.).

(2274) Example oligonucleotide comprising internucleotidic linkages comprising triazole moieties.

(2275) ##STR00948## ##STR00949##

(2276) Synthesis scheme for dimer preparation in solution phase.

(2277) ##STR00950##

(2278) Synthesis scheme for dimer preparation on solid support.

(2279) ##STR00951## ##STR00952## ##STR00953##

(2280) Triazole backbone oligonucleotides:

(2281) ##STR00954## ##STR00955##

(2282) Synthesis scheme for dimer preparation in solution phase:

(2283) ##STR00956##

(2284) Synthesis scheme for dimer preparation on solid support:

(2285) ##STR00957## ##STR00958## ##STR00959##

(2286) Alkyne backbone oligonucleotides:

(2287) ##STR00960## ##STR00961##

(2288) Synthesis scheme for dimer preparation on solid support:

(2289) ##STR00962## ##STR00963## ##STR00964##

Example 3. Example Synthesis of Phosphoramidate Internucleotidic Linkages Comprising a Guanidine Moiety

(2290) As illustrated herein, phosphoramidate internucleotidic linkages can be readily prepared from phosphite internucleotidic linkages, including stereopure phosphite internucleotidic linkages, in accordance with the present disclosure.

(2291) ##STR00965##

(2292) To a stirred solution of amidite (474 mg, 0.624 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) and TBS protected alcohol (150 mg, 0.41 mmol, pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) in dry acetonitrile (5.2 ml) was added 5-(ethylthio)-1H-tetrazole (ETT, 2.08 ml, 0.6M, 3 equiv.) under argon atmosphere at room temperature. The reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (356 mg, 1.24 mmol, 3 equiv.) in acetonitrile (1 ml) was added. Once the reaction was completed (after 5 mins, monitored by LCMS) then triethylamine (0.17 ml, 1.24 mmol, 3 equiv) was added and the reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure and then redissolved in dichloromethane (50 ml), washed with water (25 ml), saturated aq. sodium bicarbonate (25 ml), and brine (25 ml), and dried with magnesium sulfate. The solvent was removed under reduced pressure. The crude product was purified by silica gel column (80 g) using DCM (5% triethyl amine) and MeOH as eluent. Product-containing fractions were collected and the solvent was evaporated. The resulted product may contain Triethylamine trihydrochloride (TEA.Math.HCl) salt. To remove the salt, the product was re-dissolved in DCM (50 ml) and washed with saturated aq. sodium bicarbonate (20 ml) and brine (20 ml) then dried with magnesium sulfate and the the solvent was evaporated. A pale yellow solid was obtained. Yield: 440 mg (89%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.34, 1.98. MS calculated for C.sub.51H.sub.65FN.sub.7O.sub.14PSi [M].sup.+ 1078.17, Observed: 1078.57 [M+H].sup.+.

(2293) ##STR00966##
Synthesis of Stereopure (Rp) Dimer.

(2294) To a stirred solution of L-DPSE chiral amidite (1.87 g, 2.08 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) and TBS protected alcohol (500 mg, 1.38 mmol, pre-dried by co-evaporation with dry acetonitrile and under vacuum for a minimum of 12 h) in dry acetonitrile (18 mL) was added 2-(1H-imidazol-1-yl) acetonitrile trifluoromethanesulfonate (CMIMT, 5.54 mL, 0.5M, 2 equiv.) under argon atmosphere at room temperature. The resulting reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (1.18 g, 4.16 mmol, 3 equiv.) in acetonitrile (2 mL) was added. Once the reaction was completed (after 5 mins, monitored by LCMS), the reaction mixture was concentrated under reduced pressure and then redissolved in dichloromethane (70 mL), washed with water (40 mL), saturated aq. sodium bicarbonate (40 mL) and brine (40 mL), and dried with magnesium sulfate. The solvent was removed under reduced pressure. The crude product was purified by silica gel column (120 g) using DCM (5% triethyl amine) and MeOH as eluent. Product containing fractions were collected and the solvent was evaporated. The resulted product contained TEA.Math.HCl salt. To remove the salt, the product was re-dissolved in DCM (50 mL) and washed with saturated aq. sodium bicarbonate (20 mL) and brine (20 mL) and then dried with magnesium sulfate and the solvent was evaporated. A pale yellow foamy solid was obtained. Yield: 710 mg (47%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.38. MS calculated for C.sub.51H.sub.65FN.sub.7O.sub.14PSi [M].sup.+ 1078.17, Observed: 1078.19.

(2295) ##STR00967##
Synthesis of Stereopure (Sp) Dimer

(2296) The same procedure was followed as for the Rp dimer. In place of L-DPSE chiral amidite, D-DPSE chiral amidite was used. A pale yellow foamy solid was obtained. Yield: 890 mg (59%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.93. MS calculated for C.sub.51H.sub.65FN.sub.2O.sub.14PSi [M].sup.+ 1078.17, Observed: 1078.00.

(2297) In an example .sup.31P NMR (internal standard of phosphoric acid at 0.0), the stereorandom preparation showed two peaks at 1.34 and 1.98, respectively; the stereopure Rp preparation showed a peak at 1.93, and the stereopure Sp preparation showed a peak at 1.38.

Example 4A. Preparation of Oligonucleotides with Internucleotidic Linkages Comprising Neutral Guanidinium Group

(2298) In accordance with technologies described in the present disclosure, oligonucleotides with various neutral and/or cationic internucleotidic linkages (e.g., at physiological pH) can be prepared. Illustrated below are preparation of oligonucleotides comprising representative such internucleotidic linkages.

(2299) WV-11237 is an oligonucleotide comprising four internucleotidic linkages having the structure of

(2300) ##STR00968##
(n001) to introduce a neutral nature to the backbone and reduce the overall negative charges of the backbone. Expected molecular weight: 7113.4.

(2301) As an example, one preparation of WV-11237, including certain synthetic conditions and analytical results, is described below. Briefly, stereopure internucleotidic linkages were constructed using L-DPSE amidites and typical DPSE coupling cycles comprising Detritylation.fwdarw.Coupling.fwdarw.Pre-Cap.fwdarw.Thiolation.fwdarw.Post-Cap. Cycles for the n001 internucleotidic linkages were modified and comprised Detritylation.fwdarw.Coupling.fwdarw.Dimethyl imidazolium treatment.fwdarw.Post-cap. Compared to certain oxidation cycles, oxidation steps of oxidizing the P(III), e.g., with I.sub.2-Pyridine (pyr)-water, was replaced with the dimethyl imidazolium treatment.

(2302) Certain conditions and/or results of an example preparation.

(2303) Synthetic scale: 127 mol

(2304) Synthetic Conditions (Stereopure Internucleotidic Linkages)

(2305) TABLE-US-00118 Synthetic Steps Conditions Detritylation 3% DCA in Toluene; 300 cm/hr, 436 UV watch Coupling 2.5 eq. of 0.2M chiral amidite, 67% of 0.6M CMIMT Recycle time: 10 min Pre-Cap B Reagent: 20:30:50::Acetic anhydride:Lutidine:Acetonitrile 1.5 CV, 3 min CT Thiolation Reagent: 0.2M Xanthane Hydride 0.6 CV, 6 mm CT Capping (1:1 Cap A + Cap B) 0.4 CV, 0.8 min CT
Cap A=N-Methylimidazole in acetonitrile, 20/80, v/v (20%: 80%=NMI:ACN (v/v))
Cap B=Acetic anhydride/2,6-Lutidine/Acetonitrile, 20/30/50, v/v/v, 20%:30%:50%=Ac.sub.2O: 2,6-Lutidine:ACN(v/v/v)
Synthetic Conditions (Stereorandom n001)

(2306) TABLE-US-00119 Synthetic Steps Conditions Detritylation 3% DCA in Toluene; 300 cm/hr, 436 UV watch Coupling 2.5 eq. of 0.2M standard amidite, 67% of 0.6M ETT Recycle time: 8 min Dimethyl imidazolium treatment: 2.30 CV, 5 mm CT, 3.5 eq. Capping (1:1 Cap A + Cap B) 0.4 CV, 0.8 min CT
Synthesis Process Parameters: Synthesizer: AKTA Oligopilot 100 Solid Support: CPG 2Fluoro-U, (85 umol/g) Synthetic scale: 127 umol; 1.5 gm Column diameter: 20 mm Column volume: 6.3 mL
Stereopure Coupling Reagents: Monomer: 0.2M in MeCN (2Fluoro-dA-L-DPSE, 2Fluoro-dG-L-DPSE, 2-OMe-A-L-DPSE); 0.2M in 20% isobutyronitrle/MeCN (2Fluoro-dC-L-DPSE, 2Fluoro-U-L-DPSE) Deblocking: 3% Dichloroacetic acid (DCA) in Toluene Activator: 0.6M CMIMT in MeCN Sulfurization: 0.2M Xanthane Hydride in pyridine Cap A: N-Methylimidazole in acetonitrile, 20/80, v/v (20% NMI in MeCN) Cap B: Acetic anhydride/2,6-Lutidine/Acetonitrile, 20/30/50, v/v/v, (Acetic anhydride, Lutidine, MeCN (20:30:50)) Pre-Cap: Neat Cap B
Stereorandom Coupling Reagents: Monomer: 0.2M in MeCN (2OMeA and 2OMeG) Deblocking: 3% DCA in Toluene Activator: 0.6M ETT in MeCN 2-Azido-1,3-dimethylimidazolinium-hexafluorophosphate: 0.1M in MeCN Cap A: 20% NMI in MeCN Cap B: Acetic anhydride, Lutidine, MeCN
Deprotection Condition:
One pot deprotection by first treating the support with 5M Triethylamine trihydrofluoride (TEA.Math.HF) in Dimethylsulfoxid (DMSO), H2O, Triethylamine (pH 6.8). Incubation: 3 h, room temperature, 80 L/mol. Followed by addition of aqueous ammonia (200 L/mol). Incubation: 24 h, 35 C. The deprotected material was sterile filtered using 0.45 m filters.
Yield: 72 O.D./mol
Recipe for 5 Solution of TEA.Math.HF in DMSO/Water, 5/1, v/v:

(2307) TABLE-US-00120 Solvents/ Volume Total Volume Reagent Reagents (mL) (mL) (5X) TEA.HF in DMSO 55.0 100 DMSO/Water, Water 11.0 5/1, v/v Triethylamine (TEA) 9.0 Triethylamine 25.0 trihydrofluoride (TEA.3HF)

(2308) In an example crude UPLC chromatogram, there were four distinct peaks all having same desired molecular weight of 7113.2:

(2309) TABLE-US-00121 RT Area % Area Height 9 7.843 402732 16.75 212901 10 7.884 941388 39.14 327190 11 7.968 595232 24.75 275741 12 8.025 353090 14.68 150141

(2310) The example final QC UPLC chromatogram showed four distinct peaks all having the desired molecular weight of 7113.2 (% Purity 95.32). Crude LC-MS showed a single peak of desired molecular weight of 7113.2 (data not shown). The example final QC LC-MS showed a major peak with the desired molecular weight of 7113.1.

(2311) Other oligonucleotides may be prepared using similar cycle conditions or variants thereof depending on specific chemistries of each oligonucleotides. MS data of certain oligonucleotides are listed below:

(2312) TABLE-US-00122 ID Average Observed WV-11237 7113.40288 7113.1 WV-11340 6967.19736 6967.4 WV-11341 6876.08178 6875.6 WV-11342 6888.1173 6887.7 WV-11343 7072.39402 7072.4 WV-11344 6981.27844 6981.6 WV-11345 6981.27844 6981.6 WV-11346 6981.27844 6981.6 WV-11347 6981.27844 6981.6 WV-11532 6905.78632 6905 WV-11533 7098.86298 7099 WV-12116 7909.88196 7909.4 WV-12117 7909.88196 7909.8 WV-12118 7909.88196 7910.2 WV-12119 7909.88196 7909.4 WV-12120 7909.88196 7909.8 WV-12121 7909.88196 7909.8 WV-12123 7125.35748 7125 WV-12124 6967.19736 6967 WV-12125 6967.19736 6967 WV-12126 6967.19736 6967 WV-12127 7046.27742 7046 WV-12128 7046.27742 7046 WV-12129 7046.27742 7046 WV-12504 8887.86402 8887.5 WV-12505 7278.017 7278.2 WV-12506 8944.9584 8945.2 WV-12507 7335.11138 7334.4 WV-12508 7155.95736 7156.3 WV-12539 7171.78104 7171 WV-12540 7171.78104 7171 WV-12541 7457.21802 7457 WV-12542 7219.97784 7219 WV-12543 7235.97724 7236 WV-12544 7112.86454 7113 WV-12553 6872.0517 6872 WV-12555 6876.08178 6875.8 WV-12556 6888.1173 6887.8 WV-12558 6876.08178 6875.6 WV-12559 6888.1173 6887.7 WV-12876 7204.43754 7204.4 WV-12877 7113.32196 7113.5 WV-12878 7125.35748 7125.4 WV-12879 6919.00056 6919.1 WV-12880 6923.03064 6923.2 WV-12881 6935.06616 6935.3 WV-12882 7094.4195 7094.1 WV-12883 7410.73974 7411.1

Example 4B. Chirally Controlled Non-Negatively Charged Internucleotidic Linkages

(2313) Dimer Synthesis

(2314) This procedure is to make stereopure dimer phosphate backbone followed by incorporating it to the selective sites of oligonucleotides (e.g., antisense oligonucleotide or ASO, single-stranded RNAi agent or ssRNA, etc.). A second approach is to synthesize molecules using an automated oligonucleotide synthesizer to introduce a non-negatively charged internucleotidic linkage, e.g., a neutral internucleotidic linkage, at a specific site or full oligonucleotide.

(2315) ##STR00969## ##STR00970## ##STR00971##

(2316) General experimental procedure (A): To a stirred solution of stereorandom amidite (474 mg, 0.624 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) and TBS protected alcohol (150 mg, 0.41 mmol, pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) in dry acetonitrile (5.2 mL) was added 5-(Ethylthio)-1H-tetrazole (ETT, 2.08 ml, 0.6M, 3 equiv.) under argon atmosphere at room temperature. Resulting reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (356 mg, 1.24 mmol, 3 equiv.) in acetonitrile (1 mL) was added. Once the reaction was completed (after 5 mins, monitored by LCMS) then triethylamine (0.17 mL, 1.24 mmol, 3 equiv.) was added and monitored LCMS. Reaction mixture was concentrated under reduced pressure and then re-dissolved in dichloromethane (50 mL) washed with water (25 mL), saturated aq. Sodium bicarbonate (25 mL) and brine (25 mL) dried with magnesium sulfate. Solvent was removed under reduced pressure. The crude product was purified by silica gel column (80 g) using DCM (2% triethylamine) and MeOH as eluent. Product containing fractions collected and evaporated. Pale yellow solid 1001 obtained. Yield: 440 mg (89%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.34, 1.98. MS (ES) m/z calculated for C.sub.51H.sub.65FN.sub.7O.sub.14PSi [M].sup.+ 1077.40, Observed: 1078.57 [M+H].sup.+.

(2317) ##STR00972##

(2318) General experimental procedure (B) for stereopure (Rp) dimer: To a stirred solution of L (or) D-DPSE chiral amidite (1.87 g, 2.08 mmol, 1.5 equiv., pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) and TBS protected alcohol (500 mg, 1.38 mmol, pre-dried by co-evaporation with dry acetonitrile and kept it under vacuum for minimum 12 h) in dry acetonitrile (18 mL) was added 2-(1H-imidazol-1-yl) acetonitrile trifluoromethanesulfonate (CMIMT, 5.54 mL, 0.5M, 2 equiv.) under argon atmosphere at room temperature. Resulting reaction mixture was stirred for 5 mins then monitored by LCMS and then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (1.18 g, 4.16 mmol, 3 equiv.) in acetonitrile (2 mL) was added. Once the reaction was completed (after 5 mins, monitored by LCMS) then the reaction mixture was concentrated under reduced pressure and then redissolved in dichloromethane (70 mL) washed with water (40 mL), saturated aq. sodium bicarbonate (40 mL) and brine (40 mL) dried with magnesium sulfate. Solvent was removed under reduced pressure. The crude product was purified by silica gel column (120 g) using DCM (2% triethyl amine) and MeOH as eluent. Product containing fractions are evaporated. Pale yellow foamy solid 1002 was obtained. Yield: 710 mg (47%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.38. MS (ES) m/z calculated for C.sub.51H.sub.65FN.sub.7O.sub.14PSi [M].sup.+ 1077.40, Observed: 1078.19 [M+H].sup.+.

(2319) ##STR00973##

(2320) Stereopure (Sp) dimer 1003: The procedure B was followed as shown above. D-DPSE chiral amidite was used. Pale yellow foamy solid was obtained. Yield: 890 mg (59%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.93. MS (ES) m/z calculated for C.sub.51H.sub.65FN.sub.7O.sub.14PSi [M].sup.+ 1077.40, Observed: 1078.00 [M+H].sup.+.

(2321) General experimental procedure (C) for deprotection of TBS group: To a stirred solution of TBS protected compound (9.04 mmol) in trihydrofluoride (THF) (70 mL), was added TBAF (1.0 M, 13.6 mmol) at rt. The reaction mixture was stirred at room temperature for 2-4 h. LCMS showed there was no starting material left, then concentrated followed by purification using ISCO-combiflash system (330 g gold rediSep high performance silica column pre-equilibrated 3 CV with 2% TEA in DCM) and DCM/Methanol/2% TEA as a gradient eluent. Product containing column fractions were pooled together and evaporated followed by drying under high vacuum afforded the pure product.

(2322) General experimental procedure (D) for chiral amidites: The TBS deprotected compound (2.5 mmol) was dried by co-evaporation with 80 mL of anhydrous toluene (30 mL2) at 35 C. and dried under at high vacuum for overnight. Then dried it was dissolved in dry THF (30 mL), followed by the addition of triethylamine (17.3 mmol) then the reaction mixture was cooled to 65 C. [for Guanine flavors: TMS-Cl, 2.5 mmol was added at 65 C., for non-Guanine flavors no TMS-Cl was added]. The THF solution of [(1R,3S,3aS)-1-chloro-3-((methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (or) (1S,3R,3aR)-1-chloro-3-((methyldiphenylsilyl)methyl)tetrahydro-1H,3H-pyrrolo[1,2-c][1,3,2]oxazaphosphole (1.8 equiv.) was added through syringe to the above reaction mixture over 2 min then gradually warmed to room temperature. After 20-30 min, at rt, TLC as well as LCMS indicated starting material was converted to product (reaction time: 1 h). Then the reaction mixture was filtered under argon using air free filter tube, washed with THF and dried under rotary evaporation at 26 C. afforded crude solid material, which was purified by ISCO-combiflash system (40 g gold rediSep high performance silica column (pre-equilibrated 3 CV with CH.sub.3CN/5% TEA then 3 CV with DCM/5% TEA) using DCM/CH.sub.3CN/5% TEA as a solvent (compound eluted at 10-40 DCM/CH.sub.3CN/5% TEA). After evaporation of column fractions pooled together was dried under high vacuum afforded white solid to give isolated yield.

(2323) .sup.31P NMR (internal standard of Phosphoric acid at 0.0): 1001: 1.34 and 1.98. 1002: 1.93. 1003: 1.38. .sup.1H NMR of 1001, 1002, and 1003 demonstrated different chemical shifts for multiple hydrogens of the diastereomers. LCMS showed different retention times for the two diastereomers as well. Under one condition, the following retention times were observed: 1.90 and 2.15 for 1001, 1.92 for one diastereomer, and 2.17 for the other.

(2324) ##STR00974##

(2325) Compound 1004: Procedures B and C followed, Off-white foamy solid, Yield: (36%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.23. MS (ES) m/z calculated for C.sub.47H.sub.54FN.sub.8O.sub.14P [M].sup.+ 1004.34, Observed: 1043.21 [M+K].sup.+.

(2326) Compound 1005: Procedure D used, Off-white foamy solid, Yield: (81%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.43, 2.52. MS (ES) m/z calculated for C.sub.66H.sub.76FN.sub.9O.sub.15P.sub.2Si [M].sup.+ 1343.46, Observed: 1344.85 [M+H].sup.+.

(2327) ##STR00975##

(2328) Compound 1006: Procedures B, and C followed, Off-white foamy solid, Yield: (47%). .sup.31P NMR (162 MHz, CDCl.sub.3) 2.54. MS (ES) m/z calculated for C.sub.47H.sub.54FN.sub.8O.sub.14P [M].sup.+ 1004.34, Observed: 1043.12 [M+K].sup.+.

(2329) Compound 1007: Procedure D used, Off-white foamy solid, Yield: (81%). .sup.31P NMR (162 MHz, CDCl.sub.3) 153.55, 2.20. MS (ES) m/z calculated for C.sub.66H.sub.76FN.sub.9O.sub.15P.sub.2Si [M].sup.+ 1343.46, Observed: 1344.75 [M+H].sup.+.

(2330) ##STR00976##

(2331) Compound 1008: Procedures B and C followed, Off-white foamy solid, Yield: (36%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.38. MS (ES) m/z calculated for C.sub.58H.sub.63FN.sub.13O.sub.13P [M].sup.+ 1199.43, Observed: 1200.76 [M+H].sup.+.

(2332) Compound 1009: Procedure D used, Off-white foamy solid, Yield: (60%). .sup.31P NMR (162 MHz, CDCl.sub.3) 157.26, 2.86. MS (ES) m/z calculated for C.sub.77H.sub.85FN.sub.14O.sub.14P.sub.2Si [M].sup.+ 1538.55, Observed: 1539.93 [M+H].sup.+.

(2333) ##STR00977##

(2334) Compound 1010: Procedures B and C followed, Off-white foamy solid, Yield: (36%). 31p NMR (162 MHz, CDCl.sub.3) 2.82. MS (ES) m/z calculated for C.sub.58H.sub.63FN.sub.13O.sub.13P [M].sup.+ 1199.43, Observed: 1200.19 [M+H].sup.+.

(2335) Compound 1011: Procedure D used, Off-white foamy solid, Yield: (63%). .sup.31P NMR (162 MHz, CDCl.sub.3) 159.56, 2.99. MS (ES) m/z calculated for C.sub.77H.sub.85FN.sub.14O.sub.14P.sub.2Si [M].sup.+ 1538.55, Observed: 1539.83 [M+H].sup.+.

(2336) ##STR00978##

(2337) Compound 1012: Procedures B and C followed, Off-white foamy solid, Yield: (36%). [].sub.D.sup.23=25.74 (c 1.06, CHCl.sub.3). .sup.31P NMR (162 MHz, Chloroform-d) 1.83. .sup.1H NMR (400 MHz, Chloroform-d) 12.14 (s, 1H), 11.28 (s, 1H), 9.15 (s, 1H), 8.56 (s, 1H), 8.25-7.94 (m, 2H), 7.90 (s, 1H), 7.72-7.48 (m, 2H), 7.44 (dd, J=8.2, 6.7 Hz, 2H), 7.35-7.26 (m, 2H), 7.24-7.02 (m, 8H), 6.81-6.56 (m, 4H), 6.04 (d, J=5.2 Hz, 1H), 5.67 (d, J=5.5 Hz, 1H), 4.83 (dt, J=8.6, 4.4 Hz, 1H), 4.71-4.54 (m, 2H), 4.49 (dt, J=14.2, 4.8 Hz, 2H), 4.35 (ddt, J=11.0, 5.1, 3.2 Hz, 1H), 4.28-4.09 (m, 2H), 3.68 (s, 6H), 3.37 (d, J=3.3 Hz, 7H), 3.33-3.17 (m, 5H), 2.82 (s, 5H), 2.74-2.60 (m, 1H), 1.92 (s, 2H), 1.72-1.50 (m, 1H), 1.08 (d, J=6.9 Hz, 3H), 0.94 (d, J=6.9 Hz, 3H). MS (ES) m/z calculated for C.sub.59H.sub.66N.sub.13O.sub.14P 1211.45 [M].sup.+, Observed: 1212.42 [M+H].sup.+.

(2338) Compound 1013: Procedure D used, Off-white foamy solid, Yield: (78%). [].sub.D.sup.23=15.48 (c 0.96, CHCl.sub.3). .sup.31P NMR (162 MHz, Chloroform-d) 159.42, 2.47. MS (ES) m/z calculated for C.sub.78H.sub.88N.sub.14O.sub.15P.sub.2Si 1550.57 [M].sup.+, Observed: 1551.96 [M+H].sup.+.

(2339) ##STR00979##

(2340) Compound 1014: Procedures B and C followed, Off-white foamy solid, Yield: (30%). [].sub.D.sup.23=21.45 (c 0.55, CHCl.sub.3). MS (ES) m/z calculated for C.sub.59H.sub.66N.sub.13O.sub.14P 1211.45 [M].sup.+, Observed: 1212.80 [M+H].sup.+.

(2341) Compound 1015: Procedure D used, Off-white foamy solid, Yield: (68%). [].sub.D.sup.23=15.63 (c 1.44, CHCl.sub.3). MS (ES) m/z Calculated for C.sub.78H.sub.88N.sub.14O.sub.15P.sub.2Si 1550.57 [M].sup.+, Observed: 1551.77 [M+H].sup.+.

(2342) Compound 1016: Procedure D used, Off-white foamy solid, Yield: (64%). .sup.31P NMR (162 MHz, CDCl.sub.3) 156.64, 2.67. MS (ES) m/z Calculated for C.sub.78H.sub.88N.sub.14O.sub.15P.sub.2Si 1550.57 [M].sup.+, Observed: 1551.77 [M+H].sup.+.

(2343) ##STR00980##

(2344) General experimental procedure (E) for stereopure dimer using sulfonyl amidite: To a stirred solution of steropure sulfonyl amidite 1017 (259 mg, 0.275 mmol, 1.5 equiv) and TBS protected alcohol (100 mg, 0.18 mmol) in dry acetonitrile (2 mL) was added 2-(1H-imidazol-1-yl) acetonitrile trifluoromethanesulfonate (CMIMT, 0.73 mL, 0.36 mmol, 0.5M, 2 equiv.) under argon atmosphere at room temperature. Resulting reaction mixture was stirred for 5 mins and monitored by LCMS then a mixture of acetic anhydride (2M in ACN, 0.18 ml, 0.36 mmol, 2 equ) and lutidine (2M in ACN, 0.18 ml, 0.36 mmol, 2 equ) was added then stirred for 5 mins then a solution of 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (104.7 mg, 0.367 mmol, 2 equiv.) in acetonitrile (1 mL) was added. Once the reaction was completed (after 5 mins, monitored by LCMS) then triethylamine (0.13 mL, 0.91 mmol, 5 equiv.) was added and monitored by LCMS. Once the reaction was completed, it was concentrated under reduced pressure and then re-dissolved in dichloromethane (50 mL) washed with water (25 mL), saturated aq. Sodium bicarbonate (25 mL) and brine (25 mL) dried with magnesium sulfate. Solvent was removed under reduced pressure. The crude product was purified by silica gel column (80 g) using DCM (2% triethylamine) and MeOH as eluent. Product containing fractions collected and evaporated. Off white solid 1018 obtained. Yield: 204 mg (82%). .sup.31P NMR (162 MHz, CDCl.sub.3) 1.87. MS (ES) m/z calculated for C.sub.74H.sub.75FN.sub.10O.sub.14P [M].sup.+ 1359.44, Observed: 1360.39 [M+H].sup.+.

(2345) Additional phosphoramidites that may be utilized for synthesis include:

(2346) ##STR00981##
Additional useful chiral auxiliaries include:

(2347) ##STR00982##
Other phosphoramidites and chiral auxiliaries, such as those described in U.S. Pat. Nos. 9,695,211, 9,605,019, 9,598,458, US 2013/0178612, US20150211006, US20170037399, WO 2017/015555, WO 2017/062862, WO 2017/160741, WO 2017/192664, WO 2017/192679, WO 2017/210647, WO 2018/098264, WO 2018/223056, and/or WO 2018/237194, the chiral auxiliaries and phosphoramidites of each of which is incorporated by reference.

Example 4C. Synthesis of N.SUP.2.,N.SUP.6.-bis(4-sulfamoylbenzoyl)-L-lysine

(2348) ##STR00983##

(2349) Step 1. To a solution of 4-sulfamoylbenzoic acid (10.00 g, 49.70 mmol) and HOSu (6.29 g, 54.67 mmol) in DMF (300 mL) was added DCC(10.25 g, 49.70 mmol) at 0 C. The mixture was stirred at 0 C. for 16 hours. LCMS showed compound was consumed. The resulting mixture was combined and workup with another batch of crude (1 g scale). The white suspension of N,N-dicyclohexylurea (DCU) was filtered and removed white solid. The filtrate was concentrated to give an oil. This crude product was washed with hot 2-propanol (50 mL*3) to afford an off-white solid. Compound (2,5-dioxopyrrolidin-1-yl) 4-sulfamoylbenzoate (11.80 g, 38.66 mmol, 77.78% yield, 97.713% purity) (yield from conversion rate for 10 g batch) was obtained as a white solid. Compound (2,5-dioxopyrrolidin-1-yl) 4-sulfamoylbenzoate (13 g) was totally obtained as a white solid for two batches of reactions. .sup.1H NMR (400 MHz, CHLOROFORM-d) =8.30 (d, J=8.4 Hz, 2H), 8.08 (d, J=8.3 Hz, 2H), 7.70 (s, 2H), 2.96-2.87 (m, 4H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) =170.62, 161.47, 150.32, 131.40, 127.65, 127.18, 26.04; HPLC purity: 97.71%.

(2350) Step 2. To a solution of (2,5-dioxopyrrolidin-1-yl) 4-sulfamoylbenzoate (5.00 g, 16.76 mmol) and (2S)-2,6-diaminohexanoic acid (1.23 g, 8.38 mmol) in H.sub.2O (50 mL) and DMF (50.00 mL) was added NaHCO.sub.3 (2.11 g, 25.14 mmol). The mixture was stirred at 15 C. for 16 hours. LCMS showed MS with desired compound was detected. The mixture concentrated under reduced pressure to give a crude (6 g). The crude (3.5 g) was purified by prep-HPLC(column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water(0.1% TFA)-ACN]; B %: 1%-30%,20 min). N.sup.2,N.sup.6-bis(4-sulfamoylbenzoyl)-L-lysine (1.40 g, 30.40% yield, 93.268% purity) was obtained as a white solid and 2.5 g crude as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =12.64 (br s, 1H), 8.80 (br d, J=7.5 Hz, 1H), 8.65 (br t, J=5.3 Hz, 1H), 8.04 (d, J=8.2 Hz, 2H), 7.99-7.95 (m, 2H), 7.95-7.84 (m, 4H), 7.48 (br d, J=11.6 Hz, 4H), 4.44-4.32 (m, 1H), 3.28 (br d, J=6.1 Hz, 2H), 1.94-1.71 (m, 3H), 1.63-1.36 (m, 4H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) =174.04, 166.08, 165.58, 146.89, 146.57, 138.05, 137.36, 128.60, 128.26, 126.05, 53.21, 30.77, 29.11, 23.84. LCMS (MH.sup.+): 511.0 (M+H).sup.+; HPLC purity: 93.268%.

Example 4D. Example Technologies for Chirally Controlled Oligonucleotide PreparationExample Useful Chiral Auxiliaries

(2351) Among other things, the present disclosure provides technologies (e.g., chiral auxiliaries, phosphoramidites, cycles, conditions, reagents, etc.) that are useful for preparing chirally controlled internucleotidic linkages. In some embodiments, provided technologies are particularly useful for preparing certain internucleotidic linkages, e.g., non-negatively charged internucleotidic linkages, neutral internucleotidic linkages, etc., comprising PN, wherein P is the linkage. In some embodiments, the linkage phosphorus is trivalent. In some embodiments, the linkage phosphorus is pentavalent. In some embodiments, such internucleotidic linkages have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. Certain example technologies (chiral auxiliaries and their preparations, phosphoramidites and their preparations, cycles, conditions, reagents, etc.) are described in the Examples herein. Among other things, such chiral auxiliaries provide milder reaction conditions, higher functional group compatibility, alternative deprotection and/or cleavage conditions, higher crude and/or purified yields, higher crude purity, higher product purity, and/or higher (or substantially the same or comparable) stereoselectivity when compared to a reference chiral auxiliary (e.g., of formula O, P, Q, R or DPSE).

(2352) ##STR00984##

(2353) Two batches in parallel: To a solution of methylsulfonylbenzene (102.93 g, 658.96 mmol, 1.5 eq.) in THF (600 mL) was added KHMDS (1 M, 658.96 mL, 1.5 eq.) dropwise at 70 C., and warmed to 30 C. slowly over 30 min. The mixture was then cooled to 70 C. A solution of compound 1 (150 g, 439.31 mmol, 1 eq.) in THF (400 mL) was added dropwise at 70 C. The mixture was stirred at 70 C. for 3 hr. TLC(Petroleum ether: Ethyl acetate=3:1, Rf=0.1) indicated compound 1 was consumed completely and one major new spot with larger polarity was detected. Combined 2 batches. The reaction mixture was quenched by added to the sat. NH.sub.4Cl (aq. 1000 mL), and then extracted with EtOAc (1000 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give 1000 mL solution. Then added the MeOH (600 mL), concentrated under reduced pressure to give 1000 mL solution, then filtered the residue and washed with MeOH (150 mL); the residue was dissolved with THF (1000 mL) and MeOH (600 mL), then concentrated under reduced pressure to give 1000 mL solution. Then filtered to give a residue and washed with MeOH (150 mL). And repeat one more time. Compound 2 (248 g, crude) was obtained as a white solid. And the combined mother solution was concentrated under reduced pressure to give compound 3 (200 g, crude) as yellow oil.

(2354) Compound 2: .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.80 (d, J=7.5 Hz, 2H), 7.74-7.66 (m, 1H), 7.61-7.53 (m, 2H), 7.47 (d, J=7.5 Hz, 6H), 7.24-7.12 (m, 9H), 4.50-4.33 (m, 1H), 3.33 (s, 1H), 3.26 (ddd, J=2.9, 5.2, 8.2 Hz, 1H), 3.23-3.10 (m, 2H), 3.05-2.91 (m, 2H), 1.59-1.48 (m, 1H), 1.38-1.23 (m, 1H), 1.19-1.01 (m, 1H), 0.31-0.12 (m, 1H).

(2355) Preparation of Compound WV-CA-108.

(2356) ##STR00985##

(2357) To a solution of compound 2 (248 g, 498.35 mmol, 1 eq.) in THF (1 L) was added HCl (5 M, 996.69 mL, 10 eq.). The mixture was stirred at 15 C. for 1 hr. TLC(Petroleum ether: Ethyl acetate=3:1, Rf=0.03) indicated compound 2 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (500 mL3). The combined organic layers were back-extracted with water (100 mL). The combined aqueous layer was adjusted to pH 12 with 5M NaOH aq. and extracted with DCM (500 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a white solid. WV-CA-108 (122.6 g, crude) was obtained as a white solid.

(2358) .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.95 (d, J=7.5 Hz, 2H), 7.66 (t, J=7.5 Hz, 1H), 7.57 (t, J=7.7 Hz, 2H), 4.03 (ddd, J=2.6, 5.3, 8.3 Hz, 1H), 3.37-3.23 (m, 2H), 3.20-3.14 (m, 1H), 2.91-2.75 (m, 3H), 2.69 (br s, 1H), 1.79-1.54 (m, 5H); .sup.13C NMR (101 MHz, CHLOROFORM-d) =139.58, 133.83, 129.28, 127.98, 67.90, 61.71, 59.99, 46.88, 25.98, 25.84; LCMS [M+H].sup.+: 256.1. LCMS purity: 100%. SFC 100% purity.

(2359) Among other things, the present disclosure encompasses the recognition that bases utilized in reactions (e.g., from compound 1 to compound 2) can impact stereoselectivity of such reactions. Certain example results are described below:

(2360) TABLE-US-00123 Chiral Auxiliary S. No Aldehyde Nucleophile Base (Diastereoselectivity, cis/trans) 1 1 n-BuLi WV-CA-108 (87:13) 2 1 LiHMDS WV-CA-108 (1.85:1) 3 1 LDA WV-CA-108 (1.85:1) 4 1 KHMDS WV-CA-108 (10:1) 5 1 0 t-BuOK WV-CA-108 (10:1) 6 4 n-BuLi WV-CA-242 (2:1) 7 4 KHMDS WV-CA-242 (8:1) 8 4 n-BuLi WV-CA-243 (2:1) 9 4 KHMDS WV-CA-243 (8:1) 10 4 n-BuLi WV-CA-347 (5.5:1) 11 4 KHMDS WV-CA-347 (10:1) 12 4 KHMDS WV-CA-247 (43:57) 13 4 n-BuLi WV-CA-247 (~1:1) 14 4 LiHMDS WV-CA-247 (~39:51) 15 4 000 NaHMDS WV-CA-247 (~40:66)
Preparation of Compound WV-CA-237.

(2361) ##STR01001##

(2362) To a solution of compound 3 (400.00 g, 803.78 mmol) in THF (1.5 L) was added HCl (5 M, 1.61 L). The mixture was stirred at 15 C. for 2 hr. TLC indicated compound 3 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (500 mL3). The combined aqueous layer was adjusted to pH 12 with 5M NaOH aq. and extracted with DCM (500 mL1) and EtOAc (1000 mL2). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to afford as a brown solid. WV-CA-237 (100 g, crude) was obtained as a brown solid.

(2363) The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=3/1 to Ethyl acetate: Methanol=1:2) to give 24 g crude. Then the 4 g residue was purified by prep-HPLC(column: Phenomenex luna C18 25050 mm10 um; mobile phase: [water(0.05% HCl)-ACN]; B %: 2%.fwdarw.20%, 15 min) to give desired compound (2.68 g, yield 65%,) as a white solid. WV-CA-237 (2.68 g) was obtained as a white solid. WV-CA-237: .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.98-7.88 (m, 2H), 7.68-7.61 (m, 1H), 7.60-7.51 (m, 2H), 4.04 (dt, J=2.4, 5.6 Hz, 1H), 3.85 (ddd, J=3.1, 5.6, 8.4 Hz, 1H), 3.37-3.09 (m, 3H), 2.95-2.77 (m, 3H), 1.89-1.53 (m, 4H), 1.53-1.39 (m, 1H); .sup.13C NMR (101 MHz, CHLOROFORM-d) =139.89, 133.81, 133.70, 129.26, 129.16, 128.05, 127.96, 68.20, 61.77, 61.61, 61.01, 60.05, 46.67, 28.02, 26.24, 25.93; LCMS [M+H].sup.+: 256.1. LCMS purity: 80.0%. SFC dr=77.3:22.7.

(2364) ##STR01002##

(2365) To a solution of compound 4 (140 g, 410.02 mmol) in THF (1400 mL) was added methylsulfonylbenzene (96.07 g, 615.03 mmol), then added KHMDS (1 M, 615.03 mL) in 0.5 hr. The mixture was stirred at 7040 C. for 3 hr. TLC indicated compound 4 was consumed and one new spot formed. The reaction mixture was quenched by addition sat. NH.sub.4Cl aq. 3000 mL at 0 C., and then diluted with EtOAc (3000 mL) and extracted with EtOAc (2000 mL3). Dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. To the crude was added THF (1000 mL) and MeOH (1500 mL), concentrated under reduced pressure at 45 C. until about 1000 mL residue remained, filtered the solid. Repeat 3 times. Compound 5 (590 g, 72.29% yield) was obtained as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.81 (d, J=7.5 Hz, 2H), 7.75-7.65 (m, 1H), 7.62-7.53 (m, 2H), 7.48 (br d, J=7.2 Hz, 6H), 7.25-7.11 (m, 9H), 4.50-4.37 (m, 1H), 3.31-3.11 (m, 3H), 3.04-2.87 (m, 2H), 1.60-1.48 (m, 1H), 1.39-1.24 (m, 1H), 1.11 (dtd, J=4.5, 8.8, 12.8 Hz, 1H), 0.32-0.12 (m, 1H).

(2366) Preparation of Compound WV-CA-236.

(2367) ##STR01003##

(2368) To a solution of compound 5 (283 g, 568.68 mmol) in THF (1100 mL) was added HCl (5 M, 1.14 L). The mixture was stirred at 25 C. for 2 hr. TLC indicated compound 5 was consumed and two new spots formed. The reaction mixture was washed with MTBE (1000 mL3), then the aqueous phase was basified by addition NaOH (5M) until pH=12 at 0 C., and then extracted with DCM (1000 mL3) to give a residue, dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. Compound WV-CA-236 (280 g, 1.10 mol, 96.42% yield) was obtained as a yellow solid.

(2369) The crude product was added HCl/EtOAc (1400 mL, 4M) at 0 C., 2 hr later, filtered the white solid and washed the solid with MeOH (1000 mL3). LCMS showed the solid contained another peak (MS=297). Then the white solid was added H.sub.2O (600 mL) and washed with DCM (300 mL3). The aqueous phase was added NaOH (5 M) until pH=12. Then diluted with DCM (800 mL) and extracted with DCM (800 mL4). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give the product. Compound WV-CA-236 (280 g) was obtained as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =8.01-7.89 (m, 2H), 7.69-7.62 (m, 1H), 7.61-7.51 (m, 2H), 4.05 (ddd, J=2.8, 5.2, 8.4 Hz, 1H), 3.38-3.22 (m, 2H), 3.21-3.08 (m, 1H), 2.95-2.72 (m, 4H), 1.85-1.51 (m, 4H); .sup.13C NMR (101 MHz, CHLOROFORM-d) =139.75, 133.76, 129.25, 127.94, 67.57, 61.90, 60.16, 46.86, 25.86. LCMS [M+H].sup.+: 256. LCMS purity: 95.94. SFC purity: 99.86%.

(2370) ##STR01004##

(2371) To a solution of 1-methoxy-4-methylsulfonyl-benzene (36.82 g, 197.69 mmol) in THF (500 mL) was added KHMDS (1 M, 197.69 mL) at 70 C., 0.5 hr later added compound 4 (45 g, 131.79 mmol) in THF (400 mL) at 70 C. The mixture was stirred at 70.fwdarw.30 C. for 4 hr, and then the mixture was added with KHMDS (1M, 131.79 mL) at 70 C. The mixture was stirred at 70 C. for 1 hr. TLC indicated compound 4 was remained, and two new spots were detected. The reaction mixture was quenched by sat. NH.sub.4Cl (aq. 300 mL), and then extracted with EtOAc (500 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. The residue was dissolved in THF (800 mL) and MeOH (500 mL), and then concentrated under reduced pressure until 200 mL solvent left. The mixture was added with MeOH (500 mL) and concentrated under reduced pressure to 200 mL solvent left and solid appeared. The solid was filtered to give product. Repeated the trituration 2 times. Compound 6 (49.8 g, 71.61% yield) was obtained as a brown solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.73-7.66 (m, 2H), 7.46 (d, J=7.5 Hz, 6H), 7.24-7.11 (m, 9H), 7.04-6.96 (m, 2H), 4.37 (td, J=3.1, 8.3 Hz, 1H), 3.94-3.88 (m, 3H), 3.36 (s, 1H), 3.26-3.10 (m, 3H), 3.00-2.89 (m, 2H), 1.58-1.45 (m, 1H), 1.37-1.23 (m, 1H), 1.15-1.00 (m, 1H), 0.26-0.10 (m, 1H).

(2372) Preparation of Compound WV-CA-241.

(2373) ##STR01005##

(2374) To a solution of compound 6 (50 g, 94.76 mmol) in THF (250 mL) was added HCl (5 M, 189.51 mL). The mixture was stirred at 20 C. for 3 hr. TLC indicated compound 6 was consumed and two new spots formed. The reaction mixture was extracted with MTBE (200 mL3) and the MTBE phases were discarded. And then the water phase was added with 5 M NaOH (aq.) to pH=9 and extracted with DCM (200 mL5). The combined organic layers were washed with brine (100 mL), dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give the product. WV-CA-241 (27 g, 98.10% yield, LCMS purity: 98.24% purity) was obtained as a colorless oil. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.83-7.76 (m, 2H), 6.98-6.91 (m, 2H), 4.00 (ddd, J=2.9, 5.0, 8.4 Hz, 1H), 3.81 (s, 3H), 3.33-3.07 (m, 5H), 2.87-2.75 (m, 2H), 1.74-1.49 (m, 4H); .sup.13C NMR (101 MHz, CHLOROFORM-d) =163.79, 131.10, 130.21, 114.44, 67.66, 61.88, 60.25, 55.69, 46.85, 25.84, 25.81. LCMS [M+H].sup.+: 286.1. LCMS purity: 98.24%. SFC: dr=0.18:99.82. LCMS purity: 99.9%; SFC purity: 99.82%.

(2375) ##STR01006##

(2376) To a solution of 2-methylsulfonylpropane (32.21 g, 263.59 mmol) in THF (1200 mL) was added KHMDS (1 M, 263.59 mL) dropwise at 60 C., and warm to 30 C., slowly over 30 min. The mixture was then cooled to 70 C. A solution of compound 4 (60 g, 175.72 mmol) in THF (300 mL) was added dropwise at 70 C..fwdarw.60 C., over 30 min. The mixture was stirred at 70 C..fwdarw.60 C. for 2 hr. TLC showed compound 4 was consumed and new spot was detected. The reaction mixture was quenched with sat. aq. NH.sub.4Cl (800 mL), and then extracted with EtOAc (1 L3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. Compound 7 (95 g, crude) was obtained as a yellow oil.

(2377) Preparation of Compound WV-CA-242.

(2378) ##STR01007##

(2379) To a solution of compound 7 (95 g, 204.90 mmol) in THF (400 mL) was added HCl (5 M, 409.81 mL). The mixture was stirred at 0.fwdarw.25 C. for 2 hr. TLC indicated compound 7 was consumed and one new spot formed. The reaction mixture was washed with MTBE (300 mL3), then the aqueous phase was basified by addition NaOH (5 M) until pH=12 at 0 C., and then extracted with DCM (300 mL3) to give a residue dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. Compound WV-CA-242 (45 g, 99.23% yield) was obtained as a yellow oil. LCMS [M+H].sup.+: 222.0.

(2380) Purification of Compound WV-CA-242.

(2381) ##STR01008##

(2382) A solution of WV-CA-242 (45 g, 203.33 mmol), (E)-3-phenylprop-2-enoic acid (30.12 g, 203.33 mmol) in EtOH (450 mL) was stirred at 80 C. for 1 hr. The reaction was concentrated in vacuo. The residue was dissolved in TBME (400 mL), and then stirred at 80 C. for 15 min, and then to the mixture was added EtOH (20 mL) and MeCN(30 mL), and then the mixture was filtered, and the filtered cake was washed with TBME (30 mL2) and then did this for 8 times. The salt (35 g, crude) was obtained as a red solid.

(2383) To a solution of salt (34 g, 92.02 mmol) in H.sub.2O (20 mL) was added aq. 5N NaOH (5 M, 36.81 mL). The mixture was stirred at 25 C. for 10 min. The reaction was extracted with DCM (100 mL8), and then the organic phase was concentrated in vacuo. Compound WV-CA-242 (18.9 g, 91.09% yield, LCMS purity: 98.16%) was obtained as an off-white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =4.13 (ddd, J=2.1, 4.6, 9.5 Hz, 1H), 3.38 (spt, J=6.9 Hz, 1H), 3.23-3.14 (m, 2H), 3.01 (dd, J=2.1, 14.4 Hz, 1H), 2.95-2.91 (m, 2H), 1.83-1.60 (m, 4H), 1.40 (dd, J=4.0, 6.8 Hz, 6H); .sup.13C NMR (101 MHz, CHLOROFORM-d) =67.45, 61.71, 53.93, 53.42, 46.80, 25.86, 5.43, 16.03, 14.17. LCMS [M+H].sup.+: 222.1. LCMS purity: 98.17%.

(2384) ##STR01009##

(2385) To a solution 2-methyl-2-(methylsulfonyl)propane (14.96 g, 109.83 mmol) in THF (150 mL) was added KHMDS (1 M, 109.83 mL) dropwise at 70 C., and warm to 30 C. slowly over 30 min. The mixture was then cooled to 70 C. A solution of compound 4 (25.00 g, 73.22 mmol) in THF (100 mL) was added dropwise at 70 C. The mixture was stirred at 70 C. for 4 hr. TLC(Petroleum ether: Ethyl acetate=3:1 Rf=0.3) showed compound 4 was remained a little, and one major new spot with larger polarity was detected. The reaction mixture was quenched by added to the sat. NH.sub.4Cl (aq., 100 mL), and then extracted with EtOAc (100 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give 30 mL solution. Then added MeOH (30 mL), concentrated under reduced pressure to give 30 mL solution, then filtered the residue and washed with MeOH (10 mL); the residue was dissolved with THF (30 mL) and MeOH (30 mL), and then concentrated under reduced pressure to give 30 mL solution. Then filtered to give a residue and washed with MeOH (10 mL). And repeat one more time to give 21 g white solid and 20 g brown oil. Compound 8 (21 g, crude) was obtained as a white solid, and Compound 8A (20 g, crude) as a brown oil. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.56 (d, J=7.5 Hz, 6H), 7.32-7.23 (m, 6H), 7.21-7.14 (m, 3H), 4.85-4.68 (m, 1H), 3.52-3.43 (m, 4H), 3.41 (td, J=3.8, 8.1 Hz, 1H), 3.28 (td, J=8.5, 11.9 Hz, 1H), 3.09-2.91 (m, 2H), 2.78 (dd, J=2.6, 13.6 Hz, 1H), 1.65-1.50 (m, 1H), 1.37 (s, 10H), 1.16-0.98 (m, 2H), 0.39-0.21 (m, 1H). LCMS [M+H].sup.+: 235.9.

(2386) Preparation of Compound WV-CA-243.

(2387) ##STR01010##

(2388) To a solution of compound 8 (20 g, 41.87 mmol) in THF (200 mL) was added HCl (5 M, 83.74 mL). The mixture was stirred at 15 C. for 3 hr. TLC indicated compound 8 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (100 mL3). The combined aqueous layer was adjusted to pH 12 with 5M NaOH aq. and extracted with DCM (50 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a white solid. WV-CA-243 (9 g, 90.42% yield, 99% purity) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) 4.18 (ddd, J=2.8, 5.8, 8.2 Hz, 1H), 3.29-3.21 (m, 1H), 3.19 (d, J=2.6 Hz, 1H), 3.16-3.08 (m, 1H), 2.92 (t, J=6.6 Hz, 2H), 2.74 (br s, 1H), 1.92-1.81 (m, 1H), 1.81-1.61 (m, 3H), 1.42 (s, 10H); .sup.13CNMR (101 MHz, CHLOROFORM-d) =68.01, 62.00, 59.73, 49.79, 46.96, 26.77, 25.80, 23.22. LCMS [M+H].sup.+: 236.1. LCMS purity: 99.46%.

(2389) ##STR01011##

(2390) To a solution (chloromethyl)(phenyl)sulfane of Mg (17.08 g, 702.90 mmol, 4 eq.) and I.sub.2 (0.50 g, 1.97 mmol, 396.83 uL, 1.12-2 eq.) in THF (100 mL) was added with 1,2-dibromoethane (1.25 g, 6.63 mmol, 0.5 mL, 3.77-2 eq.). Once the mixture turned to be colorless, chloromethylsulfanylbenzene (111.51 g, 702.90 mmol, 4 eq.) in THF (100 mL) was dropwise added at 10-20 C. for 1 hr. After addition, the mixture was stirred at 10-20 C. for 1 hr, most of Mg was consumed. And then the mixture was added in the mixture of compound 1 (60 g, 175.72 mmol, 1 eq.) in THF (600 mL) at 78 C., the mixture was stirred at 78 C.-20 C. for 4 hr. TLC(Petroleum ether: Ethyl acetate=9:1, R.sub.f=0.26) indicated compound 1 was remained and two new spots formed. The reaction mixture was quenched by addition water (100 mL) at 0 C., and then extracted with EtOAc (100 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=200/1 to 10:1) 2 times. Compound 9 (80 g, 171.80 mmol, 97.77% yield) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.52 (d, J=7.5 Hz, 6H), 7.31-7.09 (m, 14H), 4.24-4.14 (m, 1H), 3.54-3.44 (m, 1H), 3.30-3.18 (m, 1H), 3.08-2.96 (m, 1H), 2.91 (s, 1H), 2.80 (d, J=7.0 Hz, 2H), 1.69-1.53 (m, 1H), 1.39-1.30 (m, 1H), 1.15-1.01 (m, 1H), 0.30-0.12 (m, 1H).

(2391) Preparation of Compound WV-CA-244.

(2392) ##STR01012##

(2393) To a solution of compound 9 (80 g, 171.80 mmol, 1 eq.) in EtOAc (350 mL) was added HCl (5 M, 266.30 mL, 7.75 eq.). The mixture was stirred at 15 C. for 18 hr. TLC(Petroleum ether: Ethyl acetate=9:1, R.sub.f=0.01) indicated compound 9 was consumed and new spots formed. The reaction mixture was extracted with MTBE (200 mL3) and the MTBE phases were discarded. And then the water phase was added with 2 M NaOH (aq.) to pH=9 and extracted with EtOAc (200 mL5). The combined organic layers were washed with brine (200 mL), dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give the crude product. To the crude product was added EtOAc (100 mL) at 70 C. The mixture was stirred at 70 C..fwdarw.20 C. for 1 hr. The reaction mixture was filtered, and the filter cake was dried to give the product. WV-CA-244 (31.9 g, 142.84 mmol, 94.66% yield) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.37 (d, J=7.5 Hz, 2H), 7.26 (t, J=7.7 Hz, 2H), 7.20-7.12 (m, 1H), 3.74-3.65 (m, 1H), 3.24-3.15 (m, 1H), 3.13-3.00 (m, 2H), 3.00-2.21 (m, 4H), 1.77-1.59 (m, 4H); .sup.13C NMR (101 MHz, CHLOROFORM-d) =136.04, 129.35, 128.95, 126.15, 70.75, 61.64, 46.86, 38.54, 25.86, 25.17. LCMS [M+H].sup.+: 224.1. LCMS purity: 99.57% .

(2394) ##STR01013##

(2395) To a solution of 4-methylsulfonylbenzonitrile (47.76 g, 263.59 mmol, 1.5 eq.) in THE (800 mL) was added KHMDS (1 M, 263.59 mL, 1.5 eq.) at 70 C..fwdarw.40 C., 0.5 hr later, added compound 4 (60.00 g, 175.72 mmol, 1 eq.) in THF (400 mL) at 70 C. The mixture was stirred at 70 C. for 2.5 hr. TLC(Petroleum ether: Ethyl acetate=1:1, R.sub.f=0.4) indicated compound 4 was consumed and one new spot formed. The reaction mixture was quenched by addition sat. NH.sub.4Cl (20 mL) at 0 C. and extracted with DCM (600 mL3). Dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. The residue was washed with MeOH (500 mL5) to get compound 10 (28 g, 53.57 mmol, 30.49% yield) as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) 7.84-7.74 (m, 2H), 7.73-7.65 (m, 2H), 7.32 (d, J=7.2 Hz, 6H), 7.15-6.99 (m, 9H), 4.20 (td, J=2.9, 5.6 Hz, 1H), 3.22 (ddd, J=3.1, 5.7, 8.3 Hz, 1H), 3.12-3.03 (m, 2H), 3.02-2.92 (m, 1H), 2.90-2.77 (m, 2H), 1.39-1.26 (m, 1H), 1.20-0.93 (m, 2H), 0.13-0.11 (m, 1H).

(2396) Preparation of Compound WV-CA-238.

(2397) ##STR01014##

(2398) To a solution of compound 10 (28 g, 53.57 mmol, 1 eq.) in DCM (196 mL) was added TFA (12.22 g, 107.15 mmol, 7.93 mL, 2 eq.). The mixture was stirred at 0 C. for 3 hr. TLC and LCMS indicated compound 10 was consumed and two new spots formed. The reaction mixture was washed with MTBE (100 mL3), then the aqueous phase was basified by addition NaOH (5 M) until pH=12 at 0 C., and then extracted with DCM (50 mL3) to give a residue dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give a residue. Compound WV-CA-238 (9.5 g, 33.42 mmol, 62.38% yield, 98.62% purity) was obtained as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =8.09 (d, J=8.4 Hz, 2H), 7.87 (d, J=8.4 Hz, 2H), 4.06 (ddd, J=2.9, 4.9, 8.3 Hz, 1H), 3.38-3.16 (m, 3H), 2.96-2.79 (m, 2H), 1.81-1.64 (m, 3H), 1.61-1.45 (m, 1H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =144.05, 132.88, 128.93, 117.48, 117.15, 67.63, 61.50, 60.09, 46.83, 25.88, 25.55. LCMS [M+H].sup.+: 281.1. LCMS purity: 98.62%. SFC: dr=99.75:0.25.

(2399) ##STR01015##

(2400) To a solution of methylsulfinylbenzene (25 g, 178.31 mmol, 1.5 eq.) in THF (400 mL) was added KHMDS (1 M, 178.31 mL, 1.5 eq.) dropwise at 60 C., and warm to 30 C. slowly over 30 min. The mixture was then cooled to 70 C. A solution of compound 4 (40.59 g, 118.88 mmol, 1 eq.) in THF (100 mL) was added dropwise at 70 C. The mixture was stirred at 70 C..fwdarw.50 C. for 2 hr. TLC (Petroleum ether: Ethyl acetate=3:1) showed compound 4 was remained. The reaction mixture was cooled to 70 C., additionally added KHMDS (1M, 40 mL), and stirred at 70 C..fwdarw.40 C. for 2 hr. TLC(Petroleum ether: Ethyl acetate=3:1) showed compound 4 was little remained. The reaction mixture was quenched with sat. NH.sub.4Cl (aq. 300 mL), and the separated aqueous layer was extracted with EtOAc (200 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a residue as a yellow gum, which was crystallized in MeOH (100 mL), filtered and rinsed with MeOH (50 mL) to give an off-white solid (17 g), and the filtrate was concentrated to afford a yellow gum (50 g). The white solid product (17 g) was re-dissolved in THF (150 mL), and added MeOH (80 mL), and the mixture was concentrated to remove THE, filtered and dried to give an off-white solid, which was re-dissolved in THF (150 mL), and added MeOH (80 mL), and the mixture was concentrated to remove THE, filtered and dried to give the product as an off-white solid (13 g). The filtrate was concentrated to give 4 g crude. No further purification. The product compound 11 (13 g, 26.99 mmol, 22.70% yield) was obtained as an off-white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.62-7.56 (m, 2H), 7.55-7.52 (m, 3H), 7.51-7.45 (m, 6H), 7.25-7.12 (m, 9H), 4.60 (td, J=2.4, 10.1 Hz, 1H), 3.72 (s, 1H), 3.27-3.13 (m, 2H), 3.04-2.84 (m, 2H), 2.46 (dd, J=2.2, 13.5 Hz, 1H), 1.71-1.53 (m, 1H), 1.42-1.28 (m, 1H), 1.07-0.90 (m, 1H), 0.37-0.21 (m, 1H).

(2401) Preparation of Compound WV-CA-247.

(2402) ##STR01016##

(2403) To a solution of compound 11 (13 g, 26.99 mmol, 1 eq.) in THF (45 mL) was added HCl (5 M, 52.00 mL, 9.63 eq.) aqueous. The mixture was stirred at 20 C. for 2 hr. TLC(Petroleum ether: Ethyl acetate=3:1) showed the reaction was completed. The resulting mixture was washed with MTBE (60 mL3). The combined aqueous layer was adjusted to pH 12 with 5 M NaOH aq. and extracted with DCM (80 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to afford a white solid (5.8 g). Without further purification. The compound WV-CA-247 (5.8 g, 24.17 mmol, 89.55% yield, 99.74% purity) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.67-7.60 (m, 2H), 7.55-7.42 (m, 3H), 4.17 (ddd, J=2.6, 4.2, 9.9 Hz, 1H), 3.74-3.23 (brs, 2H), 3.13 (dt, J=4.3, 7.3 Hz, 1H), 2.96-2.74 (m, 4H), 1.81-1.52 (m, 4H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =143.99, 130.93, 129.32, 123.92, 66.97, 62.23, 61.58, 46.86, 25.88, 25.3. LCMS [M+H].sup.+: 240. LCMS purity: 99.74%. SFC: dr=99.48:0.52.

(2404) ##STR01017##

(2405) To a solution of 1,3-dithiane (13.21 g, 109.83 mmol) in THF (250 mL) was added n-BuLi (2.5 M, 29.29 mL) at 20 C., 0.5 hr later added compound 1 (25 g, 73.22 mmol) in THF (250 mL) at 70 C. The mixture was stirred at 70.fwdarw.20 C. for 16 hr. TLC indicated compound 4 was remained, and one new spot was detected. The reaction mixture was quenched by sat. NH.sub.4Cl (200 mL), and then extracted with EtOAc (200 mL5). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by MPLC (SiO.sub.2, Petroleum ether/Ethyl acetate=50/1 to 10/1, 5% TEA) 2 times. Compound 12 (16 g, 47.33% yield) was obtained as a yellow oil. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.59 (d, J=7.0 Hz, 5H), 7.29-7.25 (m, 6H), 7.20-7.14 (m, 3H), 4.39 (dd, J=2.4, 10.3 Hz, 1H), 4.03 (ddd, J=2.4, 5.6, 8.2 Hz, 1H), 3.38 (d, J=10.1 Hz, 1H), 3.28 (ddd, J=7.0, 10.1, 12.3 Hz, 1H), 3.07-2.99 (m, 1H), 2.93-2.85 (m, 1H), 2.63-2.54 (m, 1H), 2.34-2.18 (m, 2H), 1.97-1.82 (m, 2H), 1.59-1.45 (m, 1H), 1.22-1.11 (m, 1H), 0.22-0.06 (m, 1H).

(2406) Preparation of Compound WV-CA-246.

(2407) ##STR01018##

(2408) To a solution of compound 12 (16 g, 34.66 mmol) in EtOAc (80 mL) was added HCl (5 M, 69.31 mL). The mixture was stirred at 15 C. for 16 hr. TLC indicated compound 12 was consumed completely and new spots formed. The reaction mixture was extracted with TBME (100 mL3) and the TBME phases were discarded. And then the water phase was added with 5 M NaOH (aq.) to pH=9 and extracted with DCM (100 mL5). The combined organic layers were washed with brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give the crude product. The residue was purified by prep-HPLC(column: Phenomenex luna C18 25050 mm10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-15%, 20 min and column: Phenomenex luna (2) C18 2505010 um; mobile phase: [water(0.1% TFA)-ACN]; B %: 0%-12%,20 min). WV-CA-246 (4.2 g, 55.25% yield) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =4.13 (d, J=7.2 Hz, 1H), 3.83 (dd, J=5.1, 7.2 Hz, 1H), 3.49 (dt, J=5.1, 7.3 Hz, 1H), 3.13-2.76 (m, 6H), 2.60 (br s, 2H), 2.20-2.05 (m, 1H), 2.04-1.90 (m, 1H), 1.89-1.62 (m, 4H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =73.76, 59.94, 50.42, 46.83, 28.95, 28.45, 25.87, 25.32. HPLC purity: 97.75%. LCMS [M+H].sup.+: 220.1. SFC: dr=0.22:99.78.

(2409) ##STR01019##

(2410) To a solution of N-methyl-N-phenyl-acetamide (18.5 g, 124.00 mmol) in THF (250 mL) was added KHMDS (1 M, 124.00 mL) dropwise at 70 C., and to warm to 30 C. slowly over 30 min. The mixture was then cooled to 70 C. A solution of compound 4 (28.23 g, 82.67 mmol) in THF (150 mL) was added dropwise at 70 C. The mixture was stirred at 70 C.50 C. for 3 hr. TLC showed the reaction was almost completed. The reaction mixture was quenched with sat. NH.sub.4Cl (aq., 30 mL), and extracted with EtOAc (25 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a residue as yellow gum. The crude was purified by column chromatography on silica gel (Petroleum ether: Ethyl acetate=10:1, 3:1, 1:1, 1:2, 5% TEA). Compound 13 (38 g, 93.7% yield) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.53 (br d, J=7.5 Hz, 6H), 7.44-7.31 (m, 4H), 7.26-7.09 (m, 12H), 4.46-4.40 (m, 1H), 3.90 (br s, 1H), 3.31-3.19 (m, 4H), 3.15-3.07 (m, 1H), 3.00-2.91 (m, 1H), 1.48-1.26 (m, 2H), 0.86-0.74 (m, 1H), 0.33-0.19 (m, 1H).

(2411) Preparation of Compound WV-CA-248.

(2412) ##STR01020##

(2413) To a solution of compound 13 (38 g, 77.45 mmol) in THF (125 mL) was added HCl (5 M, 152.00 mL) aqueous. The mixture was stirred at 20 C. for 2 hr. TLC showed the reaction was completed. The resulting mixture was washed with MTBE (80 mL3), EtOAc (100 mL3), and DCM (100 mL2) in turn. The combined aqueous layer was adjusted to pH=12 with 5M NaOH aq. and extracted with DCM (120 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a yellow gum. The crude of WV-CA-248 (15.2 g, 73.26% yield, 92.7% purity) appears a yellow gum. To a solution of WV-CA-248 (14.5 g, 58.39 mmol) in EtOH (150 mL) was added (E)-3-phenylprop-2-enoic acid (8.65 g, 58.39 mmol). The mixture was stirred at 80 C. for 1 hr. The mixture was concentrated in vacuo. The residue was dissolved in TBME (50 mL), and then the mixture was added MeCN (3 mL), the mixture was turned clear, then the solution was standed, and then solid was appeared, and the mixture was filtered, and the filtered cake was washed with TMBE (10 mL2), and the filtered cake was desired compound. The residue (6.5 g, crude) was obtained as a yellow solid. The residue was dissolved in H.sub.2O (10 mL) was added aq. NaOH (5 M, 6.56 mL, 2 eq.). The mixture was stirred at 25 C. for 10 min. The pH of the mixture was 13. The solution was extracted with DCM (40 mL6), and the organic phase was concentrated in vacuo. Compound WV-CA-248 (4 g, 91.74% yield, 93.4% purity) was obtained as a brown oil. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.49-7.31 (m, 3H), 7.21 (br d, J=7.3 Hz, 2H), 4.00 (td, J=4.3, 8.6 Hz, 1H), 3.48 (br s, 2H), 3.28 (s, 3H), 3.10-2.98 (m, 1H), 2.97-2.80 (m, 2H), 2.36-2.17 (m, 2H), 1.79-1.47 (m, 3H), 1.79-1.47 (m, 1H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =172.38, 143.42, 129.89, 128.04, 127.27, 69.90, 62.29, 46.77, 37.98, 37.23, 25.99, 25.65. LCMS [M+H].sup.+: 249.1. LCMS purity: 93.35%. SFC: SFC purity de=94.26%.

(2414) ##STR01021##

(2415) To a solution of methylsulfonylmethane (8.27 g, 87.86 mmol) in THF (150 mL) was added KHMDS (1 M, 87.86 mL) at 70 C.40 C., 0.5 hr later added compound 1 (20 g, 58.57 mmol) in THF (100 mL). The mixture was stirred at 70 C. for 1.5 hr. TLC indicated compound 4 was remained a little and one new spot formed. The reaction mixture was quenched by addition sat. NH.sub.4Cl (aq. 200 mL) at 0 C., and then diluted with EtOAc (200 mL) and extracted with EtOAc (200 mL3). Dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/0.fwdarw.0:1). Compound 14 (12 g, crude, HNMR showed cis/trans isomer ratio 10:1) was obtained as a yellow oil. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.58-7.47 (m, 7H), 7.26-7.22 (m, 5H), 7.20-7.13 (m, 3H), 4.51-4.46 (m, 1H), 3.99-3.88 (m, 1H), 3.48-3.39 (m, 1H), 3.21-2.97 (m, 4H), 2.96-2.91 (m, 3H), 2.68 (br d, J=14.6 Hz, 1H), 1.57-1.43 (m, 1H), 1.36-1.26 (m, 1H), 1.20-1.10 (m, 1H), 0.57-0.44 (m, 1H), 0.25-0.04 (m, 1H).

(2416) Preparation of WV-CA-252.

(2417) ##STR01022##

(2418) To a solution of compound 14 (18 g, 41.32 mmol) in THF (82 mL) was added HCl (5 M, 82.65 mL). The mixture was stirred at 25 C. for 3 hr. TLC indicated compound 14 was consumed and two new spots formed. The reaction mixture was washed with MTBE (50 mL3), then the aqueous phase was basified by addition NaOH (5M) until pH=12 at 0 C., and then extracted with DCM (50 mL6) to give a residue dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The crude compound WV-CA-252 (6.5 g, 81.4% yield) was obtained as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =4.13 (ddd, J=1.8, 4.0, 9.7 Hz, 1H), 3.23 (dt, J=4.2, 7.4 Hz, 1H), 3.18-3.09 (m, 1H), 3.05 (s, 4H), 3.00-2.90 (m, 3H), 1.95-1.68 (m, 4H), 1.67-1.48 (m, 1H). LCMS [M+H].sup.+: 194.0.

(2419) ##STR01023##

(2420) A mixture of compound 1A (52.24 g, 241.62 mmol) in THF (500 mL) was degassed and purged with N.sub.2 for 3 times, and then the mixture was cooled to 70 C., and then to the mixture was added LDA (2 M, 112.76 mL). The mixture was stirred at 40 C. for 30 min, and then to the mixture was added compound 1 (55 g, 161.08 mmol) in THF (250 mL) at 70 C. The mixture was stirred at 70 C. for 2 hr under N.sub.2 atmosphere. TLC indicated compound 1 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction was quenched by sat. aq. NH.sub.4Cl (300 mL) and then extracted with EtOAc (100 mL3). The combined organic phase was washed with brine (100 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was dissolved in MeOH (300 mL) and filtered; the filtered cake was the desired product. Compound 2 (53 g, crude) was obtained as a white solid.

(2421) Preparation of Compound WV-CA-245.

(2422) ##STR01024##

(2423) To a solution of compound 15 (72 g, 129.11 mmol) in THF (400 mL) was added HCl (5 M, 258.22 mL). The mixture was stirred at 25 C. for 1 hr. LC-MS showed compound 15 was consumed completely and one main peak with desired mass was detected. The reaction was extracted with TBME (100 mL3), added aq. 5 N NaOH to pH=13, and then extracted with DCM (50 mL3), and the combined organic phase was concentrated in vacuo. WV-CA-245 (38 g, 92.82% yield, 99.5% purity) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.81-7.71 (m, 4H), 7.58-7.44 (m, 6H), 4.01-3.92 (m, 1H), 3.16-3.09 (m, 1H), 2.92-2.79 (m, 2H), 2.63-2.44 (m, 2H), 1.82-1.60 (m, 4H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =133.88, 132.89, 132.86, 131.95, 131.88, 130.73, 128.74, 68.98, 68.94, 63.79, 63.67, 47.03, 34.21, 33.49, 26.37, 25.88. LCMS [M+H].sup.+: 316.1. LCMS purity: 99.45%. SFC: SFC purity de=99.5%.

(2424) ##STR01025##

(2425) To a solution of compound 1B(13.32 g, 87.86 mmol) in THF (200 mL) was added KHMDS (1 M, 82.00 mL) at 70 C. under N.sub.2, and then the mixture was stirred at 70 C. for 10 min, and then to the mixture was added compound 1 (20 g, 58.57 mmol) in THF (100 mL), the reaction was stirred at 70 C. for 30 min. TLC indicated compound 1 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was quenched with sat. aq. NH.sub.4Cl (100 mL), and then extracted with EtOAc (50 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=50:1, 20:1, 10:1, 1:1, 0:1). Compound 16 (12 g, crude) was obtained as a yellow solid.

(2426) Preparation of Compound WV-CA-249.

(2427) ##STR01026##

(2428) To a solution of compound 16 (12 g, 24.34 mmol) in THF (50 mL) was added aq. HCl (5 M, 48.68 mL). The mixture was stirred at 25 C. for 30 min. TLC indicated compound 16 was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction was extracted with TBME (100 mL3), and then to the mixture was added 5N aq. NaOH to pH=13, extracted with DCM (100 mL3), and then the organic phase was concentrated in vacuo. WV-CA-249 (5.36 g, 87.84% yield, 100.00% purity) was obtained as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.64 (s, 1H), 7.49 (d, J=0.9 Hz, 2H), 3.88 (td, J=3.6, 9.4 Hz, 1H), 3.24-3.16 (m, 1H), 3.02-2.89 (m, 3H), 2.78 (dd, J=9.4, 14.0 Hz, 1H), 1.84-1.70 (m, 4H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =143.11, 134.94, 132.60, 132.33, 130.12, 117.63, 111.52, 70.86, 62.02, 46.76, 37.90, 25.88, 24.21. LCMS [M+H].sup.+: 251.0. LCMS purity: 100.000%. SFC: SFC purity de=98.28%.

(2429) ##STR01027##

(2430) To a solution of nitromethane (30.59 g, 501.15 mmol) in THF (300 mL) was added KHMDS (1 M, 263.59 mL) at 20-25 C. and stirred for 1 hr. Compound 1 (30 g, 87.86 mmol) in THF (90 mL) was added to the mixture at 20-25 C. and stirred for 0.5 hr. TLC showed that the starting material was consumed mostly, and desired product was formed. The mixture was quenched by saturated aq. NH.sub.4Cl (300 mL) and extracted with ethyl acetate (100 mL3). The organic phase was washed by saturated aq. NaCl (100 mL3) and dried with anhydrous Na.sub.2SO.sub.4, then concentrated under reduced pressure to remove the solvent. The crude product was purified by MPLC (SiO.sub.2, Ethyl acetate/Petroleum ether=0%.fwdarw.20%) to obtain compound 17 (26.55 g, 75.08% yield) as yellow solid. The product was detected by .sup.1H NMR. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.54-7.44 (m, 6H), 7.28-7.21 (m, 6H), 7.20-7.14 (m, 3H), 4.64 (td, J=3.0, 9.4 Hz, 1H), 4.53-4.06 (m, 3H), 3.60-3.40 (m, 1H), 3.24-2.96 (m, 3H), 1.52-1.41 (m, 1H), 1.40-1.28 (m, 1H), 1.17-0.94 (m, 1H), 0.67-0.50 (m, 1H), 0.23 (quin d, J=8.8, 11.6 Hz, 1H).

(2431) Preparation of Compound WV-CA-250.

(2432) ##STR01028##

(2433) To a solution of compound 17 (7.5 g, 18.63 mmol) in EtOAc (35 mL) was added HCl/EtOAc (4 M, 50 mL) at 20-25 C. and stirred for 1 hr. TLC showed that the starting material was consumed completely. Poured the supernatant liquid of the mixture, the yellow gum on the bottle wall was concentrated under reduced pressure to remove the solvent. WV-CA-250 (2.10 g, 56.70% yield, 98.927% purity, HCl salt) was obtained as yellow gum. The product was detected by .sup.1H NMR, .sup.13C NMR and LCMS. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =9.89-9.54 (m, 1H), 9.03-8.75 (m, 1H), 8.94 (br s, 1H), 4.97-4.78 (m, 1H), 4.65-4.35 (m, 2H), 3.70-3.41 (m, 4H), 3.22-3.03 (m, 2H), 2.06-1.65 (m, 4H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) =79.42, 79.00, 67.89, 66.82, 61.53, 60.77, 45.44, 45.25, 26.93, 24.57, 23.95, 23.81. LCMS [M+H].sup.+: 161.1, purity: 98.92%.

(2434) ##STR01029##

(2435) To a solution of compound benzylamine (30 g, 279.97 mmol) and TEA (56.66 g, 559.95 mmol) in DCM (60 mL) was added MsCl (38.49 g, 335.97 mmol) in DCM (30 mL) at 0 C. The mixture was stirred at 0 C. for 2 hr. LC-MS showed compound 18A was consumed and many new peaks were detected. The reaction mixture was washed with HCl (1 M, 50 mL3) and sat. NaHCO.sub.3(aq. 50 mL x 3). The organic layer was washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. TLC showed one main spot. The residue was purified by MPLC (SiO.sub.2, Petroleum ether/Ethyl acetate=5/1 to 1:1). Compound 18A (35 g, 67.49% yield) was obtained as a light-yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.44-7.24 (m, 5H), 4.82 (br s, 1H), 4.31 (d, J=6.2 Hz, 2H), 2.85 (s, 3H).

(2436) ##STR01030##

(2437) To a solution of compound 18A (16.28 g, 87.86 mmol) in THF (60 mL) was added with LDA (2 M, 87.86 mL) at 0 C. The mixture was stirred at 0-25 C. for 0.5 hr. And then compound 1 (15 g, 43.93 mmol) in THF (60 mL) was added to above solution at 70 C. The mixture was stirred at 70-25 C. for 4 hr. TLC indicated compound 1 was consumed completely and many new spots formed. The reaction mixture was added with sat. NH.sub.4Cl (aq. 50 mL) and extracted with EtOAc (100 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC(SiO.sub.2, Petroleum ether/Ethyl acetate=5/1, 2% TEA). Compound 18 (22 g, 95.08% yield) was obtained as a yellow oil.

(2438) Preparation of Compound WV-CA-255.

(2439) ##STR01031##

(2440) To a solution of compound 18 (22 g, 41.77 mmol) in EtOAc (15 mL) was added HCl (4 M in ethyl acetate, 31.33 mL) at 0 C. The mixture was stirred at 0-25 C. for 2 hr. And solid appeared in the reaction mixture. TLC indicated compound 18 was consumed completely and many new spots formed. The reaction mixture was filtered. The filter cake was dissolved in water (10 mL), washed with MTBE (40 mL3). The water phase was added with Na.sub.2CO.sub.3 (powder) to pH=89 and extracted with DCM (50 mL5). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. WV-CA-255 (11 g, 92.60% yield) was obtained as a brown solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.46-7.25 (m, 5H), 4.65-3.72 (m, 5H), 3.14-3.01 (m, 3H), 2.95-2.77 (m, 2H), 1.89-1.34 (m, 4H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =136.99, 128.71, 128.62, 128.19, 128.09, 127.85, 69.12, 67.58, 61.98, 61.70, 55.55, 55.36, 47.36, 47.30, 46.60, 46.28, 28.05, 26.16, 25.71, 24.92. LCMS [M+H].sup.+: 285.0, LCMS purity: 99.8%. SFC: dr (trans/cis)=32.36:67.64.

(2441) ##STR01032##

(2442) To a solution of compound dibenzylamine (30 g, 152.07 mmol) in DCM (250 mL) was added TEA (15.39 g, 152.07 mmol). The mixture was cooled to 0 C., and to the mixture was added MsCl (17.42 g, 152.07 mmol) in DCM (50 mL), and then the mixture was stirred at 25 C. for 12 hours. LC-MS showed desired mass was detected. The reaction was quenched by H.sub.2O (100 mL) and the organic phase was extracted with H.sub.2O (100 mL3), the organic phase was dried by Na.sub.2SO.sub.4, and then concentrated in vacuum. No need further purification. Compound 19A (39 g, crude) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.41-7.29 (m, 9H), 4.36 (s, 4H), 2.82-2.75 (m, 3H). LCMS [M+H].sup.+: 298.0, purity: 86.6%.

(2443) ##STR01033##

(2444) To a solution of compound 19A (19.36 g, 70.29 mmol) in THF (200 mL) was added KHMDS (1 M, 76.15 mL) dropwise at 78 C. to 70 C. under N.sub.2. The mixture was warmed to 40 C. and stirred for 0.5 hr, then cooled to 78 C. To the mixture was added compound 1 (20 g, 58.57 mmol) in THF (100 mL) at 78 C. to 70 C. and stirred for 1 hr under N.sub.2. TLC showed that the starting material was consumed completely. The mixture was quenched by saturated aq. NH.sub.4Cl (200 mL) and extracted with ethyl acetate (70 mL3). The organic phase was washed by saturated aq. NaCl (70 mL3) and dried with anhydrous Na.sub.2SO.sub.4, then concentrated under reduced pressure to remove the solvent to obtain the crude product as yellow gum. The crude product was re-dissolved with methanol (200 mL) and standing at 20-25 C. for 12 hours. Compound 19 (20.4 g, 99.99% yield) was crystallized from the solvent as white solid, then filtered and dried in vacuum. The filtrate was concentrated under reduced pressure to remove the solvent to give compound 20 (28.4 g, crude) as brown gum. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.47-7.42 (m, 6H), 7.23-7.05 (m, 19H), 4.36 (td, J=3.0, 8.6 Hz, 1H), 4.23-4.12 (m, 4H), 3.29-3.19 (m, 1H), 3.29-3.19 (m, 1H), 3.11 (ddd, J=7.1, 9.5, 12.1 Hz, 1H), 2.97-2.82 (m, 2H), 2.59 (dd, J=3.1, 14.2 Hz, 1H), 1.37-1.27 (m, 1H), 1.24-1.14 (m, 1H), 1.00-0.92 (m, 1H), 0.16-0.02 (m, 1H).

(2445) Preparation of Compound WV-CA-263.

(2446) ##STR01034##

(2447) To a solution of compound 19 (20 g, 32.42 mmol) in THF (100 mL) was added HCl (5 M, 64.85 mL) at 20-25 C. and stirred for 0.5 hr. TLC showed that the starting material was consumed completely. The mixture was extracted with TBME (80 mL3), then adjusted the pH of the mixture with aq. NaOH (65 mL, 5M) to 11-13 and extracted with DCM (100 mL3). The organic phase was dried with anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure to remove the solvent. The crude product was used for the next step without any purification. WV-CA-263 (10.04 g, 82.68% yield, 100% purity) was obtained as white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.38-7.28 (m, 10H), 4.38 (s, 4H), 4.01 (ddd, J=2.6, 5.6, 8.5 Hz, 1H), 3.20-3.13 (m, 2H), 3.10-3.02 (m, 1H), 2.91 (t, J=6.5 Hz, 2H), 1.89 (br d, J=8.6 Hz, 1H), 1.82-1.66 (m, 4H), 1.62-1.52 (m, 1H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =135.62, 128.77, 128.70, 127.98, 77.35, 76.87 (d, J-31.5 Hz, 1C), 68.84, 61.51, 57.03, 50.35, 46.96, 26.27, 25.88. LCMS [M+H].sup.+: 375.1, purity: 100.00%. SFC: dr=99.55:0.45.

(2448) ##STR01035##

(2449) To a solution of 3,3-dimethylbutan-2-one (11.00 g, 109.83 mmol) in THF (125 mL) was added LDA (2 M, 54.91 mL) dropwise at 70 C., and it was stirred at 70 C. 60 C. for 1 hr. A solution of compound 1 (25 g, 73.22 mmol) in THF (125 mL) was added dropwise at 70 C.60 C. The mixture was stirred at 70 C. for 1.5 hr. TLC showed compound 1 was almost consumed. The reaction mixture was quenched with sat. NH.sub.4Cl (aq., 200 mL), and the separated aqueous layer was extracted with EtOAc (150 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a residue as a light-yellow solid. The crude was purified by column chromatography on silica gel (Petroleum ether+5% TEA; Petroleum ether: Ethyl acetate (20:1)+5% TEA). Compound 21 (17 g, 52.6% yield) was obtained as a white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.37-7.25 (m, 6H), 7.03-6.95 (m, 6H), 6.94-6.84 (m, 3H), 4.22 (td, J=2.7, 9.2 Hz, 1H), 3.09 (td, J=4.1, 7.6 Hz, 1H), 3.04-2.92 (m, 2H), 2.75 (ddd, J=2.9, 8.5, 12.0 Hz, 1H), 2.26 (dd, J=9.3, 17.0 Hz, 1H), 2.04 (dd, J=3.4, 16.9 Hz, 1H), 1.43-1.24 (m, 2H), 1.14-1.01 (m, 1H), 0.84 (s, 9H), 0.81-0.71 (m, 1H), 0.09-0.07 (m, 1H).

(2450) Preparation of Compound WV-CA-289.

(2451) ##STR01036##

(2452) To a solution of compound 21 (16 g, 36.23 mmol) in EtOAc (25 mL) was added 4 M HCl/EtOAc (100 mL). The mixture was stirred at 25 C. for 0.5 hr. TLC showed the reaction was completed. The resulting mixture was filtered, and the solid was stirred in EtOAc (150 mL), filtered and re-triturated with EtOAc/MeOH (150 mL/5 mL), filtered and dried to afford compound WV-CA-289 (7.5 g, 87.8% yield, HCl salt) as a white solid. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) =4.43 (ddd, J=3.5, 4.6, 7.8 Hz, 1H), 3.71 (dt, J=3.5, 8.0 Hz, 1H), 3.42-3.22 (m, 2H), 2.92 (dd, J=7.6, 17.7 Hz, 1H), 2.73 (dd, J=4.9, 17.7 Hz, 1H), 2.23-1.90 (m, 4H), 1.28-1.05 (m, 9H). [M+H].sup.+: 200.1, purity: 100.00%.

(2453) ##STR01037##

(2454) To a solution of methylsulfonylbenzene (13.72 g, 87.86 mmol) in THF (100 mL) was added LiHMDS (1 M, 87.86 mL) in 0.5 hr at 70 C.-0 C., then added compound 4 in THF (100 mL). The mixture was stirred at 70 C. in 2.5 hr. TLC indicated compound 4 was remained a little and two new spots formed. The reaction mixture was quenched by addition sat. NH.sub.4Cl aq. (300 mL) at 0 C., extracted with DCM (200 mL3). Dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The crude was added THF (100 mL) and MeOH (150 mL), concentrated under reduced pressure at 45 C. until about 100 mL residue remained, filtered the solid. Repeated 3 times. Got solid 20 g, the mother liquid was concentrated under reduced pressure to get compound 22 (20 g, crude) was obtained as a yellow oil. Compound (1R)-2-(benzenesulfonyl)-1-[(2R)-1-tritylpyrrolidin-2-yl]ethanol (20 g, 68.61% yield) was obtained as a white solid.

(2455) Preparation of Compound WV-CA-290.

(2456) ##STR01038##

(2457) To a solution of compound 22 (20 g, 40.19 mmol) in THF (80 mL) was added HCl (5 M, 80.38 mL) at 0 C. The mixture was stirred at 25 C. for 2 hr. TLC showed the compound 22 was consumed and two new spots formed. The reaction mixture was washed with MTBE (50 mL3), then the aqueous phase was basified by addition NaOH (5M) until pH=12 at 0 C., and then extracted with DCM (50 mL3) to give a residue dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC(column: Phenomenex luna C18 25050 mm10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-15%, 20 min). Compound WV-CA-290 (0.7 g, 6.78% yield, 99.39% purity) was obtained as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.95-7.85 (m, 2H), 7.64-7.56 (m, 1H), 7.55-7.46 (m, 2H), 3.79 (ddd, J=3.2, 5.4, 8.4 Hz, 1H), 3.28-3.05 (m, 3H), 2.92-2.72 (m, 2H), 1.84-1.54 (m, 3H), 1.51-1.37 (m, 1H). .sup.13C NMR (101 MHz, CHLOROFORM-d) =139.81, 133.74, 129.19, 128.07, 68.15, 61.55, 60.97, 46.67, 28.03, 26.27. SFC: (AD_MeOH_IPAm_10_40_25_35_6 min), 100% purity. LCMS [M+H].sup.+: 256.1. LCMS purity: 99.39%.

(2458) ##STR01039##

(2459) Two batches in parallel: To a solution of compound tert-butyl(methyl) sulfane (25 g, 239.89 mmol) in MeOH (625 mL) was added Oxone (457.18 g, 743.67 mmol) in H.sub.2O (625 mL) at 0 C. The mixture was stirred at 15 C. for 12 hr. HNMR showed compound tert-butyl(methyl) sulfane was consumed completely and desired compound was detected. Combined two batches of the reaction mixture, filtered and concentrated under reduced pressure to evaporate the MeOH, and then extracted with EtOAc (400 mL4). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. Compound 23A (55 g, crude) was obtained as a colorless oil, confirmed by HNMR. .sup.1HNMR (400 MHz, CHLOROFORM-d) =7.26 (s, 1H), 5.30 (s, 8H), 2.81 (s, 3H), 1.43 (s, 9H).

(2460) ##STR01040##

(2461) To a solution of compound 23A (50 g, 367.07 mmol) in THF (510 mL) was added KHMDS (1 M, 367.07 mL) dropwise at 70 C., and warm to 30 C. slowly over 30 min. The mixture was then cooled to 70 C. A solution of compound 1 (83.56 g, 244.72 mmol) in THF (340 mL) was added dropwise at 70 C. The mixture was stirred at 70 C. for 4 hr. TLC showed compound 1 was remained a little, and one major new spot with larger polarity was detected. The reaction mixture was quenched by added to the sat. NH.sub.4Cl (aq. 800 mL), and then extracted with EtOAc (500 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give brown oil. The crude was dissolved with THF (300 mL) then concentrated under reduced pressure (40 C.) to give 150 mL clarified solution. Then added to 300 mL MeOH and concentrated under reduced pressure to give 200 mL solution, then filtered to give a residue and washed with MeOH (10 mL). The mother solution was concentrated under reduced pressure to give 100 mL solution then filtered to give a residue and washed with MeOH (10 mL). Combined all the residue, repeated two times to give 60 g residue. Compound 23 (60 g, crude) was obtained as a white solid. .sup.1HNMR (400 MHz, CHLOROFORM-d) =7.56 (d, J=7.5 Hz, 6H), 7.32-7.23 (m, 6H), 7.21-7.14 (m, 3H), 4.85-4.68 (m, 1H), 3.41 (td, J=3.8, 8.1 Hz, 1H), 3.28 (td, J=8.5, 11.9 Hz, 1H), 3.09-2.91 (m, 2H), 2.78 (dd, J=2.6, 13.6 Hz, 1H), 1.65-1.50 (m, 1H), 1.37 (s, 9H), 1.16-0.98 (m, 2H), 0.39-0.21 (m, 1H).

(2462) Preparation of Compound WV-CA-240.

(2463) ##STR01041##

(2464) To a solution of compound 23 (59 g, 123.52 mmol) in THF (500 mL) was added HCl (5 M, 247.04 mL). The mixture was stirred at 20 C. for 3 hr. TLC indicated compound 23 was consumed completely and one major new spot with larger polarity was detected. The resulting mixture was washed with MTBE (500 mL3). The combined aqueous layer was adjusted to pH 12 with 5 M NaOH aq. and extracted with DCM (200 mL3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to afford a white solid. WV-CA-240 (23.6 g, 81.14% yield, 99.95% purity) was obtained as a white solid. .sup.1HNMR (400 MHz, CHLOROFORM-d) =4.18 (ddd, J=2.8, 5.8, 8.2 Hz, 1H), 3.29-3.21 (m, 1H), 3.19 (d, J=2.6 Hz, 1H), 3.16-3.08 (m, 1H), 2.92 (t, J=6.6 Hz, 2H), 2.74 (br s, 2H), 1.92-1.81 (m, 1H), 1.81-1.61 (m, 3H), 1.42 (s, 9H). .sup.13CNMR (101 MHz, CHLOROFORM-d) =68.01, 62.00, 59.73, 49.79, 46.96, 26.77, 25.80, 23.22. LCMS [M+H].sup.+: 236.1. LCMS purity 99.95%.

(2465) ##STR01042##

(2466) To a solution of WV-CA-108 (37 g, 144.91 mmol, 1 eq.) in MeOH (370 mL) was added prop-2-enenitrile (7.69 g, 144.91 mmol, 9.61 mL, 1 eq.). The mixture was stirred at 20 C. for 3 hr., (TLC, Petroleum ether: Ethyl acetate=1:3, Rf=0.31) showed WV-CA-108 was consumed completely and in LCMS one main peak with desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Compound 24 (44 g, crude) was obtained as a white solid. LCMS [M+H].sup.+: 308.9.

(2467) Preparation of Compound WV-CA-291.

(2468) ##STR01043##

(2469) A solution of compound 24 (44 g, 142.67 mmol, 1 eq.) in DCM (220 mL) and MeOH (220 mL) was cooled to 78 C. Then mCPBA (36.93 g, 214.01 mmol, 1.5 eq.) and K.sub.2CO.sub.3 (29.58 g, 214.01 mmol, 1.5 eq.) was added. After addition, the mixture was stirred at 78 C. for 3 hr. And the resulting mixture was stirred at 20 C. for 12 hr. LC-MS showed compound 24 was consumed completely and one main peak with desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography. The residue was purified by flash silica gel chromatography (ISCOR; 220 g SepaFlash Silica Flash Column, Eluent of 030% Ethyl acetate/Petroleum ether gradient at 100 mL/min). WV-CA-291 (12 g, 42.05 mmol, 29.47% yield, 95.08% purity) was obtained as a yellow solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =7.98-7.92 (m, 2H), 7.65 (d, J=7.5 Hz, 1H), 7.61-7.53 (m, 2H), 4.50-4.39 (m, 1H), 3.33-3.15 (m, 3H), 2.97-2.78 (m, 2H), 1.89-1.64 (m, 4H). .sup.13CNMR (101 MHz, CHLOROFORM-d) =139.61, 133.90, 129.31, 128.02, 71.21, 64.96, 60.05, 58.12, 21.23, 20.29. LCMS [M+H].sup.+: 272.0. LCMS purity 95.08%.

Example 4E. Example Technologies for Chirally Controlled Oligonucleotide PreparationExample Useful Phosphoramidites

(2470) Among other things, the present disclosure provides phosphoramidites useful for oligonucleotide synthesis. In some embodiments, provided phosphoramidites are particularly useful for preparation of chirally controlled internucleotidic linkages. In some embodiments, provided phosphoramidites are particularly useful for preparing chirally controlled internucleotidic linkages, e.g., non-negatively charged internucleotidic linkages or neutral internucleotidic linkages, etc., that comprise PN. In some embodiments, the linkage phosphorus is trivalent. In some embodiments, the linkage phosphorus is pentavalent. In some embodiments, such internucleotidic linkages have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.

(2471) General Procedure I for Chloroderivative: In some embodiments, in an example procedure, a chiral auxiliary (174.54 mmol) was dried by azeotropic evaporation with anhydrous toluene (80 mL3) at 35 C. in a rota-evaporator and dried under high vacuum for overnight. A solution of this dried chiral auxiliary (174.54 mmol) and 4-methylmorpholine (366.54 mmol) dissolved in anhydrous THF (200 mL) was added to an ice-cooled (isopropyl alcohol-dry ice bath) solution of trichlorophosphine (37.07 g, 16.0 mL, 183.27 mmol) in anhydrous THF (150 mL) placed in three neck round bottomed flask through cannula under Argon (start Temp: 10.0 C., Max: temp 0 C., 28 min addition) and the reaction mixture was warmed at 15 C. for 1 hr. After that the precipitated white solid was filtered by vacuum under argon using airfree filter tube (Chemglass: Filter Tube, 24/40 Inner Joints, 80 mm OD Medium Frit, Airfree, Schlenk). The solvent was removed with rota-evaporator under argon at low temperature (25 C.) and the crude semi-solid obtained was dried under vacuum overnight (15 h) and was used for the next step directly.

(2472) General Procedure I for Chloroderivative: In some embodiments, in an example procedure, a chiral auxiliary (174.54 mmol) was dried by azeotropic evaporation with anhydrous toluene (80 mL3) at 35 C. in a rota-evaporator and dried under high vacuum for overnight. A solution of this dried chiral auxiliary (174.54 mmol) and 4-methylmorpholine (366.54 mmol) dissolved in anhydrous THF (200 mL) was added to an ice-cooled (isopropyl alcohol-dry ice bath) solution of trichlorophosphine (37.07 g, 16.0 mL, 183.27 mmol) in anhydrous THF (150 mL) placed in three neck round bottomed flask through cannula under Argon (start Temp: 10.0 C., Max: temp 0 C., 28 min addition) and the reaction mixture was warmed at 15 C. for 1 hr. After that the precipitated white solid was filtered by vacuum under argon using airfree filter tube (Chemglass: Filter Tube, 24/40 Inner Joints, 80 mm OD Medium Frit, Airfree, Schlenk). The solvent was removed with rota-evaporator under argon at low temperature (25 C.) and the crude semi-solid obtained was dried under vacuum overnight (15 h) and was used for the next step directly.

(2473) General Procedure III for Coupling: In some embodiments, in an example procedure, a nucleoside (9.11 mmol) was dried by co-evaporation with 60 mL of anhydrous toluene (60 mL2) at 35 C. and dried under high vacuum for overnight. The dried nucleoside was dissolved in dry THF (78 mL), followed by the addition of triethylamine (63.80 mmol) and then cooled to 5 C. under Argon (for 2F-dG/2OMe-dG case 0.95 eq of TMS-Cl used). The THF solution of the crude (made from general procedure I (or) II, 14.57 mmol), was added through cannula over 3 min then gradually warmed to room temperature. After 1 hr at room temperature, TLC indicated conversion of SM to product (total reaction time 1 h), the reaction mixture was then quenched with H.sub.2O (4.55 mmol) at 0 C., and anhydrous MgSO.sub.4 (9.11 mmol) was added and stirred for 10 min. Then the reaction mixture was filtered under argon using airfree filter tube, washed with THE, and dried under rotary evaporation at 26 C. to afford white crude solid product, which was dried under high vacuum overnight. The crude product was purified by ISCO-Combiflash system (rediSep high performance silica column pre-equilibrated with Acetonitrile) using Ethyl acetate/Hexane with 1% TEA as a solvent (compound eluted at 100% EtOAc/Hexanes/1% Et.sub.3N) (for 2F-dG case Acetonitrile/Ethyl acetate with 1% TEA used). After evaporation of column fractions pooled together, the residue was dried under high vacuum to afford the product as a white solid.

(2474) Preparation of Amidites (1030-1039).

(2475) ##STR01044##

(2476) Preparation of 1030: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (73%). .sup.31P NMR (162 MHz, CDCl.sub.3) 153.32. (ES) m/z Calculated for C.sub.47H.sub.50FN.sub.6O.sub.10PS: 940.98 [M].sup.+, Observed: 941.78 [M+H].sup.+.

(2477) Preparation of 1031: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). .sup.31P NMR (162 MHz, CDCl.sub.3) 153.62. (ES) m/z Calculated for C.sub.42H.sub.43FN.sub.3O.sub.10PS: 831.85 [M].sup.+, Observed: 870.58 [M+K].sup.+.

(2478) Preparation of 1032: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (68%). .sup.31P NMR (162 MHz, CDCl.sub.3) 153.95. (ES) m/z Calculated for C.sub.44H.sub.46FN.sub.4O.sub.10PS: 872.26 [M].sup.+, Observed: 873.62 [M+H].sup.+.

(2479) Preparation of 1033: General Procedure I followed by General Procedure III used. white foamy solid. Yield: (87%). .sup.31P NMR (162 MHz, CDCl.sub.3) 151.70. (ES) m/z Calculated for C.sub.50H.sub.48 FN.sub.6O.sub.9PS: 958.29 [M].sup.+, Observed: 959.79, 960.83 [M+H].sup.+.

(2480) Preparation of 1034: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (65%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.80. (ES) m/z Calculated for C.sub.51H.sub.51N.sub.6O.sub.10PS: 971.31 [M].sup.+, Observed: 971.81 [M+H].sup.+.

(2481) Preparation of 1035: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (76%). .sup.31P NMR (162 MHz, CDCl.sub.3) 156.50. (ES) m/z Calculated for C.sub.53H.sub.55N.sub.6O.sub.11PS: 1014.33 [M].sup.+, Observed: 1015.81 [M+H].sup.+.

(2482) Preparation of 1036: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). .sup.31P NMR (162 MHz, CDCl.sub.3) 156.40. (ES) m/z Calculated for C.sub.50H.sub.57N.sub.6O.sub.12PS: 996.34 [M].sup.+, Observed: 997.90 [M+H].sup.+.

(2483) Preparation of 1037: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (73%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.87. (ES) m/z Calculated for C.sub.46H.sub.52N.sub.3O.sub.12PS: 901.30 [M].sup.+, Observed: 940.83 [M+K].sup.+.

(2484) Preparation of 1038: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (75%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.94. (ES) m/z Calculated for C.sub.53H.sub.57N.sub.4O.sub.12PS: 1004.34 [M].sup.+, Observed: 1005.86 [M+H].sup.+.

(2485) Preparation of 1039: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (80%). .sup.31P NMR (162 MHz, CDCl.sub.3) 153.52. (ES) m/z Calculated for C.sub.44H.sub.47N.sub.4O.sub.10PS: 854.28 [M].sup.+, Observed: 855.41 [M+H].sup.+.

(2486) Preparation of Amidites (1040-1049).

(2487) ##STR01045##

(2488) Preparation of 1040: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). .sup.31P NMR (162 MHz, CDCl.sub.3) 157.80. (ES) m/z Calculated for C.sub.47H.sub.50FN.sub.6O.sub.10PS: 940.98 [M].sup.+, Observed: 941.68 [M+H].sup.+.

(2489) Preparation of 1041: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). .sup.31P NMR (162 MHz, CDCl.sub.3) 157.79. (ES) m/z Calculated for C.sub.42H.sub.43FN.sub.3O.sub.10PS: 831.85 [M].sup.+, Observed: 870.68 [M+K].sup.+.

(2490) Preparation of 1042: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (78%). .sup.31P NMR (162 MHz, CDCl.sub.3) 158.07. (ES) m/z Calculated for C.sub.44H.sub.46FN.sub.4O.sub.10PS: 872.26 [M].sup.+, Observed: 873.62 [M+H].sup.+.

(2491) Preparation of 1043: General Procedure I followed by General Procedure III used. white foamy solid. Yield: (86%). .sup.31P NMR (162 MHz, CDCl.sub.3) 156.48. (ES) m/z Calculated for C.sub.50H.sub.48FN.sub.6O.sub.9PS: 958.29 [M].sup.+, Observed: 959.79, 960.83 [M+H].sup.+.

(2492) Preparation of 1044: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (65%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.80. (ES) m/z Calculated for C.sub.51H.sub.51N.sub.6O.sub.10PS: 971.31 [M].sup.+, Observed: 971.81 [M+H].sup.+.

(2493) Preparation of 1045: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (77%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.74. (ES) m/z Calculated for C.sub.53H.sub.55N.sub.6O.sub.11PS: 1014.33 [M].sup.+, Observed: 1015.81 [M+H].sup.+.

(2494) Preparation of 1046: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (76%). .sup.31P NMR (162 MHz, CDCl.sub.3) 155.05. (ES) m/z Calculated for C.sub.50H.sub.57N.sub.6O.sub.12PS: 996.34 [M].sup.+, Observed: 997.90 [M+H].sup.+.

(2495) Preparation of 1047: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (75%). .sup.31P NMR (162 MHz, CDCl.sub.3) 155.44. (ES) m/z Calculated for C.sub.46H.sub.52N.sub.3O.sub.12PS: 901.30 [M].sup.+, Observed: 940.83 [M+K].sup.+.

(2496) Preparation of 1048: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (73%). .sup.31P NMR (162 MHz, CDCl.sub.3) 155.96. (ES) m/z Calculated for C.sub.53H.sub.57N.sub.4O.sub.12PS: 1004.34 [M].sup.+, Observed: 1005.86 [M+H].sup.+.

(2497) Preparation of 1049: General Procedure I followed by General Procedure III used. Off-white foamy solid. Yield: (80%). .sup.31P NMR (162 MHz, CDCl.sub.3) 156.37. (ES) m/z Calculated for C.sub.44H.sub.47N.sub.4O.sub.10PS: 854.28 [M].sup.+, Observed: 855.31 [M+H].sup.+.

(2498) Preparation of Amidites (1051).

(2499) ##STR01046##

(2500) Preparation of 1051: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (72%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.26. (ES) m/z Calculated for C.sub.42H.sub.50FN.sub.4O.sub.10PS: 852.29 [M].sup.+, Observed: 853.52 [M+H].sup.+.

(2501) Preparation of Amidites (1052).

(2502) ##STR01047##

(2503) Preparation of 1052: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (76%). .sup.31P NMR (162 MHz, CDCl.sub.3) 156.37. (ES) m/z Calculated for C.sub.42H.sub.50FN.sub.4O.sub.10PS: 852.29 [M].sup.+, Observed: 853.52 [M+H].sup.+.

(2504) Preparation of Amidites (1053, 1054).

(2505) ##STR01048##

(2506) Preparation of 1053: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (80%). .sup.31P NMR (162 MHz, CDCl.sub.3) 156.62. (ES) m/z Calculated for C.sub.47H.sub.50FN.sub.6O.sub.9PS: 908.98 [M].sup.+, Observed: 909.36 [M+H].sup.+.

(2507) Preparation of 1054: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (79%). .sup.31P NMR (162 MHz, CDCl.sub.3) 157.62. (ES) m/z Calculated for C.sub.44H.sub.46FN.sub.4O.sub.9PS: 840.90 [M].sup.+, Observed: 841.67 [M+H].sup.+.

(2508) Preparation of Amidites (1055).

(2509) ##STR01049##

(2510) Preparation of 1055: General Procedure II followed by General Procedure III used. White foamy solid. Yield: (77%). .sup.31P NMR (162 MHz, CDCl.sub.3) 160.00. (ES) m/z Calculated for C.sub.45H.sub.45FN.sub.5O.sub.10PS: 897.26 [M].sup.+, Observed: 898.74 [M+H].sup.+.

(2511) Preparation of Amidites (1056).

(2512) ##STR01050##

(2513) Preparation of 1056: General Procedure II followed by General Procedure III used. Off-white foamy solid. Yield: (84%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.80. (ES) m/z Calculated for C.sub.45H.sub.44ClFN.sub.5O.sub.9P: 867.26 [M].sup.+, Observed: 868.69 [M+H].sup.+.

(2514) Preparation of Amidites (1057).

(2515) ##STR01051##

(2516) Preparation of 1057: General Procedure II followed by General Procedure III used. white foamy solid. Yield: (91%). .sup.31P NMR (162 MHz, CDCl.sub.3) 154.48. (ES) m/z Calculated for C.sub.52H.sub.55FN.sub.5O.sub.10PS: 991.34 [M].sup.+, Observed: 992.87 [M+H].sup.+.

Example 4F. Example Technologies for Chirally Controlled Oligonucleotide PreparationExample Cycles, Conditions and Reagents for Oligonucleotide Synthesis

(2517) In some embodiments, the present disclosure provides technologies (e.g., reagents, solvents, conditions, cycle parameters, cleavage methods, deprotection methods, purification methods, etc.) that are particularly useful for preparing chirally controlled internucleotidic linkages. In some embodiments, such internucleotidic linkages, e.g., non-negatively charged internucleotidic linkages or neutral internucleotidic linkages, etc., comprise PN, wherein P is the linkage phosphorus. In some embodiments, the linkage phosphorus is trivalent. In some embodiments, the linkage phosphorus is pentavalent. In some embodiments, such internucleotidic linkages have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. As demonstrated herein, technologies of the present disclosure can provide mild reaction conditions, high functional group compatibility, alternative deprotection and/or cleavage conditions, high crude and/or purified yields, high crude purity, high product purity, and/or high stereoselectivity.

(2518) In some embodiments, a cycle for preparing natural phosphate linkages comprises or consists of deprotection (e.g., detritylation), coupling, oxidation (e.g., using I.sub.2/Pyr/Water or other suitable methods available in the art) and capping (e.g., cap 2 described herein or other suitable methods available in the art). An example cycle is depicted below, wherein B1 and B2 are independently nucleobases. As appreciated by those skilled in the art, various modifications, e.g., sugar modifications, base modifications, etc. are compatible and may be included.

(2519) ##STR01052##

(2520) In some embodiments, a cycle for preparing non-natural phosphate linkages (e.g., phosphorothioate internucleotidic linkages) comprises or consists of deprotection (e.g., detritylation), coupling, a first capping (e.g., capping-1 as described herein), modification (e.g., thiolation using XH or other suitable methods available in the art), and a second capping (e.g., capping-2 as described herein or other suitable methods available in the art). An example cycle is depicted below, wherein B1 and B2 are independently nucleobases. As appreciated by those skilled in the art, various modifications, e.g., sugar modifications, base modifications, etc. are compatible and may be included. In some embodiments, a cycle using a DPSE chiral auxiliary is referred to as a DPSE cycle or DPSE amidite cycle.

(2521) ##STR01053##

(2522) In some embodiments, a cycle for preparing non-natural phosphate linkages (e.g., certain non-negatively charged internucleotidic linkages, neutral internucleotidic linkages, etc.), particularly those comprising PN, wherein P is the linkage phosphorus and/or those have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, III, or a salt form thereof, comprises or consists of deprotection (e.g., detritylation), coupling, a first capping (e.g., capping-1 as described herein), modification (e.g., using ADIH

(2523) ##STR01054##
2-azido-1,3-dimethyl-4,5-dihydro-1H-imidazol-3-ium hexafluorophosphate (V)) or other suitable methods available in the art), and a second capping (e.g., capping-2 as described herein or other suitable methods available in the art). An example cycle is depicted below, wherein B1 and B2 are independently nucleobases. In some embodiments, a chiral auxiliary utilized in such a cycle for preparing a chirally controlled internucleotidic linkage comprises an electron-withdrawing group as described herein, e.g., various chiral auxiliaries having a G.sup.2 comprising an electron-withdrawing group. In some embodiments, G.sup.2 comprises a SO.sub.2R group as described herein (e.g., in some embodiments, R is optionally substituted phenyl; in some embodiments, R is optionally substituted alkyl (e.g., t-butyl); in some embodiments, it was observed that R being alkyl (e.g., R being t-butyl (e.g., WV-CA-240)) can provide comparable results to R being optionally substituted phenyl(e.g., R being phenyl(PSM))). As appreciated by those skilled in the art, various modifications, e.g., sugar modifications, base modifications, etc. are compatible and may be included. In some embodiments, a cycle using a PSM chiral auxiliary is referred to as a PSM cycle or PSM amidite cycle.

(2524) ##STR01055##

(2525) Various cleavage and deprotection methods may be utilized in accordance with the present disclosure. In some embodiments, as appreciated by those skilled in the art, parameters of cleavage and deprotection (e.g., bases, solvents, temperatures, equivalents, time, etc.) can be adjusted in view of, e.g., structures of oligonucleotides to be prepared (e.g., nucleobases, sugars, internucleotidic linkages, and modifications/protections thereof), solid supports, reaction scales, etc. In some embodiments, cleavage and deprotection comprise one, or two or more, individual steps. For example, in some embodiments, a two-step cleavage and deprotection is utilized. In some embodiments, a cleavage and deprotection step comprises a fluoride-containing reagent (e.g., TEA-HF, optionally buffered with additional bases such as TEA) in a suitable solvent (e.g., DMSO/H.sub.2O) at a suitable amount (e.g., about 100 or more (e.g., 1005) mL/mmol) and is performed at a suitable temperature (e.g., about 0-100, 0-80, 0-50, 0-40, 0-30, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 C. (e.g., in one example, 272 C.)) for a suitable period of time (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more hours (e.g., in one example, 60.5 h)). In some embodiments, a cleavage and deprotection step comprises a suitable base (e.g., NR.sub.3) in a suitable solvent (e.g., water) (e.g., conc. NH.sub.4OH) at a suitable amount (e.g., about 200 or more (e.g., 2005) mL/mmol) and is performed at a suitable temperature (e.g., about 0-100, 0-80, 0-50, 0-40, 0-30, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 C. (e.g., in one example, 372 C.)) for a suitable period of time (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more hours (e.g., in one example, 241 h)). In some embodiments, cleavage and deprotection comprises or consists of two steps, wherein one step (e.g., step 1) is 1TEA-HF in DMSO/H.sub.2O, 1005 mL/mmol, 272 C. and 60.5 h, and the other step (e.g., step 2) is conc. NH.sub.4OH, 2005 mL/mmol, 372 C. and 241 h. Certain examples of cleavage and deprotection processes are described here.

(2526) As appreciated by those skilled in the art, oligonucleotide synthesis is often performed on solid support. Many types of solid support are commercially available and/or can be otherwise prepared/obtained and can be utilized in accordance with the present disclosure. In some embodiments, a solid support is CPG. In some embodiments, a solid support is NittoPhase HL. Types and sizes of solid support can be selected based on desired applications, and in some cases, for a specific use one type of solid support may perform better than the other. In some embodiments, it was observed that for certain preparations CPG can deliver higher crude yields and/or purities compared to certain polymer solid supports such as NittoPhase HL.

(2527) Amidites are typically dissolved in solvents at suitable concentrations. In some embodiments, amidites are dissolved in ACN. In some embodiments, amidites are dissolved in a mixture of two or more solvents. In some embodiments, amidites are dissolved in a mixture of ACN and IBN (e.g., 20% ACN/80% IBN). Various concentrations of amidites may be utilized, and may be adjusted in view of specific conditions (e.g., solid support, oligonucleotides to be prepared, reaction times, scales, etc.). In some embodiments, a concentration of about 0.01-0.5, 0.05-0.5, 0.1-0.5, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 M is utilized. In some embodiments, a concentration of about 0.2 M is utilized. In many embodiments, amidite solutions are dried. In some embodiments, 3 molecular sieves are utilized to dry amidite solutions (or keep amidite solutions dry). In some embodiments, molecular sieves are utilized at about 15-20% v/v.

(2528) Various equivalents of amidites may be useful for oligonucleotide synthesis. As those skilled in the art will appreciate, equivalents of amidites can be adjusted in view of specific conditions (e.g., solid support, oligonucleotides to be prepared, reaction times, scales, etc.), and the same or different equivalents may be utilized during synthesis. In some embodiments, equivalents of amidites are about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more. In some embodiments, a suitable equivalent is about 2. In some embodiments, a suitable equivalent is about 2.5. In some embodiments, a suitable equivalent is about 3. In some embodiments, a suitable equivalent is about 3.5. In some embodiments, a suitable equivalent is about 4.

(2529) A number of activators are available in the art and may be utilized in accordance with the present disclosure. In some embodiments, an activator is ETT. In some embodiments, an activator is CMIMT. In some embodiments, CMIMT is utilized for chirally controlled synthesis. As appreciated by those skilled in the art, the same or different activators may be utilized for different amidites, and may be utilized at different amounts. In some embodiments, activators are utilized at about 40-100%, e.g., 40%, 50%, 60%, 70%, 80% or 90% delivery. In some embodiments, a delivery is about 60% (e.g., for ETT). In some embodiments, a delivery is about 70% (e.g., for CMIMT). In some embodiments, molar ratio of activator/amidite is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, a molar ratio is about 3-6. In some embodiments, a molar ratio is about 1. In some embodiments, a molar ratio is about 2. In some embodiments, a molar ratio is about 3. In some embodiments, a molar ratio is about 4. In some embodiments, a molar ratio is about 5. In some embodiments, a molar ratio is about 6. In some embodiments, a molar ratio is about 7. In some embodiments, a molar ratio is about 8. In some embodiments, a molar ratio is about 9. In some embodiments, a molar ratio is about 10. In some embodiments, a molar ratio is about 2-5, 2-4 or 3-4 (e.g., for ETT). In some embodiments, a molar ratio is about 3.7 (e.g., for ETT). In some embodiments, a molar ratio is about 3-8, 4-8, 4-7, 4-6, 5-7, 5-8 or 5-6 (e.g., for CMIMT). In some embodiments, a molar ratio is about 5.8 (e.g., for CMIMT).

(2530) As appreciated by those skilled in the art, various suitable flowrates and reaction times may be utilized for oligonucleotide synthesis, and may be adjusted according to oligonucleotides to be prepared, scales, synthetic setups, etc. In some embodiments, a recycle flow rate utilized for synthesis is about 200 cm/h. In some embodiments, a recycle time is about 1-10 minutes. In some embodiments, a recycle time is about 8 minutes. In some embodiments, a recycle time is about 10 minutes.

(2531) Many technologies are available to modify P(III) linkages, e.g., after coupling. For example, various methods are available to convert a P(III) linkage to a P(V) P(O)-type linkage, e.g., via oxidation. In some embodiments, I.sub.2/Pyr/H.sub.2O is utilized. Similarly, many methods are available to convert a P(III) linkage to a P(V) P(S)-type linkage, e.g., via sulfurization. In some embodiments, as illustrated herein, XH is utilized as a thiolation reagent. Technologies for converting P(III) linkages to P(V) P(N)-type linkages are also widely available and can be utilized in accordance with the present disclosure. In some embodiments, as illustrated herein ADIH is employed. Suitable reaction parameters are described herein. In some embodiments, ADIH is used at a concentration of about 0.01-0.5, 0.05-0.5, 0.1-0.5, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 M. In some embodiments, concentration of ADIH is about 0.25 M. In some embodiments, concentration of ADIH is about 0.3 M. In some embodiments, ADIH is utilized at about 1-50, 1-40, 1-30, 1-25, 1-20, 1-10, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45 or 50 or more equivalent. In some embodiments, equivalent of ADIH is about 7.5. In some embodiments, equivalent of ADIH is about 10. In some embodiments, equivalent of ADIH is about 15. In some embodiments, equivalent of ADIH is about 20. In some embodiments, equivalent of ADIH is about 23. In some embodiments, equivalent of ADIH is about 25. In some embodiments, equivalent of ADIH is about 30. In some embodiments, equivalent of ADIH is about 35. In some embodiments, one experiment, ADIH was utilized at 15.2 equivalent, and 15 min contact time. In some embodiments, depending on amidites, concentrations, equivalents, contact times, etc. of reagents, e.g., ADIH, may be adjusted.

(2532) Technologies of the present disclosure are suitable for preparation at various scales. In some embodiments, synthesis is performed at hundreds of umol or more. In some embodiments, a scale is about 200 umol. In some embodiments, a scale is about 300 umol. In some embodiments, a scale is about 400 umol. In some embodiments, a scale is about 500 umol. In some embodiments, a scale is about 550 umol. In some embodiments, a scale is about 600 umol. In some embodiments, a scale is about 650 umol. In some embodiments, a scale is about 700 umol. In some embodiments, a scale is about 750 umol. In some embodiments, a scale is about 800 umol. In some embodiments, a scale is about 850 umol. In some embodiments, a scale is about 900 umol. In some embodiments, a scale is about 950 umol. In some embodiments, a scale is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or more mmol. In some embodiments, a scale is about 1 mmol or more. In some embodiments, a scale is about 2 mmol or more. In some embodiments, a scale is about 5 mmol or more. In some embodiments, a scale is about 10 mmol or more. In some embodiments, a scale is about 15 mmol or more. In some embodiments, a scale is about 20 mmol or more. In some embodiments, a scale is about 25 mmol or more.

(2533) In some embodiments, observed yields were 85-90 OD/umol (e.g., 85,000 OD/mmol for a 10.2 mmol synthesis, with 58.4% crude purity (% FLP)).

(2534) Technologies of the present disclosure, among other things, can provide various advantages when utilized for preparing oligonucleotides comprising chirally controlled internucleotidic linkages, e.g., those comprising PN wherein P is a linkage phosphorus (e.g., internucleotidic linkages of I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof, etc.). For example, as demonstrated herein, technologies of the present disclosure can provide high crude purities and yields (e.g., in many embodiments, about 55-60% full-length product for a 20-mer oligonucleotide) with minimal amount of shorter oligonucleotides (e.g., from incomplete coupling, decomposition, etc.). Such high crude yields and/or purities, among other things, can significantly reduce downstream purification and can significantly reduce production cost and cost of goods, and in some embodiments, greatly facilitate or make possible large scale commercial production, clinical trials and/or commercial sales.

(2535) Example Procedure for Preparing Chirally Controlled Oligonucleotide CompositionsWV-13864.

(2536) Described below are example procedures for preparing WV-13864 using controlled pore glass (CPG) low bulk density solid support (e.g., 2-fC (acetyl) via CNA linker CPG (600 LBD)). Useful phosphoramidites include 5-ODMTr-2-F-dA(N6-Bz)-(L)-DPSE phosphoramidite, 5-ODMTr-2-F-dC(N4-Ac)-(L)-DPSE phosphoramidite, 5-ODMTr-2-F-dG(N2-iBu)-(L)-DPSE phosphoramidite, 5-ODMTr-2-F-dU-(L)-DPSE phosphoramidite, 5-ODMTr-2-OMe-G(N.sup.2-iBu)-(L)-DPSE phosphoramidite, 5-ODMTr-2-F-dC(N4-Ac)-(L)-PSM phosphoramidite, 5-ODMTr-2-F-dG(N2-iBu)-(L)-PSM phosphoramidite, 5-DMT-2-OMe-A(Bz)--Cyanoethyl phosphoramidite, and 5-DMT-2-OMe-C(Ac)--Cyanoethyl phosphoramidite.

(2537) 0.1 M Xanthane hydride solution (XH) was used for thiolation. Neutral PN linkages were formed utilizing 0.3 M of 2-azido-1,3-dimethyl-imidazolinium hexafluorophosphate (ADIH) in acetonitrile. Oxidation solution was 0.04-0.06 M iodine in pyridine/water, 90/10, v/v. Cap A was N-Methylimidazole in acetonitrile, 20/80, v/v. Cap B was acetic anhydride/2,6-Lutidine/Acetonitrile, 20/30/50, v/v/v. Deblocking was performed using 3% dichloroacetic acid in toluene. NH.sub.4OH used was 28-30% concentrated ammonium hydroxide.
Detritylation.

(2538) To initiate the synthesis, the 5-ODMTr-2-F-dC(N4-Ac)-CPG solid support was subjected to acid catalyzed removal of the DMTr protecting group from the 5-hydroxyl by treatment with 3% (DCA) in toluene. The DMTr removal step was usually visualized with strong red or orange color and can be monitored by UV watch command at the wavelength of 436 nm.

(2539) DMTr removal can be repeated at the beginning of a synthesis cycle. In every case, following detritylation, the support-bound material was washed with acetonitrile in preparation for the next step of the synthesis.

(2540) Coupling.

(2541) Amidites were dissolved either in acetonitrile (ACN) or in 20% isobutyronitrile (IBN)/80% ACN at a concentration of 0.2M without density correction. The solutions were dried over molecular sieves (3 ) not less than 4 h before use (15-20%, v/v).

(2542) TABLE-US-00124 Amidite Solvent Concentration MS3 5-ODMTr-2-OMe-A(N6-Bz)-CE ACN 0.2M 15-20%, v/v 5-ODMTr-2-OMe-C(N4-Ac)-CE ACN 0.2M 15-20%, v/v 5-ODMTr-2-F-dA(N6-Bz)-(L)-DPSE ACN 0.2M 15-20%, v/v 5-ODMTr-2-F-dC(N4-Ac)-(L)-DPSE ACN 0.2M 15-20%, v/v 5-ODMTr-2-F-dU-(L)-DPSE 20% IBN/80% ACN 0.2M 15-20%, v/v 5-ODMTr-2-F-dG(N2-iBu)-(L)-DPSE ACN 0.2M 15-20%, v/v 5-ODMTr-2-OMe-G(N2-iBu)-(L)-DPSE 20% IBN/80% ACN 0.2M 15-20%, v/v 5-ODMTr-2-F-dC(N4-Ac)-(L)-PSM ACN 0.2M 15-20%, v/v 5-ODMTr-2-F-dG(N2-iBu)-(L)-PSM ACN 0.2M 15-20%, v/v

(2543) Dual activators (CMIMT and ETT) coupling approach were utilized. Both activators were dissolved in ACN at a concentration of 0.5M. CMIMT has been used for chirally controlled coupling with CMIMT to amidite molar ratio of 5.833/l. ETT was used for the coupling of standard amidites (for natural phosphate linkages) with ETT to amidite molar ratio of 3.752/l. Recycle time for all DPSE and PSM amidites was 10 min except mG-L-DPSE which was 8 min. All standard amidites were coupled for 8 min.

(2544) Cap-1 (Capping-1, First Capping).

(2545) Cap B(Ac.sub.2O/2,6-lutidine/MeCN (2:3:5, v/v/v)) was used. In some embodiments, Cap-1 capped secondary amine groups, e.g., on the chrial auxiliaries. In some embodiments, incomplete protection of secondary amines may lead side reaction resulting in a failed coupling or formation of one or more by-products. In some embodiments, Cap-1 may not be an efficient condition for esterification (e.g., a condition less efficient than Cap-2 (the second capping) for capping unreacted 5-OH).

(2546) Thiolation for DPSE Cycles.

(2547) Following Cap-1, phosphite intermediates, P(III), were modified with sulfurizing reagent. In an example preparation, 1.2 CV (6-7 equivalent) of sulfurizing reagent (0.1 M XH/pyridine-ACN, 1:1, v/v) was delivered through the synthetic column via flow through mode over 6 min contact time to form P(V).

(2548) Azide Reaction for PSM Cycles.

(2549) After Cap-1, a suitable reagent (e.g., comprising N.sub.3 such as ADIH), in ACN was used to form neutral internucleotidic linkages (PN linkages). In an example preparation, 10.3 eq. of 0.25 M ADIH over 10 min contact time for fG-L-PSM and 25.8 eq. of 0.3 M ADIH over 15 min contact time for fC-L-PSM were utilized in the respective cycles.

(2550) Oxidation for Standard Nucleotide Cycles.

(2551) Cap-1 step was not necessary for standard amidite cycle. After coupling of a standard amidite onto the solid support, the phosphite intermediate, P(III), was oxidized with 0.05 M of iodine/water/pyridine solution to form P(V). In an example preparation, 3.5 eq. of oxidation solution delivered to the column by a flow through mode over 2 min contact time for efficient oxidation.

(2552) Cap-2 (Capping-2, a Second Capping).

(2553) Coupling efficiency on the solid phase oligonucleotide synthesis for each cycle was approx. 97-100% and monitored by, e.g., release of DMTr cation. Residual uncoupled 5-hydroxyl groups, typically 1-3% by detrit monitoring, on the solid support were blocked with Cap A (20% N-Methylimidazole in acetonitrile (NMI/ACN=20/80, v/v)) and Cap B(20%: 30%: 50%=Ac.sub.2O: 2,6-Lutidine: ACN(v/v/v)) reagents (e.g., 1:1). Both reagents (e.g., 0.4 CV) were delivered to the column by flow through mode over 0.8 min contact time to prevent formation of failure sequences. Uncapped amine groups may also be protected in this step.

(2554) As illustrated herein, in some embodiments, a DPSE amidite or DPSE cycle is Detritylation.fwdarw.Coupling.fwdarw.Cap-1 (Capping-1, first capping).fwdarw.Thiolation.fwdarw.Cap-2 (Capping-1, Post-capping, second capping); in some embodiments, a PSM amidite or PSM cycle is Detritylation.fwdarw.Coupling.fwdarw.Cap-1 (Capping-1, first capping).fwdarw.Azide reaction.fwdarw.Cap-2 (Capping-1, Post-capping, second capping); in some embodiments, a standard amidite or standard cycle (traditional, non-chirally controlled) is Detritylation.fwdarw.Coupling.fwdarw.Oxidation.fwdarw.Cap-2 (Capping-1, Post-capping, second capping).

(2555) Synthetic cycles were selected and repeated until the desired length was achieved.

(2556) Amine Wash.

(2557) In some embodiments, provided technologies are particularly effective for preparing oligonucleotides comprising internucleotidic linkages that comprise PN, wherein P is the linkage phosphorus. In some embodiments, provided technologies comprise contacting an oligonucleotide intermediate with a base. In some embodiments, a contact is performed after desired oligonucleotide lengths have been achieved. In some embodiments, such a contact provides an oligonucleotide comprising internucleotidic linkages that comprise PN, wherein P is the linkage phosphorus (e.g., those of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof). In some embodiments, a contact removes a chiral auxiliary (e.g., those with a G.sup.2 that is connected to the rest of the molecule through a carbon atom, and the carbon atom is connected to at least one electron-withdrawing group (e.g., WV-CA-231, WV-CA-236, WV-CA-240, etc.)). In some embodiments, a contact is performed utilizing a base or a solution of a base which is substantially free of OH.sup. or water (anhydrous). In some embodiments, a base is an amine (e.g., N(R).sub.3). In some embodiments, an amine has the structure of NH(R).sub.2, wherein each R is independently optionally substituted C1-6 aliphatic; in some embodiments, each R is independently optionally substituted C1-6 alkyl. In some embodiments, a base is N, N-diethylamine (DEA). In some embodiments, a base solution is 20% DEA/ACN. In some embodiments, such a contact with a base lowers levels of by-products which, at one or more locations of internucleotidic linkages that comprise PN, have instead natural phosphate linkages.

(2558) In an example preparation, an on-column amine wash was performed after completion of oligonucleotide nucleotide synthesis cycles, by five column volume of 20% DEA in acetonitrile over 15 min contact time.

(2559) In some embodiments, contact with a base may also remove 2-cyanoethyl group used for construction of standard natural phosphate linkage. In some embodiments, contact with a base provide a natural phosphate linkage (e.g., in a salt form in which the cation is the corresponding ammonium salt of the amine base).

(2560) Cleavage and Deprotection.

(2561) After contact with a base, oligonucleotides are exposed to further cleavage and deprotection. In an example preparation, auxiliary removal (e.g., DPSE), cleavage & deprotection was a two steps process. In step 1, CPG solid support with oligonucleotides was treated with 1TEA-HF solution (DMSO: Water: TEA.Math.3HF: TEA=43:8.6:2.8:1=v/v/v/v, 1005 uL/umol) for 6=0.5 h at 272 C. The bulk slurry was then treated with concentrated ammonium hydroxide (28-30%, 20010 mL/mmol) for 241 h at 372 C. (step 2) to release oligonucleotide from the solid support. Crude product was collected by filtration. Filtrates were combined with washes (e.g., water) of the solid support. In some embodiments, observed yields were about 80-90 OD/umole.

(2562) Example Procedure for Preparing Chirally Controlled Oligonucleotide CompositionsWV-13835.

(2563) In an example preparation, WV-13835 was prepared at a 1.2 mmol scale starting from CPG 2-F-U. DPSE was utilized as chiral auxiliary for chirally controlled internucleotidic linkages. The preparation comprised multiple cycles comprising a de-blocking step (detritylation under an acidic condition), a coupling step (with a DPSE phosphoramidite), a pre-modification capping step (e.g., Cap B), a modification step (e.g., thiolation using 0.1M XH in Pyr/CAN), a post-modification capping step (e.g., under a cap 2 condition (1:1 Cap A+Cap B). In some embodiments, a cycle comprises a modification step which is or comprises oxidation with I.sub.2/Pyr/H.sub.2O. Cleavage and deprotection included two steps, wherein step one utilized TEA-HF at 100 mL/mmol and 272.5 C., and step 2 utilized conc. NH.sub.4OH at 200 mL/mmol and 372.5 C. Total crude yield was 91800 OD (76500 OD/mmol). Neat % FLP was 53.6% and NAP(after de-salting) % FLP was 58.3%. % FLP in crude was 1.71 g.

(2564) Example Procedure for Preparing Chirally Controlled Oligonucleotide CompositionsWV-14791.

(2565) In an example preparation, WV-14791 was prepared at a 402 umol scale starting from CPG 2-F-U. DPSE was utilized as chiral auxiliary for chirally controlled phosphorothioate internucleotidic linkages, and PSM for chirally controlled n001. The preparation comprised multiple cycles comprising a de-blocking step (detritylation under an acidic condition), a coupling step (with a DPSE (for a chirally controlled phosphorothioate internucleotidic linkage) or PSM phosphoramidites (for a chirally controlled n001 internucleotidic linkage)), a pre-modification capping step (e.g., Cap B), a modification step (e.g., thiolation using 0.1M XH in Pyr/CAN for phosphorothioate internucleotidic linkages, 2-azido-1,3-dimethyl-imidazolinium hexafluorophosphate in CAN for n001), a post-modification capping step (e.g., under a cap 2 condition (1:1 Cap A+Cap B). In some embodiments, a cycle comprises a modification step which is or comprises oxidation with I.sub.2/Pyr/H.sub.2O. Total crude yield was 27000 OD (67.1 OD/umol). Neat % FLP was 45.7% and NAP(after de-salting) % FLP was 51.8%. % FLP in crude was 445 mg.

(2566) Example Procedure for Preparing Chirally Controlled Oligonucleotide CompositionsWV-14344.

(2567) In an example preparation, WV-14344 was prepared at a 400 umol scale starting from CPG 2-F-C. DPSE was utilized as chiral auxiliary for chirally controlled phosphorothioate internucleotidic linkages, and PSM for chirally controlled n001. The preparation comprised multiple cycles comprising a de-blocking step (detritylation under an acidic condition), a coupling step (with a DPSE (for a chirally controlled phosphorothioate internucleotidic linkage) or PSM phosphoramidites (for a chirally controlled n001 internucleotidic linkage)), a pre-modification capping step (e.g., Cap B), a modification step (e.g., thiolation using 0.1M XH in Pyr/CAN for phosphorothioate internucleotidic linkages, 2-azido-1,3-dimethyl-imidazolinium hexafluorophosphate in CAN for n001), a post-modification capping step (e.g., under a cap 2 condition (1:1 Cap A+Cap B). In some embodiments, a cycle comprises a modification step which is or comprises oxidation with I.sub.2/Pyr/H.sub.2O. Total crude yield was 32000 OD (80 OD/umol). Neat % FLP was 48.8% and NAP(after de-salting) % FLP was 59.2%. % FLP in crude was 571 mg.

(2568) Example Preparation of Additional Chirally Controlled Oligonucleotide Compositions.

(2569) Various oligonucleotide compositions including chirally controlled oligonucleotide composition were prepared utilizing technologies described herein. In some embodiments, oligonucleotide compositions were prepared using automated solid-phase synthesis. Certain preparations were performed at 25 umol using TWIST columns 10 um/15 um column (GlenResearch, catalog #20-0040) filled with 325 mg of CNA linked nucleosides-CPG. Example cycles and azide modification reagents for chirally controlled internucleotidic linkages at 25 umol were shown below.

(2570) TABLE-US-00125 Waiting Step Operation Reagents Volume time 1 Deblocking (detritylation) 3% DCA/DCM 10 mL 1 min 2 Coupling 0.2M monomer/MeCN 0.5 mL 8 min 0.6M CMIMT/MeCN 1 mL 3 Pre-modification capping (cap-1) Cap-B 2 mL 2 min 4 Modification 0.2M XH/pyridine or 2 mL 6 min (sulfurization or azide reaction) 0.5M azide reagent/MeCN 2 mL 10 min 5 Post-modification capping (cap-2) Cap-A + Cap-B 2 mL 45 s Final linkage Azide Reagent n001 056 n003 057 n004 058 n006 059 n008 060

(2571) After cycles were completed, the CPG support was treated with 20% DEA in MeCN for 12 min, washed with dry MeCN and dried under argon and vacuum. The dried CPG support was transferred into a 15 mL plastic tube, treated with 1 solution (1M HF-TEA in H.sub.2O-DMSO(1:5, v/v), 100 uL/umol) for 6 h at 28 C., then added conc. NH.sub.3 (200 uL/umol) and reacted for 24 h at 37 C. The mixture was cooled to room temperature and the CPG was removed by membrane filtration, and the product was analyzed by LTQ and RP-UPLC with a linear gradient of MeCN (1-15%/15 min) in (10 mM TEA, 100 mM HFIP in water) at 55 C. at a rate of 0.8 mL/min. Crude oligonucleotides were purified by AEX-HPLC eluting with 20 mM NaOH to 2.5M NaCl, and desalted to obtain the target oligonucleotide compositions.

(2572) Example preparations were listed below, with crude UPLC purity ranging from about 9% to about 58% percent. Higher crude HPLC purities were observed for preparation of the same and/or other oligonucleotides.

(2573) TABLE-US-00126 Oligonucleotide Scale (umol) Observed Mass WV-16006 70 6912.3 WV-16007 70 7068.9 WV-24092 24 7282 WV-24098 24 7237.1 WV-24104 24 7399.1 WV-24109 24 7355.1 WV-25536 24 6729.1 WV-25537 24 6705.2 WV-25538 24 6739.1 WV-25539 24 6702 WV-25540 24 6726.9 WV-25541 25 7012.6 WV-25542 25 7014.1 WV-25543 25 6989.9 WV-25544 25 7024.2

(2574) Among other things, provided technologies provided high crude purities and/or yields. In many preparations (various scales, reagents concentrations, reaction times, etc.), about 55-60% crude purities (% FLP) were obtained, with minimal amount of shorter oligonucleotides (e.g., from incomplete coupling, decomposition, side-reactions, etc.). In many embodiments, amounts of the most significant shorter oligonucleotide are no more than about 2-10%, often no more than 2-4% (e.g., in some embodiments, as low as about 2% (the most significant shorter oligonucleotide being N-3)).

(2575) Various technologies are available for oligonucleotide purification and can be utilized in accordance with the present disclosure. In some embodiments, crude products were further purified (e.g., over 90% purity) using, e.g., AEX purification, and/or UF/DF.

(2576) Using technologies described herein, various oligonucleotides comprising diverse base sequences, modifications (e.g., nucleobase, sugar, and internucleotidic linkage modifications) and/or patterns thereof, linkage phosphorus stereochemistry and/or patterns thereof, etc. were prepared at various scales from umol to mmol. Such oligonucleotides have various targets and may function through various mechanisms. Certain such oligonucleotides were presented in the Tables of the present disclosure.

(2577) As appreciated by those skilled in the art, examples described herein are for illustration only. Those skilled in the art will appreciate that various conditions, parameters, etc. may be adjusted according to, e.g., instrumentation, scales, reagents, reactants, desired outcomes, etc. Certain results may be further improved using various technologies in accordance with the present disclosure. Among other things, provided oligonucleotides and compositions thereof can provide significantly improved properties and/or activities, e.g., in various assays and in vivo models, and may be particularly useful for preventing and/or treating various conditions, disorders or diseases. Certain data are provided in Examples herein.

Example 4G. Synthesis of Certain Reagents for Incorporation of Mod

(2578) As described in the present disclosure, oligonucleotide of the present disclosure may comprise various additional chemical moieties (e.g., various Mods) in addition to the oligonucleotide chain moiety. In some embodiments, the present disclosure provides oligonucleotide comprising a Mod described herein. In some embodiments, such additional moieties provide improved properties, activities, deliveries, etc. In some embodiments, the present disclosure provides useful additional chemical moieties, and technologies for preparing and incorporating such additional chemical moieties. Certain examples are described below. Those skilled in the art appreciates and various technologies related to additional chemical moieties (e.g., structures, preparations, incorporation, uses, etc.), e.g., those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, WO 2017/210647, WO 2018/223056, WO 2018/237194, WO 2019/055951, etc., such technologies of each of which are independently incorporated by reference, may be utilized in accordance with the present disclosure.

(2579) Synthesis of 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoic acid.

(2580) ##STR01061##

(2581) Step 1. To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (5 g, 4.95 mmol, 1 eq.) in DCM (50 mL) was added TFA (16.93 g, 148.48 mmol, 10.99 mL, 30 eq.) at 0 C. The mixture was stirred at 0-25 C. for 2 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. Then added ACN(5 mL), and MTBE (40 mL), filtered the viscous liquid. The crude benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (5.21 g, crude, 3TFA) was obtained as a yellowish oil. LCMS: (M+H.sup.+): 710.6; (M+Na.sup.+): 732.7.

(2582) Step 2. To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (5.21 g, crude, 3TFA) in DCM (35 mL) was added DIEA (6.39 g, 49.45 mmol, 8.61 mL, 10 eq.) and 2-chloro-1,3-dimethyl-4,5-dihydroimidazol-1-ium; hexafluorophosphate (4.55 g, 16.32 mmol, 3.3 eq.). The mixture was stirred at 25 C. for 15 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The crude was purified by RP-MPLC(Spec: C18, 330 g, 2035 micron, 100 ). The product benzyl 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (4.94 g, crude) was obtained as a yellow oil. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) =7.39-7.29 (m, 5H), 3.70-3.62 (m, 28H), 3.45 (q, J=6.6 Hz, 7H), 3.30-3.26 (m, 6H), 3.08-2.99 (m, 21H), 2.47-2.39 (m, 9H), 2.23 (t, J=7.4 Hz, 2H), 1.92-1.78 (m, 10H).

(2583) Step 3. To a solution of benzyl 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (2 g, 2.00 mmol, 1 eq.) in THF (10 mL) and H.sub.2O (2 mL) was added LiOH.Math.H.sub.2O (588.51 mg, 14.02 mmol, 7 eq.). The mixture was stirred at 25 C. for 3 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep-HPLC(column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-25%,20 min). 5-((1,19-bis((1,3-dimethylimidazolidin-2-ylidene)amino)-10-((3-((3-((1,3-dimethylimidazolidin-2-ylidene)amino)propyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoic acid (0.6 g, 651.84 umol, 32.54% yield, 98.66% purity) was obtained as a yellow gum. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =8.03 (br t, J=5.6 Hz, 3H), 7.75 (br t, J=5.6 Hz, 3H), 7.08 (s, 1H), 3.62-3.54 (m, 24H), 3.34 (q, J=6.6 Hz, 7H), 3.12 (q, J=6.2 Hz, 5H), 2.96 (s, 18H), 2.30 (br t, J=6.4 Hz, 6H), 2.23-2.03 (m, 4H), 1.79-1.59 (m, 8H); LCMS: (M/2+H.sup.+): 454.9; LCMS purity: 98.66%.

(2584) Synthesis of (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid.

(2585) ##STR01062##

(2586) Step 1. To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (5 g, 4.95 mmol, 1 eq.) in DCM (50 mL) was added TFA (16.93 g, 148.48 mmol, 10.99 mL, 30 eq.). The mixture was stirred at 0-25 C. for 2 hr. The reaction mixture was concentrated under reduced pressure to remove solvent, then added ACN(50 mL), and MTBE (500 mL), filtered the viscous liquid. The crude benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (5.21 g, crude, 3TFA) was obtained as a yellow oil. LCMS: (M+H.sup.+): 710.6; (M+Na.sup.+): 732.5.

(2587) Step 2. To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (3.86 g, 3.67 mmol, 1 eq., 3TFA) in DCM (35.1 mL) was added DIEA (4.73 g, 36.63 mmol, 6.38 mL, 10 eq.) and [[(Z)-(1-cyano-2-ethoxy-2-oxo-ethylidene)amino]oxy-morpholino-methylene]-dimethylammonium; hexafluorophosphate (5.18 g, 12.09 mmol, 3.3 eq.). The mixture was stirred at 25 C. for 15 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The crude was dissolved by ACN(15 mL) then input it into the reversed-phase column. The crude product was purified by reversed-phase HPLC(0.75% TFA in water, and acetonitrile). The crude compound benzyl (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (4.14 g, crude) was obtained as a yellow oil. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) =7.43-7.24 (m, 5H), 3.78 (br s, 13H), 3.72-3.64 (m, 12H), 3.50-3.36 (m, 13H), 3.27 (br d, J=8.6 Hz, 11H), 3.11-2.97 (m, 18H), 2.50-2.42 (m, 8H), 2.26 (t, J=7.4 Hz, 2H), 1.93-1.78 (m, 8H).

(2588) Step 3. To a solution of benzyl (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (2 g, 1.77 mmol, 1 eq.) in THF (1 mL) and H.sub.2O (0.2 mL) was added LiOH.Math.H.sub.2O (519.71 mg, 12.38 mmol, 7 eq.). The mixture was stirred at 25 C. for 3 hr. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by prep-HPLC(Phenomenex luna C18 250*50 mm *10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-20%, 20 min). The compound (E)-2-methyl-14,14-bis((E)-2-methyl-3-morpholino-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-3-morpholino-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (1.2 g, 1.14 mmol, 64.65% yield, 99.16% purity) was obtained as a yellow gum. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =7.99 (br s, 3H), 7.84 (br s, 3H), 7.06 (s, 1H), 3.67 (br s, 12H), 3.59-3.49 (m, 12H), 3.44-3.25 (m, 12H), 3.11 (br s, 12H), 3.02-2.81 (m, 17H), 2.31 (br t, J=6.1 Hz, 6H), 2.23-2.04 (m, 4H), 1.79-1.60 (m, 8H). LCMS: (M/2+H.sup.+): 521.0; LCMS purity: 99.16%.

(2589) Synthesis of(S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-en-34-oic acid.

(2590) ##STR01063## ##STR01064##

(2591) Step 1. To a solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (10 g, 10.94 mmol, 5 eq.) in DMF (100 mL) was added DIPEA (2.83 g, 21.88 mmol, 3.81 mL, 10 eq.) and followed by benzyl(S)-6-(2,6-diaminohexanamido)hexanoate (924.07 mg, 2.19 mmol, 1 eq., 2HCl) and then to the mixture was dropwise added HATU (1.91 g, 5.03 mmol, 2.3 eq.) in DMF (10 mL) at 0 C. The reaction mixture was stirred at 25 C. for 12 hr. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC(TFA condition). Column: Phenomenex luna C 18 250*50 mm *10 um; mobile phase: [water (0.1% TFA)-ACN]; B % CH.sub.3CN: 10%-35%, 20 min. Benzyl(S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-en-34-oate (3.7 g, crude) was obtained as a yellow oil. .sup.1H NMR (400 MHz, CHLOROFORM-d) =8.01-7.77 (m, 10H), 7.63 (br t, J=4.9 Hz, 6H), 7.40-7.29 (m, 5H), 7.07 (br d, J=16.5 Hz, 2H), 5.08 (s, 2H), 4.18-4.07 (m, 1H), 3.63-3.46 (m, 24H), 3.10 (br dd, J-3.2, 5.1 Hz, 25H), 3.00-2.78 (m, 79H), 2.39-2.23 (m, 18H), 2.15-1.98 (m, 20H), 1.72-1.13 (m, 31H). LCMS: M/4+H.sup.+=536.5.

(2592) Step 2. To a solution of compound benzyl(S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-en-34-oate (4.4 g, 2.05 mmol, 1 eq.) in THF (40 mL) and H.sub.2O (8 mL) was added LiOH.Math.H.sub.2O (603.45 mg, 14.38 mmol, 7 eq.). The mixture was stirred at 25 C. for 2 hr. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC(TFA condition). Column: Phenomenex luna C 18 250*50 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 2%-30%, 20 min. Compound(S)-3-(dimethylamino)-26-(3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-amido)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16,20,27-tetraoxo-12-oxa-2,4,8,15,21,28-hexaazatetratriacont-3-en-34-oic acid (1.4 g, 678.84 umol, 33.04% yield, 99.483% purity) was obtained as a yellow oil. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =8.00 (br t, J=5.5 Hz, 6H), 7.91 (br t, J=5.6 Hz, 1H), 7.87-7.79 (m, 2H), 7.67 (br t, J=4.8 Hz, 5H), 7.15-7.01 (m, 2H), 4.17-4.10 (m, 1H), 3.70-3.43 (m, 24H), 3.16-3.06 (m, 24H), 3.05-2.75 (m, 76H), 2.30 (br t, J=6.4 Hz, 12H), 2.18 (t, J=7.4 Hz, 2H), 2.15-1.98 (m, 8H), 1.66 (quin, J=6.6 Hz, 17H), 1.48 (quin, J=7.4 Hz, 3H), 1.41-1.31 (m, 4H), 1.28-1.17 (m, 4H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) =174.85, 172.67, 172.61, 172.40, 172.19, 170.87, 161.50, 158.77 (q, J=35.2 Hz, 1C), 118.06, 115.15, 68.72, 67.84, 60.03, 53.08, 42.36, 38.87, 38.78, 36.40, 35.95, 35.88, 35.81, 35.25, 34.91, 34.08, 29.85, 29.40, 29.19, 26.34, 24.63, 23.47, 22.14. LCMS: M/3+H.sup.+=684.7, purity: 99.48%.

Synthesis of (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanamido)hexanoic acid

(2593) ##STR01065##

(2594) Step 1. To a solution of (S)-4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanoic acid (14 g, 47.41 mmol, 1 eq.) in THF (150 mL) was added TEA (14.39 g, 142.23 mmol, 19.80 mL, 3 eq.), followed by tert-butyl 6-aminohexanoate 6-aminohexanoate (11.54 g, 61.63 mmol, 1.3 eq.) at 0-5 C. and stirred for 0.5 hour. T3P(60.34 g, 94.82 mmol, 56.39 mL, 50% purity, 2 eq.) was added to the mixture at 0-5 C. and stirred at 20-25 C. for 12 hours. TLC(Petroleum ether/Ethyl acetate=1:1, R.sub.f=0.35) showed that the starting material was consumed completely. The mixture was concentrated under reduced pressure to remove the solvent, and then re-dissolved with ethyl acetate (100 mL). The organic phase was washed by saturated aq. NaHCO.sub.3 (50 mL3) and dried over anhydrous Na.sub.2SO.sub.4. The crude product was purified by MPLC (SiO.sub.2, Petroleum ether/Ethyl acetate=1:1) to obtain tert-butyl (S)-6-(4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanamido)hexanoate (19.7 g, crude) as yellow oil.

(2595) Step 2. A mixture of tert-butyl (S)-6-(4-(((benzyloxy)carbonyl)amino)-5-methoxy-5-oxopentanamido)hexanoate (15 g, 32.29 mmol, 1 eq.) and Pd/C(10 g, 10% purity) in THF (300 mL) was evacuated in vacuo and backfilled with H.sub.2 (15 Psi) three times, then stirred at 20-25 C. for 6 hours. TLC(Petroleum ether/Ethyl acetate=1:1, R.sub.f=0) showed that the starting material was consumed completely. The mixture was filtered and concentrated under reduced pressure to remove the most solvent. The crude product was used for the next step without any purification. tert-butyl (S)-6-(4-amino-5-methoxy-5-oxopentanamido)hexanoate (10.67 g, 31.42 mmol, 97.31% yield, 97.303% purity) was obtained as colorless liquid (in solvent). LCMS: M+H.sup.+=331.2, purity: 97.70%.

(2596) Step 3. To a mixture of 4-(N-((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)-methyl)-2,2,2-trifluoroacetamido) benzoic acid (8.28 g, 25.06 mmol, 1.1 eq.) and DIPEA (8.83 g, 68.33 mmol, 11.90 mL, 3 eq.) in DMSO (20 mL) was added HATU (8.66 g, 22.78 mmol, 1 eq.) and tert-butyl (S)-6-(4-amino-5-methoxy-5-oxopentanamido)hexanoate at 20-25 C. and stirred for 12 hours. The mixture was diluted with H.sub.2O (20 mL) and extracted with ethyl acetate (20 mL3). The organic phase was concentrated under reduced pressure to remove the solvent. The crude product was purified by MPLC (SiO.sub.2, Methanol/Ethyl acetate=2:5) to obtain tert-butyl (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanamido)hexanoate (26.2 g, crude) as brown gum. LCMS: M+H.sup.+=721.2.

(2597) Step 4. To a solution of tert-butyl (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanamido)hexanoate (13.1 g, 11.39 mmol, 1 eq.) in DCM (100 mL) was added TFA (7.79 g, 68.35 mmol, 5.06 mL, 6 eq.) at 0-5 C. and the mixture was stirred at 35-40 C. for 12 hours. The mixture was concentrated under reduced pressure to remove the solvent. The crude product was detected by HPLC and purified by prep-HPLC(column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 15%-35%, 20 min) to obtain (S)-6-(4-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-5-methoxy-5-oxopentanamido) hexanoic acid (1.51 g, 1.88 mmol, 32.96% yield, 82.627% purity). .sup.1H NMR (400 MHz, DMSO-d.sub.6) =8.92 (br d, J-7.1 Hz, 1H), 8.74 (s, 1H), 7.93 (br d, J=8.4 Hz, 3H), 7.83 (br t, J=5.5 Hz, 1H), 7.66 (br d, J=8.3 Hz, 2H), 5.18 (s, 2H), 5.06-4.52 (m, 3H), 4.45-4.32 (m, 1H), 3.63 (s, 2H), 3.00 (q, J=6.2 Hz, 2H), 2.25-2.13 (m, 4H), 2.12-2.03 (m, 1H), 1.99-1.87 (m, 1H), 1.46 (quin, J=7.5 Hz, 2H), 1.35 (td, J=7.4, 14.9 Hz, 2H), 1.27-1.15 (m, 2H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) =174.91, 172.83, 171.50, 166.02, 159.47, 153.27, 149.15, 142.22, 134.71, 129.15, 128.99, 128.64, 54.27, 52.97, 52.38, 38.79, 34.05, 32.16, 29.29, 26.76, 26.40, 24.66. LCMS: M+H.sup.+=665.2.

Example 5. Synthesis of N6-stearoyl-N2-(4-sulfamoylbenzoyl)-L-lysine

(2598) ##STR01066##

(2599) Step 1. To a solution of stearic acid (8.00 g, 28.12 mmol) in DCM (210 mL) was added 1-hydroxypyrrolidine-2,5-dione (3.24 g, 28.12 mmol) followed by EDCI (5.39 g, 28.12 mmol) at 15 C. The mixture was stirred at 15 C. for 21 hr. TLC showed part of stearic acid remained. Additionally added 1-hydroxypyrrolidine-2,5-dione (0.32 g) and EDCI (1.07 g). Stirring was continued at 15 C. for 8 hr. TLC showed the reaction was completed. The solvent was evaporated under reduced pressure. The residue was dissolved in DCM (300 mL) and the solution washed with water (200 mL); the aqueous phase was then back-extracted with DCM (2*100 mL). The combined organic phase was dried (MgSO.sub.4) and the solvent evaporated under reduced pressure to yield 2,5-dioxopyrrolidin-1-yl stearate as a white solid. No further purification. The crude product 2,5-dioxopyrrolidin-1-yl stearate (10.70 g, crude) was used into the next step without further purification. TLC(Petroleum ether: Ethyl acetate=1:1)R.sub.f=0.79.

(2600) Step 2. To a solution of (tert-butoxycarbonyl)-L-lysine (4.49 g, 18.24 mmol) and 2,5-dioxopyrrolidin-1-yl stearate (5.80 g, 15.20 mmol) in DMF (20 mL) was added DIPEA (5.89 g, 45.60 mmol, 7.96 mL). The mixture was stirred at 20 C. for 20 hour. TLC and LCMS showed the reaction was completed. The resulting mixture was concentrated to dry under reduced pressure. The residue was combined with 9 g crude compound, partitioned between water (200 mL) and EtOAc (300 mL) and DCM (80 mL). The separated aqueous layer was extracted with EtOAc (300 mL*3). The combined organic layers were washed with water (100 mL*2), dried over anhydrous MgSO.sub.4, filtered and concentrated to afford the product as a white solid (14.5 g). The crude product compound N.sup.2-(tert-butoxycarbonyl)-N.sup.6-stearoyl-L-lysine (7.70 g, crude) was used into the next step without further purification. .sup.1H NMR (400 MHz, CHLOROFORM-d) =11.29 (br s, 1H), 7.97 (s, 1H), 5.88 (br s, 1H), 5.24 (br d, J=7.3 Hz, 1H), 4.21 (br d, J=5.1 Hz, 1H), 3.17 (q, J=6.5 Hz, 2H), 2.11 (t, J=7.6 Hz, 2H), 1.79 (br s, 1H), 1.64 (dt, J=7.9, 14.0 Hz, 1H), 1.58-1.42 (m, 4H), 1.41-1.28 (m, 11H), 1.18 (br s, 29H), 0.81 (t, J=6.7 Hz, 3H); LCMS: (M+Na.sup.+): 535.3; TLC(Petroleum ether: Ethyl acetate=1:1)R.sub.f=0.01.

(2601) Step 3. To a solution of N.sup.2-(tert-butoxycarbonyl)-N.sup.6-stearoyl-L-lysine (12.50 g, 24.38 mmol) in DCM (120 mL) was added TFA (46.20 g, 405.20 mmol, 30 mL). The mixture was stirred at 15 C. for 4.5 hr. LCMS showed the reaction was almost completed. The resulting mixture was concentrated under reduced pressure on a rotary evaporator with water pump to give a gray crude solid. The crude product compound N.sup.6-stearoyl-L-lysine (12.80 g, crude, TFA salt) was used into the next step without further purification. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =8.19 (br s, 3H), 7.77-7.65 (m, 1H), 3.88 (br d, J=4.9 Hz, 1H), 3.02 (br d, J=5.5 Hz, 2H), 2.03 (br t, J=7.3 Hz, 2H), 1.75 (br s, 2H), 1.56-1.34 (m, 6H), 1.24 (s, 28H), 0.86 (br t, J=6.4 Hz, 3H); LCMS: (M+H.sup.+): 413.3.

(2602) Step 4. To a solution of compound N.sup.6-stearoyl-L-lysine (5.00 g, 9.49 mmol, TFA salt) in DMF (150 mL) was added compound 2,5-dioxopyrrolidin-1-yl 4-sulfamoylbenzoate (3.98 g, 13.34 mmol) followed by DIPEA (9.40 g, 72.73 mmol, 12.70 mL). The mixture was stirred at 80 C. for 18 hr. LCMS showed the reaction was completed. The resulting mixture was concentrated under reduced pressure until 20 mL residue mixture left. To the residue was added DCM (80 mL) and petroleum ether (50 mL). After stood for 36 hr at 15 C., the precipitated solid was filtered and dried to give the product as a light yellow solid (1.9 g). The filtrate was concentrated to dry and triturated with ACN(100 mL), filtered and the filter cake was dried to give a crude (2.4 g). The filtrate was concentrated to give an oil messy crude. No further purification. N.sup.6-stearoyl-N2-(4-sulfamoylbenzoyl)-L-lysine (1.90 g, 33.60% yield) was obtained as a light yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 13.19-11.82 (m, 1H), 8.74 (br d, J=5.7 Hz, 1H), 8.04 (br d, J=6.6 Hz, 2H), 7.91 (br d, J=7.1 Hz, 2H), 7.74 (br s, 1H), 7.49 (br s, 2H), 4.35 (br s, 1H), 3.02 (br s, 2H), 2.02 (br s, 2H), 1.80 (br s, 2H), 1.23 (br s, 31H), 0.86 (br s, 3H); .sup.13C NMR (101 MHz, DMSO-d.sub.6) 174.06, 172.39, 165.94, 146.85, 137.28, 128.54, 125.99, 53.24, 38.55, 35.88, 31.76, 30.69, 29.50, 29.41, 29.24, 29.18, 25.78, 23.72, 22.55, 14.39; LCMS: (M+H.sup.+): 596.4, purity: 89.89%.

Example 6. Synthesis of 18-oxo-18-((4-sulfamoylphenethyl)amino)octadecanoic acid

(2603) ##STR01067##

(2604) To a solution of octadecanedioic acid (4.90 g, 15.58 mmol) and 4-(2-aminoethyl)benzenesulfonamide (3.12 g, 15.58 mmol) in DCM (50 mL) was added HATU (7.11 g, 18.70 mmol) and DIPEA (6.04 g, 46.74 mmol, 8.16 mL). The mixture was stirred at 10 C. for 16 hours. The resulting mixture was concentrated under reduced pressure to give a residue. The residue was washed by CH.sub.3CN(100 mL*2) to give the crude product (11 g) as white solid. 1 g crude was dissolved by DMSO/DMF (V/V=3:1, 20 mL) purified by prep-HPLC(column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 45%-75%,20 min) to give 40 mg product as a white solid. 10 g crude was added CH.sub.3CN/H.sub.2O (V/V=4:1, 100 mL) and stayed at ultrasonic instrument for 30 min, then filtered to give filter cake, filter cake was washed by petroleum ether (20 mL) and acetone (20 mL). Filter cake was concentrated under reduced pressure to give 6 g product as a yellow solid. Compound 18-oxo-18-((4-sulfamoylphenethyl)amino) octadecanoic acid (6.00 g, 77.53% yield) was obtained as a yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =7.86 (br t, J-5.3 Hz, 1H), 7.71 (d, J=8.2 Hz, 2H), 7.35 (d, J=7.9 Hz, 2H), 7.27 (s, 2H), 3.26 (q, J=6.6 Hz, 3H), 2.75 (br t, J=7.2 Hz, 2H), 2.15 (t, J=7.3 Hz, 1H), 2.00 (br t, J=7.3 Hz, 2H), 1.44 (br d, J=6.6 Hz, 4H), 1.21 (s, 23H), 1.06 (d, J=6.6 Hz, 3H). LCMS: (M+H.sup.+): 497.3, purity 67.72%.

Example 7. Synthesis of 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oic acid

(2605) ##STR01068##

(2606) Step 1. A solution of di-tert-butyl 3,3-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (4.0 g, 7.91 mmol) and dihydro-2H-pyran-2,6(3H)-dione (0.903 g, 7.91 mmol) in THF (40 mL) was stirred at 50 C. for 3 hrs and at rt for 3 hrs. LC-MS showed desired product. Solvent was evaporated to give 5-((9-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2,2,16,16-tetramethyl-4,14-dioxo-3,7,11,15-tetraoxaheptadecan-9-yl)amino)-5-oxopentanoic acid, which was directly used for next step without purification.

(2607) Step 2. To a solution of 5-((9-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2,2,16,16-tetramethyl-4,14-dioxo-3,7,11,15-tetraoxaheptadecan-9-yl)amino)-5-oxopentanoic acid (4.90 g, 7.91 mmol) and (bromomethyl)benzene (1.623 g, 9.49 mmol) in DMF was added anhydrous K.sub.2CO.sub.3 (3.27 g, 23.73 mmol). The mixture was stirred at 40 C. for 4 hrs and at room temperature for overnight. Solvent was evaporated under reduced pressure. The reaction mixture was diluted with EtOAc, washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a residue, which was purified by ISCO eluting with 10% EtOAc in hexane to 50% EtOAc in hexane to give di-tert-butyl 3,3-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (5.43 g, 7.65 mmol, 97% yield) as a colorless oil. .sup.1H NMR (400 MHz, Chloroform-d) 7.41-7.28 (m, 5H), 6.10 (s, 1H), 5.12 (s, 2H), 3.72-3.60 (m, 12H), 2.50-2.38 (m, 8H), 2.22 (t, J=7.3 Hz, 2H), 1.95 (p, J=7.4 Hz, 2H), 1.45 (s, 27H); MS (ESI), 710.5 (M+H).sup.+.

(2608) Step 3. A solution of di-tert-butyl 3,3-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoate (5.43 g, 7.65 mmol) in formic acid (50 mL) was stirred at room temperature for 48 hrs. LC-MS showed the reaction was not complete. Solvent was evaporated under reduced pressure. The crude product was re-dissolved in formic acid (50 mL) and was stirred at room temperature for 6 hrs. LC-MS showed the reaction was complete. Solvent was evaporated under reduced pressure, co-evaporated with toluene (3) under reduced pressure, and dried under vacuum to give 3,3-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoic acid (4.22 g, 7.79 mmol, 100% yield) as a white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6) 12.11 (s, 3H), 7.41-7.27 (m, 5H), 6.97 (s, 1H), 5.07 (s, 2H), 3.55 (d, J=6.4 Hz, 6H), 2.40 (t, J=6.3 Hz, 6H), 2.37-2.26 (m, 2H), 2.08 (t, J=7.3 Hz, 2H), 1.70 (p, J=7.4 Hz, 2H); MS (ESI), 542.3 (M+H).sup.+.

(2609) Step 4. A solution of 3,3-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))dipropanoic acid (4.10 g, 7.57 mmol) and HOBt (4.60 g, 34.1 mmol) in DCM (60 mL) and DMF (15 mL) at 0 C. was added tert-butyl (3-aminopropyl) carbamate (5.94 g, 34.1 mmol), EDAC HCl salt (6.53 g, 34.1 mmol) and DIPEA (10.55 ml, 60.6 mmol). The reaction mixture was stirred at 0 C. for 15 minutes and at room temperature for 20 hrs. LC-MS showed the reaction was not complete. EDAC HCl salt (2.0 g) and tert-butyl (3-aminopropyl) carbamate (1.0 g) was added into the reaction mixture. The reaction mixture was stirred at room temperature for 4 hrs. Solvent was evaporated to give a residue, which was dissolved in EtOAc (300 mL), washed with water (1), saturated sodium bicarbonate (2), 10% citric acid (2) and water, dried over sodium sulfate, and concentrated to give a residue which was purified by ISCO (80 g gold catridge) eluting with DCM to 30% MeOH in DCM to give benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate 5 (6.99 g, 6.92 mmol, 91% yield) as a white solid. .sup.1H NMR (500 MHz, Chloroform-d) 7.35 (t, J=4.7 Hz, 5H), 6.89 (s, 3H), 6.44 (s, 1H), 5.22 (d, J=6.6 Hz, 3H), 5.12 (s, 2H), 3.71-3.62 (m, 12H), 3.29 (q, J=6.2 Hz, 6H), 3.14 (q, J=6.5 Hz, 6H), 2.43 (dt, J=27.0, 6.7 Hz, 8H), 2.24 (t, J=7.2 Hz, 2H), 1.96 (p, J=7.5 Hz, 2H), 1.69-1.59 (m, 6H), 1.43 (d, J=5.8 Hz, 27H); MS (ESI): 1011.5 (M+H).sup.+.

(2610) Step 5. A solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (1.84 g, 1.821 mmol) in DCM (40 mL) was added 2,2,2-trifluoroacetic acid (7.02 ml, 91 mmol). The reaction mixture was stirred at room temperature for overnight. Solvent was evaporated to give benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate as a colorless oil. MS (ESI), 710.6 (M+H).sup.+.

(2611) Step 6. To a solution of 4-sulfamoylbenzoic acid (1.466 g, 7.28 mmol) and HATU (2.77 g, 7.28 mmol) in DCM (40 mL) followed by benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (1.293 g, 1.821 mmol) in DMF (4.0 mL). The mixture was stirred at room temperature for 5 hrs. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (40 g gold column) eluting with DCM to 50% MeOH in DCM to give benzyl 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)-propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oate (0.36 g, 0.286 mmol, 16% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.60 (t, J=5.6 Hz, 3H), 7.96-7.81 (m, 15H), 7.44 (s, 6H), 7.35-7.23 (m, 5H), 7.04 (s, 1H), 5.02 (s, 2H), 3.50 (t, J=6.9 Hz, 6H), 3.48 (s, 6H), 3.23 (q, J=6.6 Hz, 6H), 3.06 (q, J=6.6 Hz, 6H), 2.29 (t, J=7.4 Hz, 2H), 2.24 (t, J=6.5 Hz, 6H), 2.06 (t, J=7.4 Hz, 2H), 1.69-1.57 (m, 8H).

(2612) Step 7. To a round bottom flask flushed with Ar was added 10% Pd/C (80 mg, 0.286 mmol) and EtOAc (15 mL). A solution of benzyl 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oate (360 mg) in methanol (15 mL) was added followed by diethyl(methyl)silane (0.585 g, 5.72 mmol) dropwise. The mixture was stirred at room temperature for 3 hrs. LC-MS showed the reaction was complete, diluted with EtOAc, and filtered through celite, washed with 20% MeOH in EtOAc, concentrated under reduced pressure to give 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)-amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oic acid (360 mg, 100% yield) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.60 (t, J=5.6 Hz, 3H), 7.94-7.81 (m, 15H), 7.44 (s, 6H), 7.04 (s, 1H), 3.50 (t, J=6.9 Hz, 6H), 3.48 (s, 6H), 3.23 (q, J=6.6 Hz, 6H), 3.06 (q, J=6.6 Hz, 6H), 2.24 (t, J=6.4 Hz, 6H), 2.14 (t, J=7.5 Hz, 2H), 2.05 (t, J=7.4 Hz, 2H), 1.66-1.57 (m, 8H); MS (ESI), 1170.4 (M+H).sup.+.

Example 8. Synthesis of 2,5-dioxopyrrolidin-1-yl 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoate

(2613) ##STR01069##

(2614) Step 1. A solution of 4-(2-aminoethyl)benzenesulfonamide (20 g, 99.87 mmol), tetrahydrofuran-2,5-dione (9.99 g, 99.87 mmol) in THF (200 mL) was stirred at 60 C. for 16 hr. The reaction mixture was diluted with HCl (aq., 1 M, 100 mL) and extracted with EtOAc (200 mL*3). The combined organic layers were washed with brine (100 mL*2), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (17 g, 55.60 mmol, 55.67% yield, 98.228% purity) was obtained as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =7.94 (t, J=5.7 Hz, 1H), 7.72 (d, J=7.9 Hz, 2H), 7.37 (d, J=8.3 Hz, 2H), 3.30-3.20 (m, 2H), 2.75 (t, J=7.2 Hz, 2H), 2.53-2.44 (m, 4H), 2.44-2.35 (m, 3H), 2.32-2.23 (m, 2H). LCMS: (M+H.sup.+): 301.1.

(2615) Step 2. To a solution of 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (17 g, 56.60 mmol) and HOSu (10.42 g, 90.57 mmol) in DMF (200 mL) was added DCC(18.69 g, 90.57 mmol, 18.32 mL) at 0 C.-5 C. The mixture was stirred at 0-5 C. for 16 hr. LCMS showed the reaction was not complete. The mixture was stirred at 15 C. for 16 hr. LCMS showed the reaction was complete and one main peak with desired MS was detected. The white suspension of N,N-dicyclohexylurea (DCU) was filtered and removed white solid. The filtrate was concentrated to an oil. This crude product was washed with hot 2-propanol (60 mL*3), affording an off-white solid. The crude product was added THF (100 mL), and Petroleum ether (50 mL) and stirred for 30 min, then filtered to give 2,5-dioxopyrrolidin-1-yl 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoate (8 g, 16.58 mmol, 29.29% yield, 82.36% purity) as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =8.12-7.96 (m, 1H), 7.71 (br d, J=7.9 Hz, 2H), 7.37 (br d, J=8.2 Hz, 2H), 3.58 (br t, J=6.7 Hz, 1H), 3.30-3.21 (m, 2H), 2.89-2.70 (m, 8H), 2.58 (s, 1H), 2.42 (br t, J=6.7 Hz, 2H); LCMS: (M+H.sup.+)): 398.0, LCMS purity: 82.36%.

Example 9. Synthesis of 4-oxo-4-((4-sulfamoylphenyl)amino)butanoic acid

(2616) ##STR01070##

(2617) To a solid reagent of 4-aminobenezensulfonamide (2.0 g, 11.61 mmol) and tetrahydofuran-2,5-dione (1.16 g, 11.61 mmol) was added THF (30 mL). The reaction mixture was stirred at 60 C. for 4 hrs, and white solid precipitated out. The reaction mixture was cooled to room temperature, and filtered to give a white solid. The white solid was dried under vacuum to give 4-oxo-4-(4-sulfamoylanilino)butanoic acid (2.115 g, 67% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 10.31 (s, 1H), 7.74 (s, 4H), 7.23 (s, 2H), 2.65-2.51 (m, 4H).

Example 10. Synthesis of 3-(((4-nitrophenoxy)carbonyl)oxy)propyl stearate

(2618) ##STR01071##

(2619) Step 1. A mixture of propane-1,3-diol (9.80 g, 128.75 mmol, 9.33 mL), Pyridine (2.61 g, 33.01 mmol, 2.66 mL) in CHCl.sub.3 (50 mL) was degassed and purged with N.sub.2 for 3 times, and then the mixture was dropwised stearoyl chloride (10 g, 33.01 mmol) in CHCl.sub.3 (50 mL) at 0 C. and stirred at 20 C. for 20 hr under N.sub.2 atmosphere. The mixture was extracted with EtOAc (50 mL*2), and the combined organic layers were washed with 1N HCl (50 mL*2), aq. NaHCO.sub.3 (50 mL*2), H.sub.2O (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Ethyl acetate/Petroleum ether=2%, 12.5%) to afford 3-hydroxypropyl stearate (9 g) as a white gum. .sup.1H NMR (400 MHz, DMSO-d.sub.6) =4.24 (t, J=6.06 Hz, 2H), 3.69 (t, J=5.95 Hz, 2H), 2.31 (t, J=7.50 Hz, 2H), 1.87 (q, J=6.06 Hz, 2H), 1.56-1.68 (m, 2H), 1.22-1.31 (m, 24H), 0.88 (t, J=6.73 Hz, 3H); TLC(Petroleum ether: Ethyl acetate=3:1) R.sub.f=0.54.

(2620) Step 2. A mixture of 3-hydroxypropyl stearate (9 g, 26.27 mmol), TEA (3.99 g, 39.41 mmol, 5.49 mL) in DCM (160 mL) was dropwised the solution of 4-nitrophenyl carbonochloridate (6.35 g, 31.53 mmol) in DCM (20 mL), then degassed and purged with N.sub.2 for 3 times at 0 C., and then the mixture was stirred at 20 C. for 16 hr under N.sub.2 atmosphere. TLC indicated compound was consumed completely and many new spots formed. The reaction was clean according to TLC. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by column chromatography (SiO.sub.2, Ethyl acetate/Petroleum ether=0%, 5%) to afford 3-(((4-nitrophenoxy)carbonyl)oxy)propyl stearate (5.73 g, 11.29 mmol, 42.96% yield) as an off-white solid. .sup.1H NMR (400 MHz, CHLOROFORM-d) =8.29 (d, J=9.21 Hz, 2H), 7.39 (d, J=9.21 Hz, 2H), 4.39 (t, J=6.36 Hz, 2H), 4.24 (t, J=6.14 Hz, 2H), 2.32 (t, J=7.45 Hz, 2H), 2.11 (t, J=6.36 Hz, 2H), 1.57-1.68 (m, 2H), 1.21-1.32 (m, 28H), 0.88 (t, J=6.80 Hz, 3H); .sup.13C NMR (101 MHz, CHLOROFORM-d) =173.73, 155.44, 152.40, 145.37, 125.30, 121.74, 66.00, 60.22, 34.21, 31.91, 29.68, 29.67, 29.64, 29.60, 29.30, 27.92, 24.91, 22.69, 14.12; TLC(Petroleum ether: Ethyl acetate=3:1)R.sub.f=0.72.

Example 11. Synthesis of (R)-3-(((4-nitrophenoxy)carbonyl)oxy)propane-1,2-diyl didodecanoate

(2621) ##STR01072##

(2622) To a solution of 4-nitrophenyl carbonochloridate (69.51 mg, 0.34 mmol) in THF (3.0 ml) at room temperature was added (S)-3-hydroxypropane-1,2-diyl didodecanoate (1,2-dilaurin) and DIPEA (0.11 ml, 0.66 mmol). The reaction mixture was stirred at room temperature for 3 hrs. Solvent was evaporated under reduced pressure, diluted with EtOAc, washed with water, dried over sodium sulfate, concentrated to give the desired product (R)-3-(((4-nitrophenoxy)carbonyl)oxy)propane-1,2-diyl didodecanoate (204 mg, 100% yield). .sup.1H NMR (400 MHz, Chloroform-d) 8.22 (d, J=8.9 Hz, 2H), 7.32 (d, J=8.9 Hz, 2H), 5.32-. 528 (m, 1H), 4.34-4.09 (m, 4H), 2.31-2.23 (m, 4H), 1.58-0.79 (m, 42H).

Example 12. Synthesis of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid

(2623) ##STR01073##

(2624) Step 1: To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (0.95 g, 0.940 mmol) in DCM (5 mL) was added TFA (5 mL). The reaction mixture was stirred at room temperature for 4 hrs. LC-MS showed the reaction was completed. Solvent was evaporated under reduced pressure to give benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate as a colorless oil. Directly use for next step without purification.

(2625) Step 2: To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (0.46 mmol) in DCM (6 mL) was added HOBt (62.16 mg, 0.46 mmol), HBTU (558.24 mg, 1.47 mmol), DIPEA (1.2 mL, 6.9 mmol) and a solution of 4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanoic acid (1.10 g, 1.61 mmol) in acetonitrile (5 mL). The reaction mixture was stirred at rt for 3 hrs. Solvent was evaporated under reduced pressure to give a residue, which was diluted with EtOAc, washed with water, dried over anhydrous sodium sulfate to give a residue, which was purified by ISCO (24 g gold column) eluting with DCM to 20% MeOH in DCM to give 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-anoic benzyl ester (1.14 g, 91.7%). MS (ESI), 1353.6 ((M/2+H).sup.+.

(2626) Step 3. To a solution of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-anoic benzyl ester (1.09 g, 0.400 mmol) in EtOAc (50 mL) was added 10% PdC(200 mg). The reaction mixture was stirred at rt for 4 hrs under hydrogen balloon. LC-MS showed the reaction was not completed. The reaction mixture was added another 10% PdC(300 mg) and stirred at room temperature for 24 hrs under hydrogen balloon. The reaction mixture was filtered, washed with EtOAc/MeOH, concentrated to give 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid (1.055 g, 100%). MS (ESI), 1308.1 ((M/2+H).sup.+.

Example 13. Synthesis of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5- tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic acid

(2627) ##STR01074## ##STR01075## ##STR01076##

(2628) Step 1 to 2. To a solid reagent 2,4,6-trichloro-1,3,5-triazine (0.500 g, 2.71 mmol) in THF (30 mL) was added tert-butyl 3-aminopropanoate HCl salt (0.985 g, 5.42 mmol) and DIPEA (2.36 ml, 13.56 mmol). The reaction mixture was stirred at room temperature for 5 hrs. LC-MS showed the desired product; MS (ESI): 402.4 (M+H).sup.+. Solvent was evaporated under reduced pressure to give a residue, which was directly used for next step. To a solution of di-tert-butyl 3,3-((6-chloro-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionate (1.052 g, 2.71 mmol) in aceotnitrile (50 mL) was added benzyl 5-oxo-5-(piperazin-1-yl) pentanoate (1.103 g, 3.80 mmol) and K.sub.2CO.sub.3 (2.248 g, 16.27 mmol). The reaction mixture was stirred at room temperature for overnight and at 50 C. Diluted with EtOAc, filtered and concentrated under reduced pressure to give a residue, which was purified by ISCO (40 g gold) eluting with 20% EtOAc in hexane to 50% EtOAc in hexane to give di-tert-butyl 3,3-((6-(4-(5-(benzyloxy)-5-oxopentanoyl) piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionate (1.13 g, 64%) as a white solid. .sup.1H NMR (400 MHz, Chloroform-d) 7.43-7.30 (m, 5H), 5.15 (s, 2H), 3.75 (brs, 4H), 3.63 (brs, 6H), 3.43 (brs, 2H), 2.51 (q, J=7.0, 6.5 Hz, 6H), 2.42 (t, J=7.4 Hz, 2H), 2.09-1.96 (m, 2H), 1.48 (s, 18H); MS (ESI): 656.6 (M+H).sup.+.

(2629) Step 3. A solution of di-tert-butyl 3,3-((6-(4-(5-(benzyloxy)-5-oxopentanoyl) piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl))dipropionate (1.10 g, 1.68 mmol) in formic acid (20 mL) was stirred at room temperature for overnight. LC-MS showed the reaction was not completed and solvent was evaporated. Formic acid (20 mL) was added to the reaction mixture and the reaction mixture was stirred at room temperature for 5 hrs. LC-MS showed the reaction was complete. Solvent was concentrated, co-evaporated with toluene (2) and dried under vacuum for overnight to give 3,3-((6-(4-(5-(benzyloxy)-5-oxopentanoyl) piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl)) dipropionic acid (0.91 g, 100% yield) as a white solid. MS (ESI), 544.2 (M+H).sup.+.

(2630) Step 4. A solution of 3,3-((6-(4-(5-(benzyloxy)-5-oxopentanoyl) piperazin-1-yl)-1,3,5-triazine-2,4-diyl)bis(azanediyl)) dipropionic acid (0.91 g, 1.68 mmol) and HOBt (0.76 g, 4.36 mmol) in DCM (30 mL) and DMF (3 mL) at 0 C. was added tert-butyl (3-aminopropyl) carbamate (0.840 g, 4.36 mmol), EDC HCl salt (0.836 g, 4.36 mmol) and DIPEA (1.460 ml, 8.39 mmol). The reaction mixture was stirred at 0 C. for 15 minutes and at room temperature for 20 hrs. Solvent was evaporated to give a residue, which was dissolved in EtOAc (300 mL), washed with water (1), saturated sodium bicarbonate (2), 10% citric acid (2) and water, dried over sodium sulfate, and concentrated to give a residue which was purified by ISCO (80 g gold catridge) eluting with DCM to 30% MeOH in DCM to give benzyl 5-(4-(4,6-bis((3-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoate (1.11 g, 77% yield) as a white solid. MS (ESI): 857.5 (M+H).sup.+.

(2631) Step A 5. solution of benzyl 5-(4-(4,6-bis((3-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoate (75.93 mg, 0.090 mmol) in DCM (3 mL) was added TFA (0.5 mL). The reaction mixture was stirred at room temperature for 3 hrs. Solvent was evaporated under reduced pressure, use directly for next step without purification. MS (ESI): 656.3 (M+H).sup.+.

(2632) Step 6. To a solution of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid (580 mg, 0.222 mmol) in DCM (10 mL) was added HBTU (84.1 mg, 0.220 mmol), HOBt (11.99 mg, 0.09 mmol) and DIPEA (0.15 ml, 0.890 mmol). The reaction mixture was stirred at rt for 5 minutes and a solution of benzyl 5-(4-(4,6-bis((3-((3-aminopropyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoate TFA salt (0.090 mmol) in acetonitrile was added to the reaction mixture. The reaction mixture was stirred at rt for overnight. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (24 g gold) eluting with DCM to 40% MeOH in DCM to give 5-(4-(4,6-b is ((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic benzyl ester (300 mg, 57.8%). MS (ESI), 1950.6 ((M/3+H).sup.+.

(2633) Step 7. To a solution of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic benzyl ester (300 mg, 0.05 mmol) in EtOAc (10 ml) was added 10% PdC(100 mg). The reaction mixture was stirred at rt under hydrogen balloon for overnight. LC-MS showed the reaction was not complete. The reaction mixture was added MeOH (1 mL) and triethylsilane (2 mL). The reaction mixture was stirred at room temperature for 4 hrs. LC-MS showed the desired product. The reaction mixture was filtered, washed with EtOAc/MeOH, and concentrated under reduced pressure to give a residue, which was purified by ISCO (50 g C18 catridge) eluting with 1% TFA in water to 100% acetonitrile and lyophilized to give 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-29-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazanonacosyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic acid (120 mg, 40.6% yield) as a white solid. MS (ESI), 1920 ((M/3+H).sup.+.

Example 14. Synthesis of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic acid

(2634) ##STR01077## ##STR01078## ##STR01079##

(2635) Step 1. To solution a 1 of 5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (2.43 g, 5.43 mmol) in DCM was added HBTU (2.06 g, 5.43 mmol), HOBt (183.36 mg, 1.36 mmol) and DIPEA (4.73 ml, 27.14 mmol). The reaction mixture was stirred at room temperature for 10 minutes, and a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate TFA salt (1.36 mmol) in acetonitrile was added. The reaction mixture was stirred at room temperature for 3 hrs. Solvent was concentrated under reduced pressure to give a residue, which was purified by ISCO (80 g gold catridge) eluting with 5% MeOH in DCM to 60% MeOH in DCM to give 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (2.22 g, 81.8%). MS (ESI): 1002 (M/2+H).sup.+.

(2636) Step 2. To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (2.20 g, 1.1 mmol) in EtOAc (30 mL) and MeOH (3 mL) was added 10% PdC(300 mg) and triethylsilane (1.8 mL, 11.3 mmol) slowly. The reaction mixture was stirred at room temperature for 1 hr. The reaction mixture was filtered through celite and concentrated to give 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid. MS (ESI), 1912 (M+H).sup.+.

(2637) Step 3. To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (1911 mg, 0.580 mmol) in DCM (30 mL) was added HBTU (266 mg, 0.700 mmol), HOBt (31.56 mg, 0.23 mmol) and DIPEA (0.81 ml, 4.67 mmol). The reaction mixture was stirred at rt for 10 minutes and a solution of benzyl 5-(4-(4,6-bis((3-((3-aminopropyl)amino)-3-oxopropyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoate TFA salt (0.23 mmol) in acetonitrile (5 mL) was added to the reaction mixture. The reaction mixture was stirred at rt for 3 hrs. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (24 g gold) eluting with DCM to 50% MeOH in DCM to give 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic benzyl ester (430 mg, 41.4%). MS (ESI), 1482.1 (M/3+H).sup.+.

(2638) Step 4. A solution of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic benzyl ester (420 mg, 0.090 mmol) in EtOAc (15 mL) and MeOH (2 mL) was added 10% PdC(200 mg). The reaction mixture was stirred at room temperature under hydrogen balloon for overnight. The reaction mixture was filtered through celite, washed with 50% MeOH in EtOAc, and concentrated under reduced pressure to give 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic acid. MS (ESI), 1452.0 (M/3+H).sup.+.

Example 15. Synthesis of 3-(((4-nitrophenoxy)carbonyl)oxy)propyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate

(2639) ##STR01080##

(2640) Step 1. To the solution of turbinaric acid (2.00 g, 4.992 mmol) in DCM (20 mL) was added 1,3-propanediol (1.8 mL, 24.96 mmol), EDC(1.91 g, 9.984 mmol) and DMAP(30.5 mg). The reaction mixture was stirred at rt for 5 hrs. LC-MS showed the reaction was complete. The reaction mixture was concentrated, diluted with EtOAc (100 mL), washed successively with 1N HC aq solution (20 ml), saturated NaHCO.sub.3aq solution (20 mL), water (10 mL), and brine (5 mL), dried over sodium sulfate, filtered, and concentrated to give a residue, which was purified by ISCO (40 g gold catridge) using 0-100% EtOAc in hexane as the gradient to give 3-hydroxypropyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (1.129 g, 49% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 5.15-5.02 (m, 5H), 4.46 (t, J=5.1 Hz, 1H), 4.06 (t, J=6.6 Hz, 2H), 3.45 (td, J=6.3, 5.1 Hz, 2H), 2.40-2.31 (m, 2H), 2.20 (t, J=7.6 Hz, 2H), 2.08-1.90 (m, 16H), 1.70 (p, J=6.4 Hz, 2H), 1.64 (d, J=1.5 Hz, 3H), 1.56 (m, 15H); MS (ESI), 481.3 (M+Na).sup.+.

(2641) Step 2. To a solution of 3-hydroxypropyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (1.12 g, 2.4416 mmol) in anhydrous DCM (12.5 mL) at 0 C. was added TEA (0.68 mL), and a solution of 4-nitrophenyl chloroformate (738 mg) in anhydrous DCM (5 ml) slowly. The reaction mixture was stirred at 0 C. for 40 min, and at room temperature for overnight. The reaction mixture was concentrated to give a residue, which was purified by ISCO (40 gold catridge) eluting with using 0-50% EtOAc in hexane to give 3-(((4-nitrophenoxy)carbonyl)oxy)propyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (1.06 g, 70% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.34-8.29 (m, 2H), 7.58-7.51 (m, 2H), 5.13-5.01 (m, 5H), 4.32 (t, J=6.3 Hz, 2H), 4.13 (t, J=6.3 Hz, 2H), 2.44-2.34 (m, 2H), 2.21 (t, J=7.6 Hz, 2H), 2.07-1.87 (m, 18H), 1.63 (d, J=1.5 Hz, 3H), 1.55 (m, 15H).

Example 16. Preparation of Certain Chemical Moieties and Oligonucleotides Comprising Certain Chemical Moieties

(2642) In some embodiments, the present disclosure provides chemical moieties that can be incorporated into oligonucleotides. In some embodiments, a chemical moiety is a targeting moiety. In some embodiments, a chemical moiety is a carbohydrate moiety. In some embodiments, a chemical moiety is a lipid moiety. In some embodiments, chemical moieties may be incorporated into oligonucleotides to improve one or more properties, activities, and/or delivery. Certain chemical moieties, their preparation, and oligonucleotides comprising such moieties are described in the present example. Those skilled in the art appreciate that such chemical moieties may also be incorporated into oligonucleotides having other base sequences, modifications, etc.

Synthesis of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid

(2643) ##STR01081##

(2644) Step 1. To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (9.0 g, 8.91 mmo) in DCM (100 mL) was added TFA (30.47 g, 267.27 mmol, 19.79 mL) at 0 C. The mixture was stirred at 0-15 C. for 4 hr. The mixture was formed two phase. Lower phase was separated and concentrated under reduced pressure to give a crude. benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate TFA salt (13 g) was obtained as a yellow oil. .sup.1H NMR (400 MHz, METHANOL-d.sub.4) Shift=7.39-7.27 (m, 5H), 5.12 (s, 2H), 3.70-3.63 (m, 13H), 3.32-3.30 (m, 2H), 3.26 (s, 2H), 2.94 (t, J=7.3 Hz, 7H), 2.49-2.38 (m, 9H), 2.23 (t, J=7.4 Hz, 2H), 1.94-1.78 (m, 9H). LCMS: M+H.sup.+=710.2.

(2645) Step 2. To a solution of benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate TFA salt (13 g) in DCM (200 mL) was added DIPEA (15.97 g, 123.58 mmol, 21.53 mL) and HATU (15.51 g, 40.78 mmol). The mixture was stirred at 15 C. for 15 hr. LCMS showed compound 2 was consumed and desired MS was detected. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC(column: Agela innoval ods-2 250*80 mm; mobile phase: [water (0.1% TFA)-ACN]; B %: 8%-38%, 20 min) to give compound benzyl 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (6.5 g, 52.37% yield) as a brown oil. LCMS: M/2+H.sup.+=503.1.

(2646) Step 3. To a solution of compound benzyl 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oate (5.7 g, 5.68 mmol) in MeOH (30 mL) and H.sub.2O (6 mL) was added LiOH.Math.H.sub.2O (1.67 g, 39.73 mmol). The mixture was stirred at 15 C. for 2 hr. LCMS showed compound 3 was consumed and desired MS was detected. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC(column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 0%-25%, 20 min). 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (2.09 g, 2.25 mmol, 40% yield) was obtained as a yellow gum. .sup.1HNMR (400 MHz, DMSO-d.sub.6) Shift=8.07 (br t, J=5.7 Hz, 3H), 7.75 (br t, J=5.0 Hz, 3H), 7.08 (s, 1H), 3.63-3.45 (m, 12H), 3.09 (q, J=6.1 Hz, 11H), 2.88 (br d, J=15.3 Hz, 36H), 2.29 (br t, J=6.4 Hz, 6H), 2.18 (t, J=7.5 Hz, 2H), 2.12-2.06 (m, 2H), 1.65 (br t, J=6.6 Hz, 8H). .sup.13CNMR (101 MHz, DMSO-d.sub.6) Shift=173.10, 170.88, 169.27, 159.88, 157.61, 157.27, 156.93, 156.58, 119.48, 116.56, 113.63, 110.70, 67.13, 66.27, 58.46, 40.77, 34.82, 34.34, 33.88, 31.87, 28.23, 19.66, 0.00. LCMS: M+H.sup.+=915.7, purity: 98.265%.

Synthesis of 5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid

(2647) ##STR01082##

(2648) Step 1. A mixture of phenylmethanol (864.10 g, 7.99 mol), compound 1 (100 g, 998.85 mmol), and cation exchange resin (1.92 g, 998.85 mmol.) was stirred at 75 C. with N.sub.2 for 4 hr, and then the mixture was stirred at 20 C. for 12 hr under N.sub.2 atmosphere. TLC showed compound 1 was consumed completely and two main peaks were detected. The reaction mixture was filtered and then the residue was washed with DCM (500 mL). The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to 3:1) to get compound 2 as a colorless oil (62 g, 29.81% yield). .sup.1HNMR (400 MHz, CHLOROFORM-d): =7.41-7.27 (m, 5H), 5.11 (s, 2H), 3.62 (t, J=6.4 Hz, 2H), 2.39 (t, J=7.3 Hz, 2H), 1.77-1.70 (m, 2H), 1.65-1.51 (m, 2H); TLC(Petroleum ether/Ethyl acetate=3:1)Rf=0.20.

(2649) Step 2. To a solution of compound 3 (350 g, 896.66 mmol.) in DMF (2 L) was added acetic acid hydrazine (99.10 g, 1.08 mol). The mixture was stirred at 60 C. for 5 hr. TLC showed the starting material was consumed. The mixture was concentrated to move the most solvent and water (500 mL) was added, and the mixture was extracted with EtOAc (500 mL*3). The combined organic was dried over Na.sub.2SO.sub.4, filtered and concentrated to get the compound 4 as a brown oil (310 g, crude). .sup.1HNMR (400 MHz, CHLOROFORM-d): =5.49 (t, J=9.9 Hz, 1H), 5.39 (d, J=3.5 Hz, 1H), 5.06-4.99 (m, 1H), 4.84 (dd, J=3.5, 10.1 Hz, 1H), 4.25-4.17 (m, 2H), 4.13-4.02 (m, 2H), 2.04-1.96 (m, 12H); TLC (Petroleum ether/Ethyl acetate=1:1), Rf=0.43.

(2650) Step 3. To a solution of compound 4 (310 g, 890.03 mmol.) in DCM (1.5 L) was added 2,2,2-trichloroacetonitrile (1.16 kg, 8.01 mol) at 0 C. The mixture was added drop-wise DBU (271.00 g, 1.78 mol) dissolved in DCM (1 L) at 0 C. The mixture was stirred at 20 C. for 1 h. TLC showed the starting material was consumed. The mixture was concentrated to get the crude. The mixture was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=20:1, 10:1, 5:1) to get compound 5 as a yellow oil (90 g, 20.52% yield). .sup.1HNMR (400 MHz, CDCl.sub.3): =8.70 (s, 1H), 6.56 (br d, J=3.1 Hz, 1H), 5.57 (t, J=9.8 Hz, 1H), 5.24-5.08 (m, 2H), 4.35-4.15 (m, 2H), 2.11-1.99 (m, 12H); TLC (Petroleum ether/Ethyl acetate=1:1)R.sub.f=0.31.

(2651) Step 4. To a solution compound 5 (89.5 g, 181.66 mmol) and compound 2 (75.66 g, 363.31 mmol) in DCM (800 mL) was added 4A MS (90 g), the mixture was stirred at 30 C. for 30 min. TMSOTf (40.37 g, 181.66 mmol.) was added to the reaction and the mixture was stirred at 25 C. for 3 hr. LCMS and TLC showed the starting material was consumed and LCMS showed the de-Ac MS was found. Sat. NaHCO.sub.3(aq., 100 mL) was added and the mixture was extracted with DCM (150 mL*3). The combined organic was dried over Na.sub.2SO.sub.4, filtered and concentrated to get the crude. Totally got the mixture of benzyl compound 6 and compound 6A (98 g) as a yellow oil, the mixture was used next step directly. TLC(Petroleum ether/Ethyl acetate=2:1) Rf=0.38.

(2652) Step 5. The mixture compound 6 and compound 6A (98 g crude) was dissolved in the pyridine (150 mL) and then Ac.sub.2O (150 mL) was added. The mixture was stirred at 20 C. for 12 h. TLC showed the starting material was consumed. The mixture was concentrated to get the crude. The mixture was purified by MPLC(silica, Petroleum ether/Ethyl acetate=20:1, 10:1, 05:1) to get compound 6 as a yellow oil (41 g, 41.84% yield) and 12 g crude. .sup.1HNMR (400 MHz, CDCl.sub.3): =7.39-7.31 (m, 5H), 5.23-4.93 (m, 3H), 4.48 (d, J=7.9 Hz, 1H), 4.37-4.22 (m, 1H), 4.17-4.05 (m, 1H), 3.92-3.81 (m, 1H), 3.71-3.63 (m, 1H), 3.48 (td, J=6.3, 9.8 Hz, 1H), 2.44-2.32 (m, 2H), 2.09-1.98 (m, 12H), 1.75-1.53 (m, 4H); LCMS: (M+Na.sup.+): 561.0; SFC: de %: 100%; TLC(Petroleum ether/Ethyl acetate=3:1) Rf=0.14.

(2653) Step 6. To a solution of compound 7 (19.5 g, 36.21 mmol) in EtOAc (200 mL) was added Pd/C(4 g, 17.64 mmol, 10% purity) under N.sub.2 atmosphere. The suspension was degassed and purged with H.sub.2 for 3 times. The mixture was stirred under H.sub.2 (25 Psi) at 20 C. for 2 hr. LCMS and TLC showed the starting material was consumed. The mixture was filtered, the cake was washed with MeOH (50 mL*3) and the combined filter was concentrated to get the crude. The mixture was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=3:1, 1:1, 1:3) to get 5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid 7 as a white solid (23.9 g, 51.72 mmol, 71.41% yield, 97.03% LCMS purity). .sup.1HNMR (400 MHz, CHLOROFORM-d): =5.24-5.17 (m, 1H), 5.12-4.96 (m, 2H), 4.50 (d, J=7.9 Hz, 1H), 4.26 (dd, J=4.7, 12.3 Hz, 1H), 4.20-4.02 (m, 1H), 3.95-3.85 (m, 1H), 3.75-3.64 (m, 1H), 3.55-3.46 (m, 1H), 2.42-2.32 (m, 2H), 2.15-1.99 (m, 12H), 1.76-1.57 (m, 4H); .sup.13CNMR (101 MHz, CHLOROFORM-d): =178.85, 170.71, 170.30, 169.40, 169.35, 100.71, 72.81, 71.74, 71.25, 69.37, 68.42, 61.94, 33.36, 28.59, 21.09, 20.70, 20.56; LCMS: (MH.sup.+): 447.1, LCMS purity: 97.03%; TLC(Petroleum ether/Ethyl acetate=1:1) Rf=0.03.

Synthesis of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid

(2654) ##STR01083##

(2655) Step 1: To a solution of benzyl 15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2-dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate (2.15 g, 2.1282 mmol) in DCM (20 mL) was added TFA (5 mL). The reaction mixture was stirred at room temperature for 4 hrs. LC-MS showed the reaction was completed. Solvent was evaporated under reduced pressure to give benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate as a colorless oil. Directly use for next step without purification.

(2656) Step 2: To a solution of 5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (3.817 g, 8.51 mmol) in DMF (20 mL) was added DIPEA (5.66 mL, 31.92 mmol) and HATU (2.824 g, 7.45 mmol) followed by benzyl 5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15-dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate (2.1282 mmol). The reaction mixture was stirred at room temperature for 3 hrs. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (120 g gold column) eluting with DCM to 50% MeOH in DCM to give 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (5.08 g, 120%), which containing some impurities. MS (ESI), 1001.4 ((M/2+H).sup.+.

(2657) Step 3. To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic benzyl ester (5.08 g) in EtOAc (100 mL) and MeOH (10 mL) was added 10% PdC(500 mg). The reaction mixture was stirred at rt for 4 hrs under hydrogen balloon. LC-MS showed the reaction was completed. The reaction mixture was filtered, washed with EtOAc/MeOH, concentrated to give 45,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (4.60 g, 95%). MS (ESI), 1912 ((M+H).sup.+.

Synthesis of(S)-5,11,18,22-tetraoxo-16,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic acid

(2658) ##STR01084##

(2659) Step 1: To a solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (987 mg, 0.520 mmol) in acetonitrile (3 mL) and DCM (10 ml) was added DIPEA (0.27 mL, 1.55 mmol) and HATU (150 mg, 0.400 mmol) followed by L-lysine benzyl ester di-4-toluensulfonate salt (100 mg, 0.170 mmol). The reaction mixture was stirred at room temperature for overnight. Solvent was evaporated under reduced pressure to give a residue, which was purified by ISCO (40 g gold column) eluting with DCM to 30% MeOH in DCM to give (S)-5,11,18,22-tetraoxo-16,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic benzyl ester (433 mg, 63%), which containing some impurities. MS (ESI), 1342.0 ((M/3+H).sup.+.

(2660) Step 3. To a solution of(S)-5,11,18,22-tetraoxo-16,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic benzyl ester (430 mg) in EtOAc (15 mL) and MeOH (3 mL) was added 10% PdC (100 mg). The reaction mixture was stirred at rt for 4 hrs under hydrogen balloon. LC-MS showed the reaction was completed. The reaction mixture was filtered, washed with EtOAc/MeOH, concentrated to give (S)-5,11,18,22-tetraoxo-16,16-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-28-(5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanamido)-14-oxa-6,10,17,23-tetraazanonacosan-29-oic acid (400 mg, 94%). MS (ESI), 1968 ((M/2+H).sup.+.

Synthesis of WV-12567

(2661) ##STR01085##

(2662) To a solution of WV-12566 in 0.4 ml NMP and 0.57 ml water was added DIPEA (20 L) and a solution of 3-(((4-nitrophenoxy)carbonyl)oxy)propyl (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoate (20 mg) in NMP(0.40 mL). The reaction mixture was shaken for 12 hours at 35 C. LC-MS showed the starting material was disappeared. The crude product was purified on RP HPLC (C8) using 50 mM TEAA in water and acetonitrile, and desalt to obtain 1.77 mg of the conjugate WV-12567. Deconvoluted mass: 7362; Calculated molecular weight: 7360.

Synthesis of WV-12570

(2663) ##STR01086##

(2664) To a solution of (4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenoic acid (turbinaric acid) (6.4 mg, 16 mol) and HATU (5.4 mg, 14.4 mol) was added DIPEA (17 L). The mixture was shaken for 30 min at rt. The reaction mixture was added into a solution of WV 12569 (12.4 mg, 1.6 mol) in water (0.20 mL) and NMP(0.20 ml) and stirred for 2 hrs at 35 C. LC-MS showed the starting material was disappeared. The crude product was purified on RP (C-8) HPLC using 50 mM TEAA in water and acetonitrile, and desalt to obtain 2.10 mg of the conjugate WV-12570. Deconvoluted mass: 8172; Calculated molecular weight: 8170.

Synthesis of WV-14333

(2665) ##STR01087##

(2666) A solution of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid (25.4 mg, 9.72 mol) in acetonitrile (0.50 mL) was added HATU (3.32 mg, 8.75 mol) and DIPEA (8.5 L). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was added into a solution of WV-12566 (16.7 mg, 2.43 mol) in 0.5 mL water. The reaction mixture was stirred at 30 C. for 2 hrs, and LC-MS showed the reaction was complete. The reaction mixture was transferred to the pressure tube, and 4 ml 28-30% ammonium hydroxide was added. The reaction mixture was stirred at 35 C. for overnight. LC-MS showed the reaction was completely de-protected. The crude product was purified by ISCO via 30 g C18 Catridge eluting with 50 mM TEAA to acetonitrile, and desalt to obtain 12.8 mg of the conjugate WV-14333. Deconvoluted mass: 8224; Calculated molecular weight: 8221.

Synthesis of WV-14332

(2667) ##STR01088##

(2668) A solution of 4-nitrophenyl(2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl) chroman-6-yl) carbonate (7.24 mg, 12.15 mol) and DIPEA (8.50 L) in NMP(0.20 ml) was added to a solution of WV-12566 (16.7 mg, 2.43 mol) in 0.5 ml DMSO and 0.05 mL water. The reaction mixture was shaken for 3 hours at 40 C. LC-MS showed the reaction was very clean. The crude product was lyophilized, purified on RP (C-8) HPLC using 50 mM TEAA in water and acetonitrile, and desalt to obtain 10 mg of the conjugate WV-14332. Deconvoluted mass: 7335; Calculated molecular weight: 7334.

Synthesis of WV-14346

(2669) ##STR01089##

(2670) A solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (75.26 mg, 82.34 mol) in DMF (1.0 mL) was added DIPEA (123 L, 0.823 mmol) and HATU (28.1 mg, 74.12 mol). The reaction mixture was stirred at room temperature for 15 minutes. The reaction mixture was added to a solution of WV-12566 (113.22 mg, 16.47 mol) in 1.50 ml DMSO and 0.50 mL water. The reaction mixture was shaken for 2 hours at rt. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 84.3 mg of the conjugate WV-14346. Deconvoluted mass: 7772; Calculated molecular weight: 7771.

Synthesis of WV-14335 (SEQ ID NO: 3252)

(2671) ##STR01090##

(2672) Step 1. A solution of 3-(2-Pyridyldithio)-propionic acid-OSu (9.08 mg) in DMF (1.0 mL) was added into a solution of WV-12566 (100 mg, 14.54 in 1.5 ml 0.5 M sodium phosphate buffer (pH=8). The reaction mixture was stirred at room temperature for 1 hr. LC-MS showed that reaction was completed. Diluted with water, and lyophilized to give the desired product.

Synthesis of WV-14335

(2673) Step 1. A solution of 3-(2-Pyridyldithio)-propionic acid-OSu (9.08 mg) in DMF (1.0 mL) was added into a solution of WV-12566 (100 mg, 14.54 in 1.5 ml 0.5 M sodium phosphate buffer (pH=8). The reaction mixture was stirred at room temperature for 1 hr. LC-MS showed that reaction was completed. Diluted with water, and lyophilized to give the desired product.

(2674) Step 2. A solution of H-RRQPPRSISSHPC-OH (SEQ ID NO: 3252) (5.47 mg, 3.6 mol) in DMF (0.85 ml) and 0.1 M sodium bicarbonate (0.15 ml) was added to the above product (step 1) (12 mg, 1.8 mol) in 0.1 M sodium bicarbonate (0.50 mL). The reaction mixture was shaken for 1.5 hours at rt. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 3.0 mg of the conjugate WV-14335. Deconvoluted mass: 8485; Calculated molecular weight: 8482.

Synthesis of WV-14347 (SEQ ID NO: 3259)

(2675) ##STR01091##

(2676) A solution of Ac-CHAIYPRH-OH (SEQ ID NO: 3259) (3.74 mg, 3.6 mol) in DMF (0.85 mL) and 0.1 M NaHCO.sub.3 (0.15 mL) was added to SPDP oligo (step 1 product of WV-14335) (12 mg, 1.8 mol) in 0.10 M NaHCO.sub.3 (0.50 mL). The reaction mixture was shaken for 1.5 hours at room temperature. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 8.8 mg of the conjugate WV-14347. Deconvoluted mass: 8003; Calculated molecular weight: 7999.

Synthesis of WV-14348 (SEQ ID NO: 3260)

(2677) ##STR01092##

(2678) A solution of Ac-CTHRPPMWSPVWP-OH (SEQ ID NO: 3260) (5.88 mg, 3.6 mol) in DMF (0.85 mL) and 0.1 M NaHCO.sub.3 (0.15 mL) was added to SPDP oligo (step 1 product of WV-14335) (12 mg, 1.8 mol) in 0.10 M NaHCO.sub.3 (0.50 mL). The reaction mixture was shaken for 1.5 hours at room temperature. LC-MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 4.1 mg of the conjugate WV-14348. Deconvoluted mass: 8602; Calculated molecular weight: 8597.

Synthesis of WV-15074 (SEQ ID NO: 3259)

(2679) ##STR01093##

(2680) Step 1. A solution of 2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (8.25 mg, 24.71 mol) in DMF (0.30 mL) was added to WV-12566 (113.22 mg, 16.47 mol) and DIPEA (31 L, 173 mol) in DMSO(1.50 mL) and water (0.5 mL). The reaction mixture was stirred for 30 minutes at room temperature. LC-MS showed the reaction was almost complete.

(2681) Step 2. A solution of Ac-CHAIYPRH-OH (SEQ ID NO: 3259) (38.47 mg, 37.1 mol) in DMF (0.50 mL) was added to the above reaction mixture. The reaction mixture was stirred at room temperature for 2 hr. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 66.0 mg of the conjugate WV-15074. Deconvoluted mass: 8133; Calculated molecular weight: 8132.

Synthesis of WV-15075 (SEQ ID NO: 3260)

(2682) ##STR01094##

(2683) Step 1. A solution of 2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (1.3 mg, 3.99 mol) in DMF (0.10 mL) was added to a solution of WV-12566 (16.7 mg, 2.49 mol) and DIPEA (3.5 L) in DMSO(0.30 mL) and water (0.10 mL). The reaction mixture was shaken for 1 hr at room temperature. LC-MS showed the reaction was almost complete.

(2684) Step 2. A solution of Ac-CTHRPPMWSPVWP-OH (SEQ ID NO: 3260) (9.8 mg, 6.0 mol) in DMF (0.20 mL) was added to the above reaction mixture. The reaction mixture was stirred at room temperature for 3 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 8.9 mg of the conjugate WV-15075. Deconvoluted mass: 8735; Calculated molecular weight: 8730.

Synthesis of WV-15076 (SEQ ID NO: 3252)

(2685) ##STR01095##

(2686) Step 1. A solution of 2,5-dioxopyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylate (1.3 mg, 3.99 mol) in DMF (0.10 mL) was added to a solution of WV-12566 (16.7 mg, 2.49 mol) and DIPEA (3.5 L) in DMSO(0.30 mL) and water (0.10 mL). The reaction mixture was shaken for 1 hr at room temperature. LC-MS showed the reaction was almost complete.

(2687) Step 2. A solution of H-RRQPPRSISSHPC-OH (SEQ ID NO: 3252) (9.1 mg, 6.0 mol) in DMF (0.20 mL) was added to the above reaction mixture. The reaction mixture was stirred at room temperature for 3 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 4.7 mg of the conjugate WV-15076. Deconvoluted mass: 8735; Calculated molecular weight: 8730.

Synthesis of WV-15367

(2688) ##STR01096##

(2689) A solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (13.9 mg, 7.29 mol) in DMF (0.50 mL) was added DIPEA (6.3 L, 36.4 mol) and HATU (2.3 mg, 6.0 mol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was added to a solution of WV-12566 (16.7 mg, 2.43 mol) in 0.30 ml DMSO and 0.10 mL water. The reaction mixture was shaken for 2 hours at rt. LC_MS showed the reaction was complete. The reaction mixture was added 28-30% ammonium hydroxide, stirred at 40 C. for 3 hrs. LC MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 9.2 mg of the conjugate WV-15367. Deconvoluted mass: 8269; Calculated molecular weight: 8263.

Synthesis of WV-15368

(2690) ##STR01097##

(2691) A solution of 5-(4-(4,6-bis((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)-5-oxopentanoic acid (31.7 mg, 7.29 mol) in DMF (0.50 mL) was added DIPEA (6.3 L 36.4 mol) and HATU (2.3 mg, 6.0 mol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was added to a solution of WV-12566 (16.7 mg, 2.43 mol) in 0.30 ml DMSO and 0.10 mL water. The reaction mixture was shaken for 2 hours at rt. LC_MS showed the reaction was complete. The reaction mixture was added 28-30% ammonium hydroxide (1.0 mL), stirred at 40 C. for 5 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 7.5 mg of the conjugate WV-15368. Deconvoluted mass: 10206; Calculated molecular weight: 10200.

Synthesis of WV-15882

(2692) ##STR01098##

(2693) A solution of 5,12,18-trioxo-7,7-bis((3-oxo-3-((3-(5-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-22-(((2R,3R,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-9-oxa-6,13,17-triazadocosanoic acid (102 mg, 53.43 mol) in DMF (1.0 mL) was added DIPEA (46.8 L, 266.5 mol) and HATU (13.5 mg, 35.68 mol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was added to a solution of WV-12566 (122.65 mg, 17.84 mol) in 1.5 ml DMSO and 0.50 mL water. The reaction mixture was shaken for 1.5 hours at rt. LC MS showed the reaction was completed. The reaction mixture was added 28-20% ammonium hydroxide (5.0 mL) and stirred at 35 C. for 1.5 hrs. LC_MS showed the reaction was complete. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 83.8 mg of the conjugate WV-15882. Deconvoluted mass: 8263; Calculated molecular weight: 8264.

(2694) Some of the examples reference oligonucleotides which target Malat1. Some of these oligonucleotides are described elsewhere herein and/or below.

(2695) TABLE-US-00127 Oligo- SEQID nucleotide ModifiedSequence NO: NakedSequence Stereo-chemistry WV-2809 L001*Geo*Geo*Geo*Teo*m5Ceo 3261 GGGTCAGCTGC XXXXXXXXXXX *A*G*C*T*G*C*C*A*A*T CAATGCTAG XXXXXXXXX *Geo*m5Ceo*Teo*Aeo*Geo WV-3356 L001Geo*Geo*Geo*Teo*m5Ceo* 3262 GGGTCAGCTGC OXXXXXXXXXXX A*G*C*T*G*C*C*A*A*T* CAATGCTAG XXXXXXXX Geo*m5Ceo*Teo*Aeo*Geo WV-7430 ModO43L001Geo*Geo*Geo*Teo* 3263 GGGTCAGCTGC OXXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A* CAATGCTAG XXXXXXXX A*T*Geo*m5Ceo*Teo*Aeo*Geo WV-7519 Mod009L001*Geo*Geo*Geo*Teo* 3264 GGGTCAGCTGC XXXXXXXXXXX m5Ceo*A*G*C*T*G*C*C*A* CAATGCTAG XXXXXXXXX A*T*Geo*m5Ceo*Teo*Aeo*Geo WV-7557 L001mU*Geo*Geo*Geo*Teo* 3265 UGCCAGGCTG OXXXXXXXXXXX *C*T*G*G*T*T*A*T*mG* GTTATGACUC XXXXXXXX mA*mC*mU*mC WV-7558 Mod027L001mU*mG*mC*mC*mA 3266 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-7559 Mod028L001mU*mG*mC*mC*mA 3267 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-7560 Mod007L001mU*mG*mC*mC*mA 3268 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-8448 Mod059L001mU*mG*mC*mC*mA 3269 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-8927 Mod053L001mU*mG*mC*mC*mA 3270 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-8929 Mod057L001mU*mG*mC*mC*mA 3271 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-8930 Mod058L001mU*mG*mC*mC*mA 3272 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-8931 Mod009L001mU*mG*mC*mC*mA 3273 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-8934 Mod050L001mU*mG*mC*mC*mA 3274 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-9385 Mod066L001mU*mG*mC*mC*mA 3275 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-9390 Mod074L001m1U*mG*mC*mC*mA 3276 UGCCAGGCTG OXXXXXXXXXXX *G*G*C*T*G*G*T*T*A*T* GTTATGACUC XXXXXXXX mG*mA*mC*mU*mC WV-13809 Mod0971001mU* 3277 UGCCAGGCTG OSOOOSSRS SGeom5Ceom5CeomA*SG*SG*RC* GTTATGACUC SRSSRSSSSSS ST*SG*RG*ST*ST*RA*ST* SmG*SmA*SmC*SmU*SmC WV-27145 mU*SGCCmA*SG*SG*RC* 3278 UGCCAGGCTG SOOOSSRSnXR STn001G*RG*ST*ST*RA*ST GTTATGACUC SSRSSSSSSS *SmG*SmA*SmC*SmU*SmC* U SfU
The Modifications (e.g., designated by Mod followed by a number, such as Mod097, Mod074, etc.) are described in the legend to Table A1 or elsewhere herein.

Synthesis of WV-13809

(2696) ##STR01099##

(2697) A solution of 4-nitrophenyl(2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl) chroman-6-yl) carbonate (activated vitamin E) (15 mg, 25 mol) and DIPEA (21 L) in NMP(0.20 ml) was added to a solution of WV-9696 in 0.5 ml DMSO and 0.05 ml water. The reaction mixture was shaken for 2 hrs at 50 C. LC-MS showed the reaction was completed. The crude product was lyophilized, purified on RP (C-8) HPLC using 50 mM TEAA in water and acetonitrile, and desalt to obtain 4.90 mg of the conjugate WV-13809. Deconvoluted mass: 7451; Calculated molecular weight: 7451.

Synthesis of WV-14349

(2698) ##STR01100##

(2699) A solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (19.61 mg, 21.45 mol) in DMF (0.30 mL) was added DIPEA (75 L) and HATU (7.32 mg, 19.31 mol). The reaction mixture was stirred at room temperature for 20 minutes. The reaction mixture was added to a solution of WV-9696 (30 mg, 4.29 mol) in 0.4 ml DMSO and 0.10 mL water. The reaction mixture was shaken at rt for overnight. LC_MS showed the reaction was not complete. A solution of 3-(dimethylamino)-14,14-bis(3-(dimethylamino)-2-methyl-9-oxo-12-oxa-2,4,8-triazatridec-3-en-13-yl)-2-methyl-9,16-dioxo-12-oxa-2,4,8,15-tetraazaicos-3-en-20-oic acid (10 mg) in DMF (0.10 mL) was added DIPEA (38 L) and HATU (3.7 mg). The reaction mixture was stirred at room temperature for 20 minutes. The reaction mixture was added into the above the reaction mixture with WV-9696. The reaction mixture was stirred at 30 C. for 2 hrs. LC MS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 9.1 mg of the conjugate WV-14349. Deconvoluted mass: 7893; Calculated molecular weight: 7889.

Synthesis of WV8448

(2700) ##STR01101##

(2701) To a soluition of 4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)propyl)amino)propoxy)methyl)-1-(((2R,3R,5R,6R)-3,4,5-tris(benzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenicosan-21-oic acid (57 mg, 21.8 mol), HATU (7.5 mg, 19.6 mol) and DIPEA (14.6 mg, 109 mol) in DMF (2.0 mL) was stirred at room temperature for 15 minutes. To this solution was added 75 mg (10.9 mol) of WV7557 in 1 ml water. Reaction mixture was stirred for 60 minutes to obtain the desired product. This product was heated at 40 C. with NH.sub.4OH for 3 hrs. LC_MS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 39.73 mg of the conjugate WV-8448. Deconvoluted mass: 8233; Calculated molecular weight: 8227.

Synthesis of WV8927

(2702) ##STR01102##

(2703) To a solution of gambogic acid (21 mg, 33.6 mol) in 2 ml dry DMF was added HATU (11.5 mg, 30.2 mol) and DIPEA (3.6 mg, 28 mol) and vortexed well. This solution was added WV7557 (42 mg, 5.6 mol) in water (1 ml) and shaken for 4 hours. LC-Analysis indicated product formation, but starting material remained. Another 6 six equivalents of Gambogic acid-HATU complex (same amount used initially) was added and shaken well for 2 hours. LC analysis indicated more product formation. The reaction mixture was diluted with water (10 ml). Excess gambogic acid precipitated out. This precipitate was filtered off and the crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 19 mg of the conjugate WV-8927. Deconvoluted mass: 7496; Calculated molecular weight: 7492.

Synthesis of WV-7558

(2704) ##STR01103##

(2705) To a solution of 4-sulfamoylbenzoic acid (7.3 mg, 36 mol) in DMF (2.0 mL) was added HATU (12.4 mg, 32.7 mol) and DIPEA (46 mg, 360 mol) and vortexed. After 2 minutes WV7557 (50 mg, 7.27 mol) in 1 ml water was added and shaken well. After 60 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product (17 mg). Mass calculated: 7064; Deconvoluted Mass: 7068.

Synthesis of WV-7559

(2706) ##STR01104##

(2707) To a solution of 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (8.7 mg, 29 mol) in DMF (2.0 mL) was added HATU (9.9 mg, 26 mol) and DIPEA (37 mg, 290 mol) and vortexed. After 2 minutes WV7557 (40 mg, 5.81 mol) in 1 ml water was added and shaken well. After 30 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product (13 mg). Mass calculated: 7163; Deconvoluted Mass: 7166.

(2708) ##STR01105##

(2709) To a solution of WV7557 (62 mg, 9 mol) in water (0.5 ml) and DMF (2.5 ml) was added DIPEA (11.6 mg, 90 mol) and stirred well. To this solution was added 3-(2-Pyridyldithio)-propionic acid-OSu (4 mg, 12.6 mol) and stirred well for 2 h. The crude product was diluted with water and purified on ISCO (C18 column) using 50 mM TEAA and acetonitrile. Amount of product obtained: 46 mg.

Synthesis of WV-8929

(2710) ##STR01106##

(2711) To a solution of the oligo (WV7557 derivative, 23.5 mg, 3.3 mol) in water-DMF (2 ml+1 ml) mixture was added DIPEA (8.52 mg, 66 mol), and vortexed for 5 minutes. To this solution was added H-RRQPPRSISSHPC-OH (SEQ ID NO: 3252) (10 mg, 6.6 mol) and again vortexed for 5 minutes. After 12 hours, the reaction mixture was analyzed by LC-MS. LC_MS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 14 mg of the conjugate WV-8929. Deconvoluted mass: 8496; Calculated molecular weight: 8490.

Synthesis of WV-8930

(2712) ##STR01107##

(2713) To a solution of the oligo (WV7557 derivative, 23.5 mg, 3.3 mol) in water-DMF (2 ml+1 ml) mixture was added DIPEA (8.52 mg, 66 mol) and vortexed for 5 minutes. To this solution was added H-Arg-Arg-Cys-OH (4 mg, 10 mol) and vortexed for 5 minutes. After 12 hours, the reaction mixture was analyzed by LC-MS. LC_MS showed the reaction was completed. The reaction mixture was diluted with water, and speed-vacuum to dry. The crude product was purified by RP-HPLC eluting with 50 mM TEAA in water to acetonitrile, and desalt to obtain 5 mg of the conjugate WV-8930. Deconvoluted mass: 7405; Calculated molecular weight: 7401.

Synthesis of WV8931

(2714) ##STR01108##

(2715) To a solution of WV7557 (20 mg, 2.91 mol) in 0.47 ml water was treated with DIPEA (3.76 mg, 29.1 mol) and vortexed well for 5 minutes. To this solution was added a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl (4-nitrophenyl) carbonate (activated cholesterol derivative) (10.50 mg, 19 mol) in NMP(1.0 ml). The solution turned slightly yellowish. It was shaken at 40 degrees for 12 hours. A bright yellow solution was obtained. LC-MS analysis indicated product formation. This solution was diluted to 10 ml using water, filtered and purified on a RP-HPLC using a C-8 column and desalted. Amount of product obtained: 18 mg; Deconvoluted mass: 7298; Calculated molecular weight: 7293.

Synthesis of WV8934

(2716) ##STR01109##

(2717) L-carnitine (3 mg, 17.5 mol) and HATU (6 mg, 16 mol) were mixed together and made in to a 1 ml solution in DMF. DIPEA (5.7 mg, 44 mol) was added and stirred well for 3 minutes. To this solution was added a solution of WV-7557 (30 mg, 4.4 mmol) in 0.5 ml water and stirred well for 30 minutes. LC-MS analysis of the solution indicated product formation. But starting oligo was present in the reaction mixture. 4 equivalents more L-carnitine/HATU complex was added again and stirred well for 2 h. The reaction mixture was diluted with water and the crude product was purified on a RP (C-18) column to obtain the product. Amount of product obtained: 12 mg, Calculated mass: 7025; De-convoluted mass: 7029.

Synthesis of WV-9390

(2718) ##STR01110##

(2719) To a solution of 5-oxo-5-(4-(4-((2,8,12,19,25-pentaoxo-14,14-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-29-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-16-oxa-3,7,13,20,24-pentaazanonacosyl)amino)-6-((3,9,13,20,26-pentaoxo-15,15-bis((3-oxo-3-((3-(5-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)amino)propoxy)methyl)-30-(((2S,3S,4S,5R,6R)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-17-oxa-4,8,14,21,25-pentaazatriacontyl)amino)-1,3,5-triazin-2-yl) piperazin-1-yl)pentanoic acid (15 mg, 3.5 mol) and HATU (1.33 mg, 3.5 mol) in DMF (1.0 ml) was added DIPEA (4.5 mg, 35 mol) and vortexed for 2 minutes. To this solution was added WV7557 (12 mg, 1.74 mol) in water (0.5 ml) and shaken for 60 minutes. 5 ml water was added to it and the solvent was removed under vacuum. The crude product was purified on a RP column (C-8) obtain acetylated product (Mass calculated: 10207, Deconvoluted mass: 10212). This product was dissolved in 5 ml 30% ammonium hydroxide solution and heated at 40 degrees Celsius for 6 hours. Solvent was removed under vacuum and the crude product was purified on a RP column (C-8) to obtain the product. Amount of product obtained (10 mg). Calculated Mass: 10205; Deconvoluted Mass obtained: 10205.

Synthesis of WV 9430

(2720) ##STR01111##

(2721) To a solution of 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oic acid (5.14 mg, 1.45 mol) in DMF was added HATU (1.5 mg, 3.96 mol) and DIPEA (2 mg, 15 mol). The reaction mixture was stirred at room temperature for 2 minutes. A solution of WV7557 in 0.4 ml water was added and shaken well. After 30 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product WV-9430 (6 mg). Mass calculated: 8032; Deconvoluted Mass: 8031.

Synthesis of WV-9385

(2722) ##STR01112##

(2723) WV7557 (48 mg, 6.9 mol) was dissolved in 1 ml NMP and 0.5 ml water. DIPEA (14 mg, 103.5 mol) was added to this solution. Vortexed for 5 minutes. To this solution was added 3-(((4-nitrophenoxy)carbonyl)oxy)propyl stearate (14 mg, 27.6 mol) in 1 ml NMP. The reaction mixture was filtered and the filtrate was purified by RP column chromatography (C-8) to obtain the product. The purified material was desalted and 11 mg of product was obtained. Mass calculated: 7250; Deconvoluted Mass: 7254.

Synthesis of WV-7560

(2724) ##STR01113##

(2725) 12,12-bis((3-((3-(4-methoxybenzamido)propyl)amino)-3-oxopropoxy)methyl)-1-(4-methoxyphenyl)-1,7,14-trioxo-10-oxa-2,6,13-triazapentacosan-25-oic acid (triantennary anisamide) (32.5 mg, 29 mol), HATU (10 mg, 26.1 mol) and DIPEA (28 mg, 58 mol) were dissolved in 2 ml DMF. After 2 minutes WV7557 (100 mg, 15 mol) in 1 ml water was added and shaken well. After 60 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-8) and desalted to obtain the product (55 mg). Mass calculated: 7983; Deconvoluted Mass: 7987.

Synthesis of WV-7408

(2726) ##STR01114##

(2727) A suspension of WV 3356 (40 mg, 5.3 mol) and DIPEA (7 mg, 53 mol) in 2 ml DMF was vortexed for five minutes. To this suspension was added a solution of 2,5-dioxopyrrolidin-1-yl 4-sulfamoylbenzoate (8 mg, 26.5 mol)] in 1 ml DMF. The reaction mixture was shaken for 12 hours. Afterwards, the reaction mixture was diluted with 5 ml water and filtered. The filtrate was purified by RP (C-18) column chromatography and desalted to obtain the product (20 mg). Mass calculated: 7596; Deconvoluted mass: 7594.

Synthesis of WV7409

(2728) ##STR01115##

(2729) To a solution of 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (2.16 mg, 7.2 mol), HATU (2.32 mg, 6.1 mol) and DIPEA (3.1 mg, 24 mol) were dissolved in 1 ml DMF and vortexed. After 2 minutes WV3356 (18 mg, 2.4 mol) in 0.5 ml water was added and shaken well. After 60 minutes the reaction mixture was diluted with water (5 ml) and filtered. The filtrate was purified by RP column chromatography (C-18) and desalted to obtain the product (9 mg). Mass calculated: 7694; Deconvoluted Mass: 7695.

Synthesis of WV-7430

(2730) ##STR01116##

(2731) To a solution of WV3356 (32 mg, 4.3 mol) in DMF (2.0 mL) was added DIPEA (5.8 mg, 43 mol) was added a solution of (R)-3-(((4-nitrophenoxy)carbonyl)oxy)propane-1,2-diyl didodecanoate (11 mg, 17.6 mol) in acetonitrile (1.0 mL). Reaction mixture was shaken at 40 C. for 12 hours. LC-MS analysis indicated formation of product. The reaction mixture was diluted with water and filtered. The filtrate was purified by RP column chromatography (C-8) to obtain the product. The purified material was desalted and 11 mg of product was obtained. Mass calculated: 7895, Deconvoluted Mass: 7896.

Synthesis of WV-7419

(2732) ##STR01117##

(2733) To a suspension of WV-2809 (56 mg, 7.5 mol, 125 mg support) in DMF (2.0 mL) was added DIPEA (19.3 mg, 150 mol) and vortexed well for 5 minutes. To this suspension was added perfluorophenyl 18-oxo-18-((4-(N-(2,2,2-trifluoroacetyl)sulfamoyl)phenethyl)amino)octadecanoate (12 mg, 15 mol) and shaken for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml3) and dried. The solid support was heated with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-8) and desalted to obtain the product (7 mg). Mass calculated: 7906, Deconvoluted Mass: 7909.

Synthesis of WV-7519

(2734) ##STR01118##

(2735) To a suspension of WV2809 (60 mg, 8 mol, 150 mg support) in 2 ml NMP was added DIPEA (11 mg, 80 mol) and vortexed well for 5 minutes. To this suspension was added (8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl carbonochloridate (15 mg, 33 mol) and shaken for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml3) and dried. The solid support was heated at 50 C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-8) and desalted to obtain the product (20 mg). Mass calculated: 7840, Deconvoluted mass: 7841.

Synthesis of WV-7422

(2736) ##STR01119##

(2737) To a suspension of WV2809 (56 mg, 7.5 mol, 125 mg support) in 2 ml DMF was added DIPEA (19.3 mg, 150 mol) and vortexed well for 5 minutes. To this suspension was added perfluorophenyl 3-(4-(N-(2,2,2-trifluoroacetyl)sulfamoyl)phenyl)propanoate (37 mg, 75 mol) and shaken for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml3) and dried. The solid support was heated at 50 C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-8) and desalted to obtain the product (18 mg). Mass calculated: 7638, Deconvoluted Mass: 7641.

Synthesis of WV-7421

(2738) ##STR01120##

(2739) 2-(4-sulfamoylphenyl) acetic acid (17.2 mg, 80 mol), HATU (28 mg, 76 mol) and DIPEA (20.6 mg, 160 mol) in 2 ml NMP was vortexed well for 2 minutes. To this suspension was added WV2809 (60 mg, 8 mol, 150 mg support) and shaken well for 12 hours at room temperature. The solid support was washed with acetonitrile (20 ml3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml3) and dried. The solid support was heated at 50 C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-18) and desalted to obtain the product (20 mg). Mass calculated: 7624, Deconvoluted Mass: 7627.

Synthesis of WV-7417

(2740) ##STR01121##

(2741) A suspension of 1,7,14-trioxo-12,12-bis((3-oxo-3-((3-(4-sulfamoylbenzamido)propyl)amino)propoxy)methyl)-1-(4-sulfamoylphenyl)-10-oxa-2,6,13-triazaoctadecan-18-oic acid (40 mg, 34 mol), HATU (12 mg, 76 mol) and DIPEA (44 mg, 340 mol) in 2 ml NMP was vortexed well for 3 minutes. To this suspension was added WV2809 (60 mg, 8 mol, 150 mg support) and shaken well for 12 hours at 40 C. The solid support was washed with acetonitrile (20 ml3) and dried. This support was treated with 20% DEA in acetonitrile (1 ml) for 10 minutes. The DEA solution was removed by filtration. The solid support was washed with acetonitrile (20 ml3) and dried. The solid support was heated at 50 C. with 2 ml of 30% ammonium hydroxide for 12 hours. The support was filtered off and the filtrate was lyophilized to remove the solvent. The crude product was purified by RP column chromatography (C-18) and desalted to obtain the product (10 mg). Mass calculated: 8579, Deconvoluted Mass: 8577.

Example 17. General Procedure for the Deprotection of Amine

(2742) ##STR01122##

(2743) 15.2 g of NHBoc amine was dissolved in dry DCM (100 ml) then TFA (50 ml) was added dropwise at RT. Reaction mixture was stirred at RT overnight. Solvents were removed under reduced pressure then co-evaporated with toluene (250 mL) then used for the next step without any further purification. NMR in CD3OD confirmed the NHBoc deprotection.

Example 18. General Procedure for the Anisamide Formation

(2744) ##STR01123##

(2745) Procedure-A: The crude amine from the previous step was dissolved in a mixture of DCM (100 ml) and Et.sub.3N(10 equ.) at RT. During this process, the reaction mixture was cooled with a water bath. Then 4-Methoxybenzoyl chloride (4 equ) was added dropwise to the reaction mixture under argon atmosphere at RT, stirring continued for 3 h. Reaction mixture was diluted with water and extracted with DCM. Organic layer was extracted with aq. NaHCO.sub.3, 1N HCl, brine then dried with magnesium sulfate evaporated to dryness. The crude product was purified by silica column chromatography using DCM-MeOH as eluent.

(2746) Procedure-B: The crude amine (0.27 equ), acid and HOBt (1 equ) were dissolved in a mixture of DCM and DMF (2:1) in an appropriate sized RBF under argon. EDAC.Math.HCl (1.25 equ) was added portion wise to the reaction mixture under constant stirring. After 15 mins, the reaction mixture was cooled to 10 C. then DIEA (2.7 equ) was added over a period of 5 mins. Slowly warmed the reaction mixture to ambient temperature and stirred under argon for overnight. TLC indicated completion of the reaction TLC condition, DCM: MeOH (9.5:0.5). Solvents were removed under reduced pressure, then water was added to the residue, and a gummy solid separated out. The clear solution was decanted, and the solid residue was dissolved in EtOAc and washed successively with water, 10% aqueous citric acid, aq. NaHCO.sub.3, followed by saturated brine. The organic layer was separated and dried over magnesium sulfate. Solvent was removed under reduced pressure then the crude product was purified with silica column to get the pure product.

(2747) ##STR01124##

(2748) Anisamide was obtained from the amine in 32% yield over 2 steps using the above procedure-B: .sup.1H NMR (CDCl.sub.3): =7.74 (d, 6H), 7.44 (t, 2H), 7.34 (t, 1H), 7.26 (m, 5H), 7.05 (m, 3H), 6.83 (d, 6H), 6.46 (s, 1H), 5.01 (s, 2H), 3.75 (s, 9H), 3.57 (m, 12H), 3.37 (m, 6H), 3.25 (m, 6H), 2.31 (m, 8H), 2.11 (m, 2H), 1.84 (m, 2H), 1.62 (m, 6H) ppm.

(2749) ##STR01125##

(2750) Anisamide was obtained from the amine in 57% yield over 2 steps using the above procedure-A: .sup.1H NMR (CDCl.sub.3): =7.75 (m, 3H), 7.73 (d, 6H), 7.43 (t, 3H), 7.25 (m, 5H), 6.80 (d, 6H), 6.51 (brs, 1H), 5.01 (s, 2H), 3.72 (s, 9H), 3.58 (m, 6H), 3.21 (m, 12H), 2.33 (t, 3H), 2.25 (t, 2H), 2.02 (t, 2H), 1.64 (q, 6H), 1.52 (p, 2H), 1.41 (q, 2H), 1.12 (m, 12H) ppm.

(2751) General Procedure for Debenzylation.

(2752) ##STR01126##

(2753) The benzyl ester (10 g) was dissolved in a mixture of ethyl acetate (100 ml) and methanol (25 ml) then Pd/C, 1 g (10% palladium content) was added under argon atmosphere then the reaction mixture was vacuumed and flushed with hydrogen and stirred at RT under H.sub.2 atmosphere for 3 h. TLC indicated completion of the reaction, filtered through pad of celite and washed with methanol, evaporated to dryness to yield a foamy white solid.

(2754) ##STR01127##

(2755) Yield 98%, .sup.1H NMR (CD.sub.3OD): =8.35 (t, 1H), 8.01 (t, 1H), 7.82 (d, 6H), 7.27 (d, 1H), 6.99 (d, 6H), 3.85 (s, 9H), 3.68 (m, 12H), 3.41 (m, 6H), 3.29 (m, 6H), 2.42 (m, 6H), 2.31 (q, 2H), 2.21 (td, 2H), 1.80 (m, 8H) ppm.

(2756) ##STR01128##

(2757) Yield 94%, .sup.1H NMR (CD.sub.3OD): =8.36 (t, 2H), 8.02 (t, 2H), 7.82 (d, 6H), 7.23 (d, 1H), 6.98 (d, 6H), 3.85 (s, 9H), 3.70 (s, 6H), 3.67 (t, 6H), 3.41 (q, 4H), 3.28 (m, 8H), 2.42 (t, 6H), 2.27 (t, 2H), 2.13 (t, 2H), 1.79 (p, 6H), 1.54 (dp, 4H), 1.25 (m, 12H) ppm.

Example 19. Timelines for Pre-Differentiation of Patient Myoblasts for Gymnotic Dosing

(2758) Various technologies, e.g., those described in U.S. Pat. Nos. 9,394,333, 9,744,183, 9,605,019, 9,598,458, US 2015/0211006, US 2017/0037399, WO 2017/015555, WO 2017/192664, WO 2017/015575, WO 2017/062862, WO 2017/160741, WO 2017/192679, and WO 2017/210647, etc., can be utilized in accordance with the present disclosure to assess properties and/or activities of technologies of the present disclosure. In some embodiments, technologies of the present disclosure, e.g., oligonucleotides and compositions and methods of use thereof, demonstrate unexpectedly superior results compared to a suitable reference technology (e.g., a technology based on a stereorandom composition of oligonucleotides having the same base sequence but no neutral and/or cationic internucleotidic linkages at physiological pH). Described below are example technologies that can be useful for assessing properties and/or activities of oligonucleotides described in the present disclosure. Those skilled in the art understand that conditions illustrated below may be varied/modified, and additionally and/or alternatively, other suitable reagents, temperatures, conditions, time periods, amounnts, etc., may be utilized in accordance with the present disclosure.

(2759) Maintenance of Patient Derived Myoblast Cell Lines:

(2760) DMD 52 and DMD 45-52 myoblast cells were maintained in complete Skeletal Muscle Growth Medium (Promocell, Heidelberg, Germany) supplemented with 5% FBS, 1 Penicillin-Streptomycin and 1 L-Glutamine. Flasks or plates were coated with Matrigel:DMEM solution (1:100) for a suitable period of time, e.g., 30 mins, after which Matrigel:DMEM solution was removed via aspiration before seeding of cells in complete Skeletal Muscle Growth Medium.

(2761) Standard Dosing Procedure (0 Days Pre-Differentiation)

(2762) On Day 1: Coat suitable cell growth containers, e.g., 6-well plates or 24-well plates, with Matrigel: DMEM Solution. Incubate at a condition, e.g., 37 C., 5% CO.sub.2 for a suitable period of time, e.g., 30 mins. Aspirate, and seed a suitable number of cells to cell growth containers, e.g., 150K cells/well in a total of 1500 l of complete growth medium in 6-well plate, and 30K cells/well in 500 l of growth medium in a 24-well plate. Incubate at a suitable condition for a suitable period of time, .e.g., 37 C., 5% CO.sub.2 overnight.

(2763) On Day 2: Prepare a suitable Differentiation medium, e.g., DMEM+5% Horse Serum+10 g/ml Insulin. Prepare suitable oligonucleotide dilutions in Differentiation Medium, e.g., serial dilutions of 30 M, 10 M, 3.33 M, 1.11 M, 0.37 M. Aspirate growth medium off of adherent cells, and add oligonucleotide: Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

(2764) On Day 6: Obtain RNA. In a typical procedure, a suitable number of cells, e.g., cells from wells of a 24-well plate, were washed, e.g., with cold PBS, followed by addition of a suitable amount of a reagent for RNA extraction and storage of sample/RNA extraction, e.g., 500 l/well TRIZOL in 24-well plate and freezing plate at 80 C. or continuing with RNA extraction to obtain RNA.

(2765) On Day 8: Obtain protein. In a typical procedure, a suitable number of cells, e.g., cells in wells of 6-well plate, were washe, e.g., with cold PBS. A suitable amount of a suitable lysis buffer was then addede.g., in a typical procedure, 200 ul/well of RIPA supplemented with protease inhibitors for a 6-well plate. After lysis the sample can be stored, e.g., freezing at 80 C., or continue with protein extraction.

(2766) Other suitable procedures may be employed, for example, those described below. As appreciated by those skilled in the art, many parameters, such as reagents, temperatures, conditions, time periods, amounnts, etc., may be modified.

(2767) 4 Days Pre-Differentiation Dosing Procedure

(2768) On Day 1: Coat 6-well plates or 24-well plates with Matrigel: DMEM Solution. Incubate at 37 C., 5% CO.sub.2 for 30 mins. Aspirate, seed 150K cells/well in a total of 1500 l of complete growth medium in 6-well plate, and 30K cells/well in 500 ul of growth medium in a 24-well plate. Incubate at 37 C., 5% CO.sub.2 overnight.

(2769) On Day 2: Prepare Differentiation medium as follows: DMEM+5% Horse Serum+10 g/ml Insulin. Aspirate Growth Media and replace with Differentiation Media.

(2770) On Day 6: Cells have differentiated for 4 days. Prepare oligonucleotide dilutions in Differentiation Medium, for example serial dilutions of 30 uM, 10 uM, 3.33 uM, 1.11 uM, 0.37 uM. Aspirate Differentiation medium off of adherent cells, and add oligonucleotide: Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

(2771) On Day 10: Wash cells in 24-well plate with cold PBS, add 500 ul/well TRIZOL in 24-well plate and freeze plate at 80 C. or continue with RNA extraction.

(2772) On Day 12: Wash cells in 6-well plate with cold PBS. Add 200 ul/well of RIPA supplemented with protease inhibitors. Freeze plate at 80 C. or continue with protein extraction.

(2773) 7 Days Pre-Differentiation Dosing Procedure

(2774) On Day 1: Coat 6-well plates or 24-well plates with Matrigel: DMEM Solution. Incubate at 37 C., 5% CO.sub.2 for 30 mins. Aspirate, seed 150K cells/well in a total of 1500 l of complete growth medium in 6-well plate, and 30K cells/well in 500 ul of growth medium in a 24-well plate. Incubate at 37 C., 5% CO.sub.2 overnight.

(2775) On Day 2: Prepare Differentiation medium as follows: DMEM+5% Horse Serum+10 g/ml Insulin. Aspirate Growth Media and replace with Differentiation Media.

(2776) On Day 9: Cells have differentiated for 7 days. Prepare oligonucleotide dilutions in Differentiation Medium, for example serial dilutions of 30 uM, 10 uM, 3.33 uM, 1.11 uM, 0.37 uM. Aspirate Differentiation medium off of adherent cells, and add oligonucleotide: Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

(2777) On Day 13: Wash cells in 24-well plate with cold PBS, add 500 ul/well TRIZOL in 24-well plate and freeze plate at 80 C. or continue with RNA extraction.

(2778) On Day 15: Wash cells in 6-well plate with cold PBS. Add 200 ul/well of RIPA supplemented with protease inhibitors. Freeze plate at 80 C. or continue with protein extraction. 10 days Pre-Differentiation Dosing Procedure

(2779) On Day 1: Coat 6-well plates or 24-well plates with Matrigel: DMEM Solution. Incubate at 37 C., 5% CO.sub.2 for 30 mins. Aspirate, seed 150K cells/well in a total of 1500 l of complete growth medium in 6-well plate, and 30K cells/well in 500 ul of growth medium in a 24-well plate. Incubate at 37 C., 5% CO.sub.2 overnight.

(2780) On Day 2: Prepare Differentiation medium as follows: DMEM+5% Horse Serum+10 g/ml Insulin. Aspirate Growth Media and replace with Differentiation Media.

(2781) On Day 12: Cells have differentiated for 10 days. Prepare oligonucleotide dilutions in Differentiation Medium, for example serial dilutions of 30 uM, 10 uM, 3.33 uM, 1.11 uM, 0.37 uM. Aspirate Differentiation medium off of adherent cells, and add oligonucleotide: Differentiation Medium solution to cells. Oligonucleotides remain on cells (no media change) until cell harvesting.

(2782) On Day 16: Wash cells in 24-well plate with cold PBS, add 500 ul/well TRIZOL in 24-well plate and freeze plate at 80 C. or continue with RNA extraction.

(2783) On Day 18: Wash cells in 6-well plate with cold PBS. Add 200 ul/well of RIPA supplemented with protease inhibitors. Freeze plate at 80 C. or continue with protein extraction.

Example 20. Multi-Exon Skipping Assay

(2784) The assay described herein can be adapted to detect any gene's splice-variants with frequency of each variant (quantification). DMD Exon43-Exon64 is used as an example.

(2785) Among other things, a unique feature of this assay is that an unique-molecular-identifier (UMI) is introduced in the reverse transcription primers with an unique PCR handler sequence (this can be any sequence without homology to genomic or transcriptome sequences). Therefore, each cDNA has its unique UMI (bar-code) that can be used in later sequencing analysis to eliminate PCR and sequencing bias toward smaller amplicons.

(2786) In a typical procedure, the steps include: Reverse RT primer containing a PCR handle at 5-end, then 8-16 sequences of randomly incorporated nucleotides that create UMI/bar code and reverse complement sequence in exon 64 (Reverse RT primer in table), was used to prime the reverse transcription by a RT kit (e.g., SuperScript IV, ThermoFisher, Cambridge, MA). Then primary and nested PCR were run to amplify gene-specific fragments used for PacBio long range sequencing or Oxford Nanopore MinION platform.

(2787) The NGS sequences (BAM files) were mapped to reference sequence (DMD for example) to identify splice variants (exon junctions). The UMI were counted in each splice variant, and frequency of variant was calculated by UMI counts in each variant divided by total UMI counts in all variants.

(2788) An illustration of this process is shown in FIG. 2.

(2789) Example Reverse RT Primer:

(2790) TABLE-US-00128 (SEQIDNO:3279) 5'-CAGTGGTATCAACGCAGAGTACG-NNNNNNNN- ctgagaatctgacacagg-3' 5-capital letter=N1 binding sequence (nested secondary) N . . . N=UMI underline=gene specific sequence in exon64
Forward Primer (Exon 43): Fnest=5-gaagctctctcccagcttgat-3 (SEQ ID NO: 3280)
Among other things, the present disclosure provides the following Example Embodiments:
1. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and the oligonucleotide composition being characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
2. The composition of any one of the preceding embodiment, wherein the transcript is a Dystrophin transcript.
3. The composition of any one of the preceding embodiments, wherein splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.
4. The composition of any one of the preceding embodiments, wherein each chiral internucleotidic linkage of the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.
5. The composition of any one of the preceding embodiments, wherein each chiral modified internucleotidic linkage independently has a stereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus.
6. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A.sup.1.
7. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.
8. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.
9. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.
10. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).
11. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.
12. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications, which composition is chirally controlled and it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence, pattern of backbone linkages and pattern of backbone phosphorus modifications, for oligonucleotides of the particular oligonucleotide type,
wherein: the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of skipping of an exon is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
13. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.
14. The composition of any one of the preceding embodiments, wherein the exon is DMD exon 45, 51 or 53 or multiple DMD exons, and wherein the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.
15. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Sp.
16. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least one Rp.
17. The composition of any one of the preceding embodiments, wherein the composition is a chirally pure composition.
18. The composition of any one of the preceding embodiments, wherein each chiral modified internucleotidic linkage independently has a stereopurity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% at its chiral linkage phosphorus.
19. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.
20. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.
21. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.
22. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.
23. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).
24. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.
25. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; and 3) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages; the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of skipping of an exon is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
26. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.
27. The composition of any one of the preceding embodiments, wherein the exon is DMD exon 45, 51, or 53 or multiple DMD exons, and the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.
28. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently an internucleotidic linkage at least 50% of which exists in its non-negatively charged form at pH 7.4.
29. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage is independently a neutral internucleotidic linkage, wherein at least 50% of the internucleotidic linkage exists in its neutral form at pH 7.4.
30. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.
31. The composition of any one of the preceding embodiments, wherein the neutral form of each non-negatively charged internucleotidic linkage, when the units which it connects are replaced with CH.sub.3, independently has a pKa no less than 8, 9, 10, 11, 12, 13, or 14.
32. The composition of any one of the preceding embodiments, wherein the reference condition is absence of the composition.
33. The composition of any one of the preceding embodiments, wherein the reference condition is presence of a reference composition.
34. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no chirally controlled internucleotidic linkages.
35. The composition of any one of the preceding embodiments, wherein the reference composition is an otherwise identical composition wherein the oligonucleotides of the plurality comprise no non-negatively charged internucleotidic linkages.
36. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises one or more backbone linkages selected from phosphodiester, phosphorothioate and phosphodithioate linkages.
37. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise one or more sugar modifications.
38. The composition of any one of the preceding embodiments, wherein the sugar modifications comprise one or more modifications selected from: 2-O-methyl, 2-MOE, 2-F, morpholino and bicyclic sugar moieties.
39. The composition of any one of the preceding embodiments, wherein one or more sugar modifications are 2-F modifications.
40. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 5-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety.
41. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a 3-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety.
42. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality each comprise a middle region between the 5-end region and the 3-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.
43. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.
44. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.
45. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.
46. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.
47. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).
48. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.
49. A composition comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; and 3) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise: 1) a 5-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety; 2) a 3-end region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety; and 3) a middle region between the 5-end region and the 3-region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotidic units comprising a phosphodiester linkage.
50. The composition of embodiment 43 or 49, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered in that level of skipping of an exon is increased relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
51. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.
52. The composition of any one of the preceding embodiments, wherein the exon is DMD exon 45, 51, or 53 or multiple DMD exons, and the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.
53. The composition of any one of the preceding embodiments, wherein the 5-end region comprises 1 or more nucleoside units not comprising a 2-F modified sugar moiety.
54. The composition of any one of the preceding embodiments, wherein the 3-end region comprises 1 or more nucleoside units not comprising a 2-F modified sugar moiety.
55. The composition of any one of the preceding embodiments, wherein the middle region comprises 1 or more nucleotidic units comprising no phosphodiester linkage.
56. The composition of any one of the preceding embodiments, wherein the first of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety and a modified internucleotidic linkage of the 5-end is the first, second, third, fourth or fifth nucleoside unit of the oligonucleotide from the 5-end, and the last of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleoside units comprising a 2-F modified sugar moiety and a modified internucleotidic linkage of the 3-end is the last, second last, third last, fourth last, or fifth last nucleoside unit of the oligonucleotide.
57. The composition of any one of the preceding embodiments, wherein the 5-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
58. The composition of any one of the preceding embodiments, wherein the 5-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
59. The composition of any one of the preceding embodiments, wherein the 3-end region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
60. The composition of any one of the preceding embodiments, wherein the 3-end region comprising 5, 6, 7, 8, 9, 10 or more consecutive nucleoside units comprising a 2-F modified sugar moiety.
61. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2-F modified sugar moiety in the 5-end region is independently a modified internucleotidic linkage.
62. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage between two nucleoside units comprising a 2-F modified sugar moiety in the 3-end region is independently a modified internucleotidic linkage.
63. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is independently a chiral internucleotidic linkage.
64. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.
65. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is a phosphorothioate internucleotidic linkage.
66. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is a chirally controlled phosphorothioate internucleotidic linkage.
67. The composition of embodiment 61 or 62, wherein each modified internucleotidic linkage is a Sp chirally controlled phosphorothioate internucleotidic linkage.
68. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages.
69. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages each independently between a nucleoside unit comprising a 2-OR.sup.1 modified sugar moiety and a nucleoside unit comprising a 2-F modified sugar moiety, or between two nucleoside units each independently comprising a 2-OR.sup.1 modified sugar moiety, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl.
70. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
71. The composition of any one of the preceding embodiments, wherein the middle region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages each independently between a nucleoside unit comprising a 2-OR.sup.1 modified sugar moiety and a nucleoside unit comprising a 2-F modified sugar moiety, or between two nucleoside units each independently comprising a 2-OR.sup.1 modified sugar moiety, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl.
72. The composition of embodiment 69 or 71, wherein 2-OR.sup.1 is 2-OCH.sub.3.
73. The composition of embodiment 69 or 71, wherein 2-OR.sup.1 is 2-OCH.sub.2CH.sub.2OCH.sub.3.
74. The composition of any one of the preceding embodiments, wherein the 5-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.
75. The composition of any one of the preceding embodiments, wherein the 5-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.
76. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 5-end region is a chiral modified internucleotidic linkage.
77. The composition of any one of the preceding embodiments, wherein the 3-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.
78. The composition of any one of the preceding embodiments, wherein the 3-end region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.
79. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the 3-end region is a chiral modified internucleotidic linkage.
80. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 chiral modified internucleotidic linkages.
81. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive chiral modified internucleotidic linkages.
82. The composition of any one of embodiments 74-81, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage.
83. The composition of any one of embodiments 74-81, wherein each chiral modified internucleotidic linkage is independently a chirally controlled internucleotidic linkage wherein its chirally controlled linkage phosphorus has a Sp configuration.
84. The composition of any one of embodiments 74-83, wherein each chiral modified internucleotidic linkage is independently a chirally controlled phosphorothioate internucleotidic linkage.
85. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-negatively charged internucleotidic linkages.
86. The composition of any one of the preceding embodiments, wherein the middle region comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 neutral internucleotidic linkages.
87. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chiral internucleotidic linkage.
88. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is a chirally controlled internucleotidic linkage independently of Rp or Sp at its linkage phosphorus.
89. The composition of any one of the preceding embodiments, wherein the base sequence comprises a sequence having no more than 5 mismatches from a 20 base long portion of the dystrophin gene or its complement.
90. The composition of any one of the preceding embodiments, wherein the length of the base sequence of the oligonucleotides of the plurality is no more than 50 bases.
91. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chirally controlled centers independently of Rp or Sp.
92. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 5 chirally controlled centers independently of Rp or Sp.
93. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 6 chirally controlled centers independently of Rp or Sp.
94. The composition of any one of the preceding embodiments, wherein the pattern of backbone chiral centers comprises at least 10 chirally controlled centers independently of Rp or Sp.
95. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular oligonucleotide type are capable of mediating skipping of one or more exons of the dystrophin gene.
96. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45, 51 or 53 of the dystrophin gene.
97. The composition of embodiment 96, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 45 of the dystrophin gene.
98. The composition of embodiment 96, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 51 of the dystrophin gene.
99. The composition of embodiment 96, wherein the oligonucleotides of the plurality are capable of mediating the skipping of exon 53 of the dystrophin gene.
100. The composition of embodiment 97, wherein the base sequence comprises a sequence having no more than 5 mismatches from the sequence of any oligonucleotide disclosed herein.
101. The composition of embodiment 97, wherein the base sequence comprises or is the sequence of any oligonucleotide disclosed herein.
102. The composition of embodiment 97, wherein the base sequence is that of any oligonucleotide disclosed herein.
103. The composition of embodiment 97, wherein the base sequence comprises a sequence having no more than 5 mismatches from the sequence of any oligonucleotide disclosed herein.
104. The composition of embodiment 97, wherein the base sequence comprises or is any oligonucleotide disclosed herein.
105. The composition of embodiment 97, wherein the base sequence is any oligonucleotide disclosed herein.
106. The composition of any of the preceding embodiments, wherein the oligonucleotides of the plurality are any oligonucleotide disclosed herein.
107. The composition of embodiment 18, wherein oligonucleotides of the particular oligonucleotide type are any oligonucleotide disclosed herein.
108. The composition of any one of the preceding embodiments, wherein the base sequence is or comprises or comprises 15 contiguous bases of the base sequence of any oligonucleotide in Table A1.
109. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage.
110. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
111. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.
112. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.
113. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.
114. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure.
115. The composition of any one of the preceding embodiments, wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
116. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.
117. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.
118. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a wing comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.
119. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise or consist of a wing-core-wing structure, and wherein only one wing comprise one or more non-negatively charged internucleotidic linkages.
120. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
121. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chirally controlled non-negatively charged internucleotidic linkages.
122. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive non-negatively charged internucleotidic linkages.
123. The composition of any one of the preceding embodiments, wherein the oligonucleotides comprise a wing-core-wing, core-wing, or wing-core structure, and wherein a core comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive chirally controlled non-negatively charged internucleotidic linkages.
124. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage or a Rp chiral internucleotidic linkage.
125. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage or a natural phosphate internucleotidic linkage.
126. The composition of any one of the preceding embodiments, wherein 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of internucleotidic linkages of a wing is independently a non-negatively charged internucleotidic linkage.
127. The composition of any one of embodiments 124-126, wherein the percentage is 50% or more.
128. The composition of any one of embodiments 124-126, wherein the percentage is 60% or more.
129. The composition of any one of embodiments 124-126, wherein the percentage is 75% or more.
130. The composition of any one of embodiments 124-126, wherein the percentage is 80% or more.
131. The composition of any one of embodiments 124-126, wherein the percentage is 90% or more.
132. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.
133. The composition of any one of the preceding embodiments, wherein the oligonucleotides each comprise a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.
134. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage and a natural phosphate internucleotidic linkage.
135. The composition of any one of the preceding embodiments, wherein a wing comprises a non-negatively charged internucleotidic linkage, a natural phosphate internucleotidic linkage and a Rp chiral internucleotidic linkage.
136. The composition of any one of the preceding embodiments, wherein a core comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-negatively charged internucleotidic linkages.
137. The composition of any one of the preceding embodiments, wherein all non-negatively charged internucleotidic linkages of the same oligonucleotide have the same constitution.
138. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.
139. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.
140. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof. 141. The composition of any one of the preceding embodiments, wherein each of the non-negatively charged internucleotidic linkages independently has the structure of formula II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof.
142. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one non-negatively charged internucleotidic linkage which is a neutral internucleotidic linkage.
143. The composition of any one of the preceding embodiments, wherein the pattern of backbone linkages comprises at least one neutral internucleotidic linkage which is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.
144. The composition of any one of the preceding embodiments, wherein the oligonucleotide type comprises any of: cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Gambogic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).
145. The composition of any one of the preceding embodiments, wherein the oligonucleotide type is any oligonucleotide listed in Table A1.
146. The composition of any one of the preceding embodiments, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a carbohydrate moiety, a peptide moiety, a receptor ligand moiety, or a moiety having the structure of N(R.sup.1).sub.2, N(R.sup.1).sub.3, or NC(N(R.sup.1).sub.2).sub.2.
147. The composition of any one of the preceding embodiments, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises a guanidine moiety.
148. The composition of any one of the preceding embodiments, wherein each of the oligonucleotides comprises a chemical moiety conjugated to the oligonucleotide chain of the oligonucleotide optionally through a linker moiety, wherein the chemical moiety comprises NC(N(CH.sub.3).sub.2).sub.2.
149. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.
150. The composition of any one of the preceding embodiments, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the oligonucleotides in the composition that have the base sequence, pattern of backbone linkages, and pattern of backbone phosphorus modifications of the particular oligonucleotide type are oligonucleotides of the particular oligonucleotide type.
151. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the particular type are structurally identical.
152. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage is a phosphoramidate linkage.
153. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage comprises a guanidine moiety.
154. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I:

(2791) ##STR01129##
or a salt form thereof, wherein: P.sup.L is P(W), P, or P.fwdarw.B(R).sub.3; W is O, N(-L-R.sup.5), S or Se; each of R.sup.1 and R.sup.5 is independently H, -L-R, halogen, CN, NO.sub.2, -L-Si(R).sub.3, OR, SR, or N(R).sub.2; each of X, Y and Z is independently O, S, N(-L-R.sup.5), or L; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
155. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I or a salt form thereof.
156. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-1 or a salt form thereof:

(2792) ##STR01130##
157. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-1 or a salt form thereof.
158. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-2 or a salt form thereof:

(2793) ##STR01131##
159. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof:

(2794) ##STR01132##
160. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof.
161. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
162. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
163. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.
164. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula I-n-3 or a salt form thereof, wherein one R from one N(R).sub.2 and one R from the other N(R).sub.2 are taken together with their intervening atoms to form an optionally substituted 5-membered monocyclic ring having no more than two nitrogen atoms.
165. The composition of any one of embodiments 159-162, wherein the ring formed is a saturated ring.
166. The composition of any one of embodiments 159-162, wherein the ring formed is a partially unsaturated ring.
167. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula I-n-4 or a salt form thereof:

(2795) ##STR01133##
168. The composition of embodiment 167, wherein L.sup.a is a covalent bond.
169. The composition of embodiment 167, wherein L.sup.a is N(R.sup.1).
170. The composition of embodiment 167, wherein L.sup.a is N(R).
171. The composition of embodiment 167, wherein L.sup.a is N(R).
172. The composition of embodiment 167, wherein L.sup.a is S(O).
173. The composition of embodiment 167, wherein L.sup.a is S(O).sub.2.
174. The composition of embodiment 167, wherein L.sup.a is S(O).sub.2N(R).
175. The composition of any one of embodiments 167-174, wherein L.sup.b is a covalent bond.
176. The composition of any one of embodiments 167-174, wherein L.sup.b is N(R.sup.1).
177. The composition of any one of embodiments 167-174, wherein L.sup.b is N(R).
178. The composition of any one of embodiments 167-174, wherein L.sup.b is N(R).
179 The composition of any one of embodiments 167-174, wherein L.sup.b is S(O).
180. The composition of any one of embodiments 167-174, wherein L.sup.b is S(O).sub.2.
181. The composition of any one of embodiments 167-174, wherein L.sup.b is S(O).sub.2N(R).
182. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II:

(2796) ##STR01134##
or a salt form thereof, wherein: P.sup.L is P(W), P, or P.fwdarw.B(R).sub.3; W is O, N(-L-R.sup.5), S or Se; each of X, Y and Z is independently O, S, N(-L-R.sup.5), or L; R.sup.5 is H, -L-R, halogen, CN, NO.sub.2, -L-Si(R).sub.3, OR, SR, or N(R).sub.2; Ring A.sup.L is an optionally substituted 3-20 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms; each R.sup.s is independently H, halogen, CN, N.sub.3, NO, NO.sub.2, -L-R, -L-Si(R).sub.3, -L-OR, -L-SR, -L-N(R).sub.2, O-L-R, O-L-Si(R).sub.3, O-L-OR, O-L-SR, or O-L-N(R).sub.2; g is 0-20; each L is independently a covalent bond, or a bivalent, optionally substituted, linear or branched group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with C.sub.1-6 alkylene, C.sub.1-6 alkenylene, CC, a bivalent C.sub.1-C.sub.6 heteroaliphatic group having 1-5 heteroatoms, C(R).sub.2, Cy, O, S, SS, N(R), C(O), C(S), C(NR), C(O)N(R), N(R)C(O)N(R), N(R)C(O)O, S(O), S(O).sub.2, S(O).sub.2N(R), C(O)S, C(O)O, P(O)(OR), P(O)(SR), P(O)(R), P(O)(NR), P(S)(OR), P(S)(SR), P(S)(R), P(S)(NR), P(R), P(OR), P(SR), P(NR), P(OR)[B(R).sub.3], OP(O)(OR)O, OP(O)(SR)O, OP(O)(R)O, OP(O)(NR)O, OP(OR)O, OP(SR)O, OP(NR)O, OP(R)O, or OP(OR)[B(R).sub.3]O, and one or more CH or carbon atoms are optionally and independently replaced with Cy.sup.L; each Cy is independently an optionally substituted bivalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each Cy.sup.L is independently an optionally substituted trivalent or tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms; each R is independently R, C(O)R, C(O)OR, or S(O).sub.2R; each R is independently H, or an optionally substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms, and 3-30 membered heterocyclyl having 1-10 heteroatoms, or two R groups are optionally and independently taken together to form a covalent bond, or two or more R groups on the same atom are optionally and independently taken together with the atom to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the atom, 0-10 heteroatoms, or two or more R groups on two or more atoms are optionally and independently taken together with their intervening atoms to form an optionally substituted, 3-30 membered, monocyclic, bicyclic or polycyclic ring having, in addition to the intervening atoms, 0-10 heteroatoms.
183. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II, or a salt form thereof.
184. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-1:

(2797) ##STR01135##
or a salt form thereof.
185. The composition any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-a-2:

(2798) ##STR01136##
or a salt form thereof.
186. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-a-1 or II-a-2, or a salt form thereof.
187. The composition of any one of embodiments 182-186, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-b-1:

(2799) ##STR01137##
or a salt form thereof, wherein g is 0-18.
188. The composition of any one of embodiments 182-187, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-b-2:

(2800) ##STR01138##
or a salt form thereof, wherein g is 0-18.
189. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-b-1 or II-b-2, or a salt form thereof.
190. The composition of any one of embodiments 182-188, wherein Ring A.sup.L is an optionally substituted 3-20 membered monocyclic ring having 0-10 heteroatoms (in addition to the two nitrogen atoms for formula II-b-1 or II-b-2).
191. The composition of any one of embodiments 182-188, wherein Ring A.sup.L is an optionally substituted 5-membered monocyclic saturated ring.
192. The composition of any one of embodiments 182-191, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-c-1:

(2801) ##STR01139##
or a salt form thereof, wherein g is 0-4.
193. The composition of any one of embodiments 182-193, wherein a non-negatively charged internucleotidic linkage has the structure of formula II-c-2:

(2802) ##STR01140##
or a salt form thereof, wherein g is 0-4.
194. The composition of any one of the preceding embodiments, wherein each non-negatively charged internucleotidic linkage independently has the structure of formula II-c-1 or II-c-2, or a salt form thereof.
195. The composition of any one of embodiments 182-193, wherein each non-negatively charged internucleotidic linkage has the same structure.
196. The composition of any one of the preceding embodiments, wherein, if applicable, each internucleotidic linkage in the oligonucleotides of the plurality that is not a non-negatively charged internucleotidic linkage independently has the structure of formula I.
197. The composition of any one of the preceding embodiments, wherein each internucleotidic linkage in the oligonucleotides of the plurality independently has the structure of formula I.
198. The composition of any one of the preceding embodiments, wherein one or more P.sup.L is P(W).
199. The composition of any one of the preceding embodiments, wherein each P.sup.L is independently P(W).
200. The composition of any one of the preceding embodiments, wherein one or more W is O.
201. The composition of any one of the preceding embodiments, wherein each W is O.
202. The composition of any one of the preceding embodiments, wherein one or more W is S.
203. The composition of any one of the preceding embodiments, wherein one or more W is independently N(-L-R.sup.5).
204. The composition of any one of the preceding embodiments, wherein one or more internucleotidic linkage independently has the structure of formula III or salt form thereof:

(2803) ##STR01141##
205. The composition of embodiment 204, wherein P.sup.N is P(N-L-R.sup.5).
206. The composition of embodiment 204, wherein P.sup.N is

(2804) ##STR01142##
207. The composition of embodiment 204, wherein P.sup.N is

(2805) ##STR01143##
208. The composition of embodiment 207, wherein L.sup.a is a covalent bond.
209. The composition of embodiment 207, wherein L.sup.a is N(R.sup.1).
210. The composition of embodiment 207, wherein L.sup.a is N(R).
211. The composition of embodiment 207, wherein L.sup.a is N(R).
212. The composition of embodiment 207, wherein L.sup.a is S(O).
213. The composition of embodiment 207, wherein L.sup.a is S(O).sub.2.
214. The composition of embodiment 207, wherein L.sup.a is S(O).sub.2N(R).
215. The composition of embodiment 204, wherein P.sup.N is

(2806) ##STR01144##
216. The composition of embodiment 204, wherein P.sup.N is

(2807) ##STR01145##
217. The composition of embodiment 204, wherein P.sup.N is

(2808) ##STR01146##
218. The composition of any one of the preceding embodiments, wherein one or more Y is O.
219. The composition of any one of the preceding embodiments, wherein each Y is O.
220. The composition of any one of the preceding embodiments, wherein one or more Z is O.
221. The composition of any one of the preceding embodiments, wherein each Z is O.
222. The composition of any one of the preceding embodiments, wherein one or more X is O.
223. The composition of any one of the preceding embodiments, wherein one or more X is S.
224. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

(2809) ##STR01147##
225. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

(2810) ##STR01148##
226. The composition of any one of the preceding embodiments, wherein a non-negatively charged internucleotidic linkage has the structure of

(2811) ##STR01149##
227. The composition of any one of the preceding embodiments, wherein for each internucleotidic linkage of formula I or a salt fore thereof that is not a non-negatively charged internucleotidic linkage, X is independently O or S, and -L-R.sup.1 is H (natural phosphate linkage or phosphorothioate linkage, respectively).
228. The composition of any one of the preceding embodiments, wherein each phosphorothioate linkage, if any, in the oligonucleotides of the plurality is independently a chirally controlled internucleotidic linkage.
229. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled internucleotidic linkage.
230. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a chirally controlled internucleotidic linkage.
231. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a targeting moiety wherein the targeting moiety is independently connected to an oligonucleotide backbone through a linker.
232. The composition of embodiment 231, wherein the targeting moiety is a carbohydrate moiety.
233. The composition of embodiment 231 or 232, wherein the targeting moiety comprises or is a GalNac moiety.
234. The composition of any one of the preceding embodiments, wherein the oligonucleotides of the plurality comprise a lipid moiety wherein the lipid moiety is independently connected to an oligonucleotide backbone through a linker.
235. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more non-neutral internucleotidic linkages at the condition of the composition independently exist as a salt form.
236. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a salt form.
237. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein one or more negatively-charged internucleotidic linkages at the condition of the composition independently exist as a metal salt.
238. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as a metal salt.
239. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage at the condition of the composition independently exists as sodium salt.
240. The composition of any one of the preceding embodiments, wherein oligonucleotides of the plurality exist as salts, wherein each negatively-charged internucleotidic linkage is independently a natural phosphate linkage (the neutral form of which is OP(O)(OH)O) or phosphorothioate internucleotidic linkage (the neutral form of which is OP(O)(SH)O).
241. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications,
wherein: oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages; and oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 non-negatively charged internucleotidic linkages.
242. The composition of any one of the preceding embodiments, wherein at least one non-negatively charged internucleotidic linkage is a neutral internucleotidic linkage.
243. The composition of any one of the preceding embodiments, wherein a neutral internucleotidic linkage is or comprises a triazole, neutral triazole, alkyne, or a cyclic guanidine.
244. The oligonucleotide composition of any one of the preceding embodiments, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
245. The oligonucleotide composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.
246. The oligonucleotide composition of any one of the preceding embodiments, wherein the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.
247. The oligonucleotide composition of any one of the preceding embodiments, wherein the oligonucleotide composition is capable of mediating knockdown of a target gene.
248. An oligonucleotide composition, comprising a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) pattern of backbone linkages; 3) pattern of backbone chiral centers; and 4) pattern of backbone phosphorus modifications,
wherein:
the oligonucleotides of the plurality comprise cholesterol; L-carnitine (amide and carbamate bond); Folic acid; Cleavable lipid (1,2-dilaurin and ester bond); Insulin receptor ligand; Gambogic acid; CPP; Glucose (tri- and hex-antennary); or Mannose (tri- and hex-antennary, alpha and beta).
249. The composition of embodiment 248, wherein the oligonucleotides of the plurality comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 chirally controlled internucleotidic linkages.
250. The composition of any one of the preceding embodiments, wherein the oligonucleotide composition is characterized in that, when it is contacted with a transcript in a transcript splicing system, splicing of the transcript is altered relative to that observed under a reference condition selected from the group consisting of absence of the composition, presence of a reference composition, and combinations thereof.
251. The composition of any one of the preceding embodiments, wherein the transcript is a Dystrophin transcript.
252. The composition of any one of the preceding embodiments, wherein the splicing of the transcript is altered such that the level of skipping of exon 45, 51, or 53, or multiple exons is increased.
253. The composition of any one of the preceding embodiments, wherein the oligonucleotide composition is capable of mediating knockdown of a target gene.
254. The composition of any one of the preceding embodiments, wherein each heteroatom is independently boron, nitrogen, oxygen, silicon, sulfur, or phosphorus.
255. A pharmaceutical composition comprising an oligonucleotide composition of any one of the preceding embodiments and a pharmaceutically acceptable carrier.
256. A method for altering splicing of a target transcript, comprising administering an oligonucleotide composition of any one of the preceding embodiments.
257. The method of embodiment 256, wherein the splicing of the target transcript is altered relative to absence of the composition.
258. The method of any one of the preceding embodiments, wherein the alteration is that one or more exon is skipped at an increased level relative to absence of the composition.
259. The method of any one of the preceding embodiments, wherein the target transcript is pre-mRNA of dystrophin.
260. The method of any one of the preceding embodiments, wherein exon 45 of dystrophin is skipped at an increased level relative to absence of the composition.
261. The method of any one of the preceding embodiments, wherein exon 51 of dystrophin is skipped at an increased level relative to absence of the composition.
262. The method of any one of embodiments 256-259, wherein exon 53 of dystrophin is skipped at an increased level relative to absence of the composition.
263. The method of any one of the preceding embodiments, wherein a protein encoded by the mRNA with the exon skipped provides one or more functions better than a protein encoded by the corresponding mRNA without the exon skipping.
264. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition of any one of the preceding embodiments.
265. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising administering to a subject susceptible thereto or suffering therefrom a composition comprising any oligonucleotide disclosed herein.
266. A method for treating muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD), comprising (a) administering to a subject susceptible thereto or suffering therefrom a composition comprising any oligonucleotide disclosed herein, and (b) administering to the subject additional treatment which is capable of preventing, treating, ameliorating or slowing the progress of muscular dystrophy, Duchenne (Duchenne's) muscular dystrophy (DMD), or Becker (Becker's) muscular dystrophy (BMD).
267. The method of embodiment 266, wherein the additional treatment is a second oligonucleotide.
268. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast or myotubule.
269. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell.
270. The composition of any of the preceding embodiments, wherein the transcript splicing system comprises a myoblast cell, which is contacted with the composition after 0, 4 or 7 days of pre-differentiation.
271. A composition comprising a combination comprising: (a) a first composition of any of the preceding embodiments; (b) a second composition of any of the preceding embodiments; and, optionally (c) a third composition of any of the preceding embodiments, wherein the first, second and third compositions are different.
272. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a compound having the structure of:

(2812) ##STR01150##
or a salt thereof.
273. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a compound having the structure of:

(2813) ##STR01151##
or a salt thereof.
274. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a compound having the structure of

(2814) ##STR01152##
or a salt thereof.
275. The method of any one of embodiments 272-274, wherein the compound is stereochemically pure.
276. The method of any one of embodiments 272-275, wherein the compound is a compound of Tables CA-1, CA-2, CA-3, CA-4, CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, or CA-12, or a related diastereomer or enantiomer thereof.
277. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-2 or a related diastereomer or enantiomer thereof.
278. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-3 or a related diastereomer or enantiomer thereof.
279. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-4 or a related diastereomer or enantiomer thereof.
280. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-5 or a related diastereomer or enantiomer thereof.
281. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-6 or a related diastereomer or enantiomer thereof.
282. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-7 or a related diastereomer or enantiomer thereof.
283. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-8 or a related diastereomer or enantiomer thereof.
284. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-9 or a related diastereomer or enantiomer thereof.
285. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-10 or a related diastereomer or enantiomer thereof.
286. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-11 or a related diastereomer or enantiomer thereof.
287. The method of any one of embodiments 272-275, wherein the compound is a compound of Table CA-12 or a related diastereomer or enantiomer thereof.
288. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a phosphoramidite compound comprising a chiral auxiliary moiety having the structure of

(2815) ##STR01153##
289. A method for preparing an oligonucleotide or an oligonucleotide composition thereof, comprising providing a phosphoramidite compound having the structure of:

(2816) ##STR01154## ##STR01155##
or a salt thereof.
290. The method of any one of embodiments 272-289, wherein W.sup.1 is NG.sup.5-.
291. The method of any one of embodiments 272-290, wherein G.sup.5 and one of G.sup.3 and G.sup.4 are taken together to form an optionally substituted 3-8 membered saturated ring having 0-3 heteroatoms in addition to the nitrogen of NG.sup.5-.
292. The method of any one of embodiments 272-290, wherein G.sup.5 and one of G.sup.3 and G.sup.4 are taken together to form an optionally substituted 5-membered saturated ring having no heteroatoms in addition to the nitrogen of NG.sup.5-.
293. The method of any one of embodiments 272-292, wherein W.sup.2 is O.
294. The method of any one of embodiments 272-293, wherein G.sup.2 comprises an electron-withdrawing group.
295. The method of any one of embodiments 272-293, wherein G.sup.2 is methyl substituted with one or more electron-withdrawing groups.
296. The method of any one of embodiments 294-295, wherein an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2, or aryl or heteroaryl substituted with one or more of CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.
297. The method of any one of embodiments 294-295, wherein an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2, or phenyl substituted with one or more of CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.
298. The method of any one of embodiments 294-295, wherein an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.
299. The method of any one of embodiments 272-294, wherein G.sup.2 is -L-LR, wherein L is C(R).sub.2 or optionally substituted CH.sub.2, and L is a covalent bond, P(O)(R), P(O)(R)O, P(O)(OR), P(O)(OR)O, P(O)[N(R)], P(O)[N(R)]O, P(O)[N(R)][N(R)], P(S)(R), S(O).sub.2, S(O).sub.2, S(O).sub.2O, S(O), C(O), or C(O)N(R).
300. The method of any one of embodiments 272-294, wherein G.sup.2 is -L-LR, wherein L is C(R).sub.2 or optionally substituted CH.sub.2, and L is P(O)(R), P(O)(R)O, P(O)(OR), P(O)(OR)O, P(O)[N(R)], P(O)[N(R)]O, P(O)[N(R)][N(R)], P(S)(R), S(O).sub.2, S(O).sub.2, S(O).sub.2O, S(O), C(O), or C(O)N(R).
301. The method of any one of embodiments 272-300, wherein G.sup.2 is -LS(O).sub.2R.
302. The method of embodiment 301, wherein R is optionally substituted C.sub.1-6 aliphatic.
303. The method of embodiment 301, wherein R is optionally substituted C.sub.1-6 alkyl.
304. The method of embodiment 301, wherein R is methyl, isopropyl or t-butyl.
305. The method of embodiment 301, wherein R is optionally substituted phenyl.
306 The method of embodiment 301, wherein R is phenyl.
307. The method of embodiment 301, wherein R is substituted phenyl.
308. The method of any one of embodiments 272-300, wherein G.sup.2 is -LP(O)(R).sub.2.
309. The method of embodiment 308, wherein one R is optionally substituted C.sub.1-6 aliphatic.
310. The method of embodiment 308, wherein one R is optionally substituted C.sub.1-6 alkyl.
311. The method of embodiment 308, wherein one R is optionally substituted phenyl.
312. The method of embodiment 308, wherein one R is phenyl.
313. The method of embodiment 308, wherein one R is substituted phenyl.
314. The method of any one of embodiments 309-313, wherein the other R is optionally substituted C.sub.1-6 aliphatic.
315. The method of any one of embodiments 309-313, wherein the other R is optionally substituted C.sub.1-6 alkyl.
316. The method of any one of embodiments 309-313, wherein the other R is optionally substituted phenyl.
317. The method of any one of embodiments 309-313, wherein the other R is phenyl.
318. The method of any one of embodiments 309-313, wherein the other R is substituted phenyl.
319. The method of any one of embodiments 299-318, wherein L is C(R).sub.2.
320. The method of any one of embodiments 299-318, wherein L is optionally substituted CH.sub.2.
321. The method of any one of embodiments 299-318, wherein L is CH.sub.2.
322. The method of any one of embodiments 272-321, comprising providing one or more additional compounds, wherein each compound is independently a compound of any one of embodiments 272-321.
323. The method of embodiment 322, wherein an additional compound has a different structure than the compound.
324. The method of embodiment 322, wherein in an additional compound, G.sup.2 is -LSi(R).sub.3, wherein each R is independently not H.
325. The method of embodiment 322, wherein in an additional compound, G.sup.2 is CH.sub.2SiCH.sub.3Ph.sub.2.
326. The method of any one of embodiments 272-325, comprising one or more cycles, each of which independently comprises or consisting of: 1) deblocking; 2) coupling; 3) optionally a first capping; 4) modifying; and 5) optionally a second capping.
327. A method for preparing an oligonucleotide or a composition thereof, comprising one or more cycles, each of which independently comprises or consisting of: 1) deblocking; 2) coupling; 3) optionally a first capping; 4) modifying; and 5) optionally a second capping.
328. The method of any one of embodiments 326-327, wherein at least one cycle comprises or consists of 1) to 5).
329. The method of any one of embodiments 326-328, wherein the steps are performed sequentially from 1) to 5).
330. The method of any one of embodiments 326-329, wherein the cycles are performed until a desired length of an oligonucleotide is achieved.
331. The method of any one of embodiments 326-330, wherein deblocking removes a protection group on 5-OH and provides a free 5-OH.
332. The method of embodiment 331, wherein the protection group is RC(O).
333. The method of embodiment 331, wherein the protection group is DMTr.
334. The method of any one of embodiments 331-333, comprising contacting the oligonucleotides to be de-blocked with an acid.
335. The method of any one of embodiments 272-334, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a PO bond is formed between the phosphorus of the phosphoramidite and the 5-OH of the oligonucleotide.
336. The method of any one of embodiments 272-335, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a PO bond is formed between the phosphorus of the phosphoramidite and the 5-OH of the oligonucleotide, wherein the phosphoramidite is a compound of any one of embodiments 288-321.
337. The method of any one of embodiments 272-336, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a PO bond is formed between the phosphorus of the phosphoramidite and the 5-OH of the oligonucleotide, wherein the phosphoramidite is a compound of any one of embodiments 288-293, wherein G.sup.2 is -LSi(R).sub.3, wherein each R is independently not H.
338. The method of embodiment 337, wherein G.sup.2 is CH.sub.2SiCH.sub.3Ph.sub.2.
339. The method of any one of embodiments 336-338, wherein the coupling forms an internucleotidic linkage with a stereoselectivity of 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
340. The method of embodiment 339, wherein the internucleotidic linkage formed is an internucleotidic linkage of formula I or a salt form thereof.
341. The method of embodiment 340, wherein X-L-R.sup.1 is

(2817) ##STR01156##
342. The method of embodiment 340 or 341, wherein P.sup.L is P.
343. The method of any one of embodiments 272-342, comprising a coupling that comprises: 1) providing a phosphoramidite; and 2) reacting the phosphoramidite with an oligonucleotide, wherein a PO bond is formed between the phosphorus of the phosphoramidite and the 5-OH of the oligonucleotide, wherein the phosphoramidite is a standard phosphoramidite for oligonucleotide synthesis wherein the phosphorus atom is bonded to a protected nucleoside, N(i-Pr).sub.2, and 2-cyanoethyl.
344. The method of any one of embodiments 272-343, comprising a first capping comprises: 1) providing an acylating reagent, and 2) contacting an oligonucleotide with the acylating reagent, wherein the first capping caps an amino group of an internucleotidic linkage.
345. The method of any one of embodiments 272-344, comprising a first capping which forms an internucleotidic linkage of formula I or a salt form thereof, wherein X-L-R.sup.1 is

(2818) ##STR01157##
346. The method of embodiment 345, wherein P.sup.L is P and R.sup.1 is C(O)R.
347. The method of any one of embodiments 272-346, wherein a first capping is performed after each coupling of embodiment 339.
348. The method of any one of embodiments 272-347, comprising a modifying step which is or comprises sulfurization.
349. The method of embodiment 348, wherein the sulfurization installs S on a linkage phosphorus.
350. The method of embodiment 348 or 349, wherein the sulfurization forms an internucleotidic linkage of formula I or a salt form thereof, wherein P.sup.L is P(S).

(2819) ##STR01158##
351. The method of embodiment 350, wherein X-L-R.sup.1 is
352. The method of embodiment 351, wherein R.sup.1 is C(O)R.
353. The method of any one of embodiments 272-352, comprising a modifying step which is or comprises oxidation.
354. The method of embodiment 348, wherein the sulfurization installs O on a linkage phosphorus.
355. The method of any one of embodiments 272-354, comprising a modifying step which installs N-L-R.sup.5 on a linkage phosphorus.
356. The method of any one of embodiments 272-354, comprising a modifying step which converts a linkage phosphorus into

(2820) ##STR01159##
357. The method of any one of embodiments 272-356, comprising a modifying step which comprises contact the oligonucleotide with an azido imidazolinium salt.
358. The method of any one of embodiments 272-356, comprising a modifying step which comprises contact the oligonucleotide with a compound comprising

(2821) ##STR01160##
359. The method of any one of embodiments 272-356, comprising a modifying step which comprises contact the oligonucleotide with a compound having the structure of

(2822) ##STR01161##
wherein Q.sup. is an anion.
360. The method of embodiment 359, wherein Q.sup. is F.sup., Cl.sup., Br.sup., BF.sub.4.sup., PF.sub.6.sup., TfO.sup., Tf.sub.2N.sup., AsF.sub.6.sup., ClO.sub.4.sup., or SbF.sub.6.sup..
361. The method of embodiment 360, wherein Q.sup. is PF.sub.6.sup..
362. The method of any one of embodiments 272-362, wherein a modifying step forms an internucleotidic linkage of formula I or a salt form thereof, wherein P.sup.L is P(N-L-R.sup.5).
363. The method of any one of embodiments 272-362, wherein a modifying step forms an internucleotidic linkage of formula III or a salt form thereof.
364. The method of embodiment 362 or 363, wherein X-L-R.sup.1 is

(2823) ##STR01162##
365. The method of embodiment 364, wherein R.sup.1 is C(O)R.
366. The method of any one of embodiments 272-365, comprising a second capping which caps free 5-OH.
367. The method of any one of embodiments 272-366, comprising a second capping which caps free 5-OH, wherein a second capping is performed in each cycle.
368. The method of any one of embodiments 272-366, comprising a second capping which caps free 5-OH, wherein a second capping is performed in each cycle that is followed by another cycle.
369. The method of any one of embodiments 366-368, wherein a 5-OH is capped as OAc.
370. The method of any one of embodiments 272-369, wherein the oligonucleotide is attached to a solid support.
371. The method of embodiment 370, wherein the solid support is CPG.
372. The method of any one of embodiments 370-371, comprising a contact in which the oligonucleotide is contacted with a base.
373. The method of embodiment 372, wherein the contact is performed substantially absent of water.
374. The method of embodiment 372 or 373, wherein the contact is after the oligonucleotide length is achieved before deprotection and cleavage of oligonucleotide.
375. The method of any one of embodiments 372-374, wherein the base is an amine base having the structure of NR.sub.3.
376. The method of embodiment 375, wherein the base is triethylamine.
377. The method of embodiment 375, wherein the base is N, N-diethylamine.
378. The method of any one of embodiments 372-377, wherein the contact removes a chiral auxiliary.
379. The method of any one of embodiments 372-378, wherein the contact removes a X-L-R.sup.1 group.
380. The method of embodiment 379, wherein X-L-R.sup.1 is

(2824) ##STR01163##
381. The method of any one of embodiments 372-380, wherein the contact forms an internucleotidic linkage of formula I-n-1, I-n-2, I-n-3, I-n-4, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, wherein P.sup.L is P(O).
382. The method of any one of embodiments 364-381, wherein G.sup.2 comprises an electron-withdrawing group.
383. The method of any one of embodiments 364-382, wherein G.sup.2 is methyl substituted with one or more electron-withdrawing groups.
384. The method of any one of embodiments 382-383, wherein an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2, or aryl or heteroaryl substituted with one or more of CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.
385. The method of any one of embodiments 382-383, wherein an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2, or phenyl substituted with one or more of CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.
386. The method of any one of embodiments 382-383, wherein an electron-withdrawing group is CN, NO.sub.2, halogen, C(O)R.sup.1, C(O)OR, C(O)N(R).sub.2, S(O)R.sup.1, S(O).sub.2R.sup.1, P(W)(R.sup.1).sub.2, P(O)(R.sup.1).sub.2, P(O)(OR).sub.2, or P(S)(R.sup.1).sub.2.
387. The method of any one of embodiments 364-386, wherein G.sup.2 is -L-L-R, wherein L is C(R).sub.2 or optionally substituted CH.sub.2, and L is a covalent bond, P(O)(R), P(O)(R)O, P(O)(OR), P(O)(OR)O, P(O)[N(R)], P(O)[N(R)]O, P(O)[N(R)][N(R)], P(S)(R), S(O).sub.2, S(O).sub.2, S(O).sub.2O, S(O), C(O), or C(O)N(R).
388. The method of any one of embodiments 364-386, wherein G.sup.2 is -L-L-R, wherein L is C(R).sub.2 or optionally substituted CH.sub.2, and L is P(O)(R), P(O)(R)O, P(O)(OR), P(O)(OR)O, P(O)[N(R)], P(O)[N(R)]O, P(O)[N(R)][N(R)], P(S)(R), S(O).sub.2, S(O).sub.2, S(O).sub.2O, S(O), C(O), or C(O)N(R).
389. The method of any one of embodiments 364-388, wherein G.sup.2 is -LS(O).sub.2R.
390. The method of embodiment 389, wherein R is optionally substituted C.sub.1-6 aliphatic.
391. The method of embodiment 389, wherein R is optionally substituted C.sub.1-6 alkyl.
392. The method of embodiment 389, wherein R is methyl, isopropyl or t-butyl.
393. The method of embodiment 389, wherein R is optionally substituted phenyl.
394. The method of embodiment 389, wherein R is phenyl.
395. The method of embodiment 389, wherein R is substituted phenyl.
396. The method of any one of embodiments 364-388, wherein G.sup.2 is -L-P(O)(R).sub.2.
397. The method of embodiment 396, wherein one R is optionally substituted C.sub.1-6 aliphatic.
398. The method of embodiment 396, wherein one R is optionally substituted C.sub.1-6 alkyl.
399. The method of embodiment 396, wherein one R is optionally substituted phenyl.
400. The method of embodiment 396, wherein one R is phenyl.
401. The method of embodiment 396, wherein one R is substituted phenyl.
402. The method of any one of embodiments 397-401, wherein the other R is optionally substituted C.sub.1-6 aliphatic.
403. The method of any one of embodiments 397-401, wherein the other R is optionally substituted C.sub.1-6 alkyl.
404. The method of any one of embodiments 309-313, wherein the other R is optionally substituted phenyl.
405. The method of any one of embodiments 309-313, wherein the other R is phenyl.
406. The method of any one of embodiments 309-313, wherein the other R is substituted phenyl.
407. The method of any one of embodiments 387-406, wherein L is C(R).sub.2.
408 The method of any one of embodiments 387-406, wherein L is optionally substituted CH.sub.2.
409. The method of any one of embodiments 387-406, wherein L is CH.sub.2.
410. The method of any one of embodiments 372-409, wherein the contact removes 2-cyanoethyl.
411. The method of any one of embodiments 372-410, wherein the contact forms a natural phosphate linkage or a salt form thereof.
412. The method of any one of embodiments 272-410, comprising removing of another chiral auxiliary or group that having a different structure than that of any one of embodiments 378-410.
413. The method of any one of embodiments 272-410, comprising removing of

(2825) ##STR01164##
wherein G.sup.2 is -L-Si(R).sub.3, wherein each R is independently not H.
414. The method of embodiment 413, wherein G.sup.2 is CH.sub.2SiCH.sub.3Ph.sub.2.
415. The method of any one of embodiments 412-414, comprising contacting an oligonucleotide with a fluoride.
416. The method of any one of embodiments 412-414, comprising contacting an oligonucleotide with a solution comprising TEA-HF and a base.
417. The method of any one of embodiments 272-416, comprising cleaving oligonucleotide from a solid support.
418. The method of any one of embodiments 272-417, wherein the oligonucleotide or a composition thereof is an oligonucleotide or composition of any one of embodiments 1-254.
419. The compound of any one of embodiments 272-321, or a related diastereomer or enantiomer.
420. An oligonucleotide, wherein the oligonucleotide is, WV-20104, WV-20103, WV-20102, WV-20101, WV-20100, WV-20099, WV-20098, WV-20097, WV-20096, WV-20095, WV-20094, WV-20106, WV-20119, WV-20118, WV-13739, WV-13740, WV-9079, WV-9082, WV-9100, WV-9096, WV-9097, WV-9106, WV-9133, WV-9148, WV-9154, WV-9898, WV-9899, WV-9900, WV-9906, WV-9907, WV-9908, WV-9909, WV-9756, WV-9757, WV-9517, WV-9714, WV-9715, WV-9519, WV-9521, WV-9747, WV-9748, WV-9749, WV-9897, WV-9898, WV-9900, WV-9899, WV-9906, WV-9912, WV-9524, WV-9912, WV-9906, WV-9900, WV-9899, WV-9899, WV-9898, WV-9898, WV-9898, WV-9898, WV-9898, WV-9897, WV-9897, WV-9897, WV-9897, WV-9897, WV-9747, WV-9714, WV-9699, WV-9517, WV-9517, WV-13409, WV-13408, WV-12887, WV-12882, WV-12881, WV-12880, WV-12880, WV-WV12880, WV-12878, WV-12877, WV-12877, WV-12876, WV-12873, WV-12872, WV-12559, WV-12559, WV-12558, WV-12558, WV-12557, WV-12556, WV-12556, WV-12555, WV-12555, WV-12554, WV-12553, WV-12129, WV-12127, WV-12125, WV-12123, WV-11342, WV-11342, WV-11341, WV-11341, WV-11340, WV-10672, WV-10671, WV-10670, WV-10461, WV-10455, WV-9897, WV-9898, WV-13826, WV-13827, WV-13835, WV-12880, WV-14344, WV-13864, WV-13835, WV-14791, WV-14344, WV-13754, WV-13766, WV-11086, WV-11089, WV-17859, WV-17860, WV-20070, WV-20073, WV-20076, WV-20052, WV-20099, WV-20049, WV-20085, WV-20087, WV-20034, WV-20046, WV-20052, WV-20061, WV-20064, WV-20067, WV-20092, WV-20091, WV-20093, WV-20084, WV-9738, WV-9739, WV-9740, WV-9741, WV-15860, WV-15862, WV-11084, WV-11086, WV-11088, WV-11089, WV-14522, WV-14523, WV-17861, WV-17862, WV-13815, WV-13816, WV-13817, WV-13780, WV-17862, WV-17863, WV-17864, WV-17865, WV-17866, WV-20082, WV-20081, WV-20080, WV-20079, WV-20076, WV-20075, WV-20074, WV-20073, WV-20072, WV-20071, WV-20064, WV-20059, WV-20058, WV-20057, WV-20056, WV-20053, WV-20052, WV-20051, WV-20050, WV-20049, WV-20094, WV-20095, or a salt form thereof.

EQUIVALENTS

(2826) Having described some illustrative embodiments of the disclosure, it should be apparent to those skilled in the art that the foregoing is merely illustrative and not limiting, having been presented by way of example only. Numerous modifications and other illustrative embodiments are within the scope of one of ordinary skill in the art and are contemplated as falling within the scope of the disclosure. In particular, although many of the examples presented herein involve specific combinations of method acts or system elements, it should be understood that those acts and those elements may be combined in other ways to accomplish the same objectives. Acts, elements, and features discussed only in connection with one embodiment are not intended to be excluded from a similar role in other embodiments. Further, for the one or more means-plus-function limitations, if any, recited in the following claims, the means are not intended to be limited to the means disclosed herein for performing the recited function, but are intended to cover in scope any means, known now or later developed, for performing the recited function.

(2827) Use of ordinal terms such as first, second, third, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

(2828) The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present disclosure is not to be limited in scope by examples provided. Examples are intended as illustration of one or more aspect of an invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.