Compositions and methods for inhibiting cytochrome P450 family 7 subfamily a member 1 (CYP7A1) expression

Abstract

RNAi agents (e.g., CYP7A1 RNAi agents) for inhibiting the expression of the CYP7A1 gene, compositions including the RNAi agents conjugated to a targeting moiety, and methods of use are described. Also disclosed are pharmaceutical compositions including one or more RNAi agents (e.g., CYP7A1 RNAi agents). Delivery of the RNAi agent(s) to liver cells in vivo inhibits CYP7A1 gene expression and treats a CYP7A1 disease or a CYP7A1-associated disease.

Claims

1. An RNAi agent for inhibiting expression of Cytochrome P450 family 7 subfamily A member 1 (CYP7A1) in a cell, wherein the RNAi agent comprises a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises the nucleobase sequence (5.fwdarw.3) of UCAAGAAUAAGCCAUAGACAAAG (SEQ ID NO: 1180), and wherein the sense strand is 19-23 nucleosides in length and is at least substantially complementary to the antisense strand.

2. The RNAi agent of claim 1, wherein the sense strand is 21 nucleosides in length and the antisense strand is 23 nucleosides in length.

3. The RNAi agent of claim 1, wherein the antisense strand comprises an overhang of at least 1 nucleoside at the 3 end.

4. The RNAi agent of claim 1, wherein the sense strand comprises the nucleobase sequence (5.fwdarw.3) of UUGUCUAUGGCUUAUUCUUGA (SEQ ID NO: 404).

5. The RNAi agent of claim 4, wherein the RNAi agent comprises one or more modified nucleosides.

6. The RNAi agent of claim 5, wherein each nucleoside of the antisense strand is a modified nucleoside and each nucleoside of the sense strand is a modified nucleoside.

7. The RNAi agent of claim 5, wherein each nucleoside of the antisense strand is selected from a 2-F modified nucleoside and a 2-O-Me modified nucleoside, and each nucleoside of the sense strand is a 2-modified nucleoside selected from a 2-F modified nucleoside and a 2-O-Me modified nucleoside.

8. The RNAi agent of claim 5, wherein the nucleosides at one or more of positions 9, 10, 11, and 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides.

9. The RNAi agent of claim 5, wherein the nucleosides at one or more of positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides.

10. The RNAi agent of claim 5, wherein the RNAi agent comprises one or more phosphorothioate internucleoside linkages in at least one strand.

11. The RNAi agent of claim 10, wherein the first two internucleoside linkages in the sense strand from 5.fwdarw.3 are phosphorothioate internucleoside linkages.

12. The RNAi agent of claim 10, wherein the first two internucleoside linkages and the last two internucleoside linkages in the antisense strand from 5.fwdarw.3 are phosphorothioate internucleoside linkages.

13. The RNAi agent of claim 1, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) selected from the group consisting of [mUs][fCs][mA][fA][mG][fA][mA][fU][mA][fA][mG][mC][mC][fA][mU][fA][mG][fA][mC][fA][mAs][mAs][mG] (SEQ ID NO: 2055), [mUs][fCs][fA][fA][mG][fA][mA][fU][mA][fA][mG][fC][mC][fA][mU][fA][mG][fA][mC][fA][mAs][mAs][mG] (SEQ ID NO: 2056), [mUs][fCs][mA][mA][mG][fA][mA][fU][fA][mA][mG][mC][mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2057), [mUs][fCs][mA][mA][mG][fA][mA][mU][mA][mA][mG][mC][mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2058), and [mU][fC][mA][mA][mG][fA][gA][fU][fA][mA][mG][mC][mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2059), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; gA is GNA adenosine; and s is a phosphorothioate linkage.

14. The RNAi agent of claim 13, wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of TABLE-US-00066 (SEQIDNO:2060) [fUs][mUs][fG][mU][fC][mU][fA][mU][fG][fG][fC][mU] [fU][mA][fU][mU][fC][mU][fU][mG][fAs], (SEQIDNO:2062) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][mG][fC][mU] [fU][mA][fU][mU][mC][mU][mU][mG][mAs], and (SEQIDNO:2061) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][fG][fC][mU] [mU][mA][mU][mU][mC][mU][mU][mG][mAs], wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage.

15. The RNAi agent of claim 1, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) selected from the group consisting of TABLE-US-00067 (SEQIDNO:2029) [mUs][fCs][fA][mA][fG][fA][fA][fU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG], (SEQIDNO:2031) [mUs][fCs][fA][mA][fG][mA][fA][fU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG], (SEQIDNO:2032) [mUs][fCs][fA][mA][fG][fA][fA][mU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG], and (SEQIDNO:2030) [mUs][fCs][fA][mA][mG][mA][fA][mU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG], wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.

16. The RNAi agent of claim 15, wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of [mUs][mUs][mG][mU][mC][mU][mA][mU][fG][fG][fC][fU][mU][mA][mU][mU][mC][mU][mU][mG][mAs] (SEQ ID NO: 1202), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.

17. The RNAi agent of claim 16, further comprising a targeting moiety.

18. The RNAi agent of claim 17, wherein the targeting moiety is covalently linked to the 3 end of the sense strand of the RNAi agent.

19. The RNAi agent of claim 17, wherein the targeting moiety comprises N-acetyl-galactosamine (GalNAc).

20. The RNAi agent of claim 17, wherein the targeting moiety comprises one or more instances of GalNAc attached through a monovalent, bivalent, trivalent, or tetravalent branched linker.

21. The RNAi agent of claim 18, wherein the targeting moiety is of formula: ##STR00166## or a pharmaceutically acceptable salt thereof, wherein the ##STR00167## indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand.

22. A conjugate comprising a Cytochrome P450 family 7 subfamily A member 1 (CYP7A1) RNAi agent covalently linked to a targeting moiety, wherein the CYP7A1 RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][fA][mA][fG][fA][fA][fU][mA][fA][mG][mC][mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2029); and wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of [mUs][mUs][mG][mU][mC][mU][mA][mU][fG][fG][fC][fU][mU][mA][mU][mU][mC][mU][mU][mG][mAs] (SEQ ID NO: 1202); wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage; wherein the targeting moiety is covalently linked to the 3 terminal nucleoside of the sense strand and the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of a first repeat unit of the targeting moiety, wherein the conjugate comprises a structure of: ##STR00168## or a pharmaceutically acceptable salt thereof.

23. A pharmaceutical composition for inhibiting expression of a gene encoding CYP7A1 comprising the conjugate of claim 22, or a pharmaceutically acceptable salt thereof.

24. A method of inhibiting expression of a CYP7A1 gene in a cell, the method comprising contacting the cell with the conjugate of claim 22, thereby inhibiting expression of the CYP7A1 gene in the cell.

25. A method of treating a subject having a CYP7A1 disease or CYP7A1-associated disease, comprising administering to the subject a therapeutically effective amount of the conjugate of claim 22, thereby treating the subject having the CYP7A1 disease or CYP7A1-associated disease.

26. The method of claim 25, wherein the CYP7A1 disease or CYP7A1-associated disease is primary sclerosing cholangitis (PSC).

27. The method of claim 25, wherein the subject is human.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The foregoing and other features and advantages of the present invention, as well as the invention itself, will be more fully understood from the following description of embodiments when read together with the accompanying drawings, in which:

(2) FIG. 1 is a schematic that illustrates 4 modification patterns (MOD 1, MOD 2, MOD 3, and MOD 4) used in siRNA synthesized for in vivo study. As used in FIG. 1, mN=a 2-O-methyl (2-O-Me) modified nucleoside, fN=a 2-fluoro (2-F) modified nucleoside, The bolded | between two nucleosides means phosphorothioate internucleoside linkage. Chemically modified siRNAs having the modification patterns illustrated in FIG. 1 may further be conjugated to targeting moieties (e.g., targeting moieties comprising GalNAc). For example, the targeting moiety having a structure of Formula (Z.sup.6) may be conjugated to the 3 end nucleoside of the sense strand of such an siRNA.

DETAILED DESCRIPTION OF INVENTION

(3) Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedence over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of or means and/or unless stated otherwise. The use of the term including, as well as other forms of the term, is not limiting.

(4) Generally, nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as otherwise described herein. The nomenclatures, laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients. Any of the methods for gene therapy available in the art can be used in the methods provided herein. For general reviews of the methods of gene therapy, see Goldspiel et al. (1993) Clin. Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) Science 260:926-932; Morgan and Anderson (1993) Ann. Rev. Biochem. 62:191-217; and May (1993) TIBTECH 11(5):155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed-description of various methods of gene therapy are disclosed in US Patent Publication No. US20050042664.

Definitions

(5) In order that the present disclosure may be more readily understood, certain terms are first defined. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

(6) In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure.

(7) As used herein, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise. And as used herein is interchangeably used with or unless expressly stated otherwise. The terms comprising, having, including, and containing are to be construed as open-ended terms (i.e., meaning including, but not limited to) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited.

(8) The term about or approximately, as applied to one or more values provided herein, refers to a value that is similar to a stated reference value. In some embodiments, the term about or approximately refers to a range of values that fall within and include 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context. In some embodiments, about or approximately can be understood as about 2 standard deviations from the mean. In some embodiments, about or approximately means up to and including 10% (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less). In some embodiments, about or approximately means 5%. When about or approximately is present before a series of numbers or a range, it is understood that it can modify each of the numbers in the series or range.

(9) The term administering or administration of, as used herein, means to provide an agent, e.g., RNAi agent or conjugate to a subject. In some embodiments, administering or administration of means to provide an RNAi agent or conjugate to a subject in a manner that is physiologically and/or (e.g., and) pharmacologically useful (e.g., to treat a condition in the subject). Non-limiting examples of routes of administration include intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the route of administration is subcutaneous.

(10) The term antisense strand or guide strand, as used herein, refers to a single stranded nucleic acid molecule, which is one strand of a double stranded RNAi molecule, and which comprises a region of complementarity to a target sequence (e.g., a target gene sequence, RNA sequence, or mRNA sequence). The antisense strand may contain modified nucleosides with base analogs and is not necessarily 100% complementary over its entire length to the target sequence, but must at least be sufficiently complementary to hybridize with a target RNA and result in RNA interference.

(11) The term at least prior to a number or series of numbers is understood to include the number adjacent to the term at least, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides or nucleosides in a nucleic acid molecule must be an integer. For example, at least 19 nucleotides of a 21 nucleotide nucleic acid molecule means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that at least can modify each of the numbers in the series or range.

(12) The term biological activity means any biological property of a molecule, whether present naturally in vivo, or provided or enabled by recombinant means. Biological activities include, but are not limited to, binding to a receptor, inducing cell proliferation, inhibiting cell growth, inducing other cytokines, inducing apoptosis, and enzymatic activity.

(13) Blunt or blunt end means that there are no unpaired nucleosides at that end of a double stranded RNAi agent, i.e., no nucleoside overhang. A blunt ended double stranded RNAi agent is double stranded over its entire length, i.e., no nucleoside overhang at either end of the molecule. The RNAi agents of the disclosure include RNAi agents with no nucleoside overhang at one end (i.e., agents with one overhang and one blunt end) or with no nucleoside overhangs at either end.

(14) The term complementary, as used herein, refers to the capacity for base pairing between two nucleobases or two nucleobase sequences. In particular, complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleobases or two nucleobase sequences. For example, if a base at one position of a nucleobase sequence (e.g., antisense strand of an RNAi agent) is capable of hydrogen bonding with a base at the corresponding position of another nucleobase sequence (e.g., RNAi agent sense strand or target mRNA), then the bases are considered to be complementary to each other at that position. The nucleic acid molecules (e.g., antisense strand and sense strand of an RNAi agent) whose nucleobase sequences are complementary may comprise one or more modified nucleosides and modified internucleoside linkages, which do not affect the capacity of base paring between the nucleobases and do not affect the complementarity between two nucleobase sequences. The nucleic acid molecules (e.g., antisense strand and sense strand of an RNAi agent) whose nucleobase sequences are complementary may also comprise nucleobase analogous that result in bases at certain positions not being complementary, but the nucleobase sequences of the two molecules must be sufficiently complementary over the entire length to result in a desired biological activity (e.g., RNA interference).

(15) Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs (e.g., Wobble base pairs and Hoogsteen base pairs) and may include natural or modified nucleosides or nucleoside mimics. For example, in some embodiments, for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.

(16) Complementarity is independent of modifications in the sugar of a nucleoside. For example, 2-modified A, as defined herein, are complementary to U (or T) and identical to A for the purposes of determining identity or complementarity.

(17) The term perfectly complementary or fully complementary means that all (100%) of the nucleobases, nucleosides, or nucleotides in a contiguous sequence of a first nucleotide sequence (e.g., antisense strand of an RNAi agent) will hybridize with the same number of nucleobases, nucleosides, or nucleotides in a contiguous sequence of second nucleotide sequence (e.g., RNAi agent sense strand or target mRNA). The contiguous sequence may comprise all or a part of a first or second nucleotide sequence. The term partially complementary means that in a hybridized pair of nucleobase, nucleosides, or nucleotide sequences, at least 70%, but not all, of the bases in a contiguous sequence of a first nucleotide sequence (e.g., antisense strand of an RNAi agent) will hybridize with the same number of bases in a contiguous sequence of a second nucleotide sequence (e.g., RNAi agent sense strand or target mRNA). The term sufficiently complementary or substantially complementary means that in a hybridized pair of nucleobase, nucleosides, or nucleotide sequences, at least 85%, but not all, of the bases in a contiguous sequence of a first nucleotide sequence (e.g., antisense strand of an RNAi agent) will hybridize with the same number of bases in a contiguous sequence of a second nucleotide sequence (e.g., RNAi agent sense strand or target mRNA). The terms complementary, fully complementary, partially complementary, and sufficiently/substantially complementary herein are used with respect to the nucleobase, nucleosides, or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a target mRNA sequence (e.g., CYP7A1 mRNA).

(18) The term conjugate, as used herein, refers to an RNAi agent described herein (e.g., a CYP7A1 RNAi agent) linked (e.g., covalently linked) to a conjugate group (e.g., a targeting moiety). In general, conjugate groups modify one or more properties of the RNAi agent to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

(19) The term contiguous in the context of an oligonucleotide (e.g., RNAi agent) refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, contiguous nucleobases means nucleobases that are immediately adjacent to each other in a sequence.

(20) The term control or reference, when referring to a substance, means a composition used as a standard or a point of comparison against which other test results are measured. In some embodiments, a control or reference is a composition known to not contain analyte (negative control) or to contain analyte (positive control). A positive control can comprise a known concentration of analyte. Control, and positive control, may be used to refer to a composition comprising a known concentration of analyte. A positive control can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (e.g., analytes). In some embodiments, an appropriate control or reference is where only one element is changed in order to determine the effect of the one element. In some embodiments, a control is a level of a target gene (e.g., in a cell or in a subject) before treatment (e.g., with an RNAi agent described herein).

(21) The term control or reference also means a baseline level of a measurement depending upon the context, in which the term is used. A baseline level of a measurement is a standard or a point of comparison against which the measurement is compared. In some embodiments, a control or a reference refers to a level of a measurement for certain biological activity or substance in a cell, a tissue, an organ, or a subject, e.g., the expression level of a gene, copy number of mRNA for such gene, or level of protein encoded by such gene, without treatment of the cell, the tissue, the organ, or the subject, with an agent, e.g., an RNAi agent. In some embodiments, a control or a reference refers to a level of an average measurement for a certain biological activity or substance in a cell, a tissue, an organ, or a subject, e.g., certain enzyme activity of the liver, among a group of healthy subjects, e.g., the general population within in certain geographic or demographic limits or any other limits that may be appropriate for the study of certain disease or disorder, that does not have certain disease or disorder, e.g., liver disease.

(22) The term reference may also be used in reference sequence. The term reference sequence refers to a sequence, e.g., a nucleic acid sequence or an amino acid sequence, used as a basis for sequence comparison. In certain embodiments, a reference sequence is the mRNA sequence, e.g., human CYP7A1 mRNA sequence (NM_000780.4, SEQ ID NO: 1), upon which the design of the siRNA is based.

(23) The term cross-reactive means the ability of a binding molecule (e.g., an RNAi agent) to bind a target molecule (e.g., an mRNA) other than that against which it was designed or generated. For example, the binding molecule is capable of specifically binding to more than one target molecule of a similar type or class (e.g., mRNA variants or mRNA homologus from closely related species) with similar affinity. Generally, a binding molecule will bind its target molecule with an appropriately high affinity, but can bind to the same target molecule of another species or display a low affinity for non-target molecules. In some embodiments, an RNAi agent that is cross-reactive against human and non-human primate CYP7A1 comprises an antisense strand comprising a region of complementarity to human and non-human primate CYP7A1 mRNA, and/or inhibits the expression of human and non-human primate CYP7A1. Individual binding molecules are generally selected to meet two criteria: (1) tissue staining appropriate for the known expression of the target or (2) similar staining pattern between human and tox species (mouse and cynomolgus monkey) tissues from the same organ. These and other methods of assessing cross-reactivity are known to one skilled in the art.

(24) The term effective amount or therapeutically effective amount, as used herein, refers to that amount of an RNAi agent to produce a molecular (e.g., reduced expression of CYP7A1), biological (e.g., reduced accumulation of toxic bile acids), pharmacological, therapeutic (e.g., treatment of a CYP7A1 associated disease), or preventive result. The amount administered will likely depend on such variables as the overall health status of the patient, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can, in some instances, be increased beyond the above upper level to rapidly achieve the desired blood-level or tissue level, or the initial dosage can, in some instances, be smaller than the optimum.

(25) The term GalNAc refers to N-Acetylgalactosamine (GalNAc), which is a monosaccharide and amino sugar derivative of galactose. GalNAc may also be referred to in the art as 2-(Acetylamino)-2-deoxy-D-galactopyranose, 2-(Acetylamino)-2-deoxy-D-galactose, N-Acetylchondrosamine, and N-Acetyl-D-galactosamine. Galactose derivatives such as GalNAc have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor expressed on the surface of hepatocytes. Binding of asialoglycoprotein receptor ligands to the asialoglycoprotein receptor(s) facilitates cell-specific targeting to target cells (e.g., hepatocytes) and endocytosis of the molecule into the target cells (e.g., hepatocytes). In some embodiments, any one of the targeting moieties described herein includes an asialoglycoprotein receptor ligand comprising GalNAc. In some embodiments, the asialoglycoprotein receptor ligand comprises a GalNAc trimer. Asialoglycoprotein receptor ligands can be monomeric (e.g., having a single GalNAc) or multimeric (e.g., having multiple GalNAcs). The targeting moiety may comprise one or more GalNAcs attached to the 3 or 5 end of the sense or antisense strand of the RNAi agent using methods known in the art. In some embodiments, the targeting moiety comprises one or more (e.g., 1, 2, 3, 4, or more) GalNAc, each of which are linked via phosphorothioate linkages. GalNAc targeting moieties, which comprise one or more GalNAc, have been described, for example, in the following references:

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The disclosures in these references related to GalNAc are hereby incorporated herein by reference.

(27) The terms hybridize and hybridization refer to the pairing of complementary compounds (e.g., an RNAi agent and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Wobble, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

(28) The term internucleoside linkage, as used herein, means a covalent linkage between adjacent nucleosides in an oligonucleotide (e.g., RNAi agent such as a CYP7A1 RNAi agent described herein). An internucleoside linkage may be a natural phosphodiester internucleoside linkage, or may be a modified (non-natural) internucleoside linkage. Modified internucleoside that may be used in an RNAi agent disclosed herein include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3-amino phosphoramidate and aminoalkylphosphoramidates, mesyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3-5 linkages, 2-5 linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3-5 to 5-3 or 2-5 to 5-2; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

(29) The term linker in the context of a conjugate refers to a bond (e.g., covalent bond) or chemical moiety between two components of the conjugate, e.g., between an RNAi agent and a targeting moiety. In some embodiments, the linker facilitates covalent linkage of an RNAi agent to a targeting moiety. In some embodiments, the linker is conjugated to the 5 or 3end of the sense strand or antisense strand of an RNAi agent. In some embodiments, the linker is conjugated to the 5 or 3 end the sense strand of an RNAi agent. In some embodiments, a linker is conjugated to the 5 end of the sense strand of an RNAi agent. In some embodiments, a linker is conjugated to the 3 end of the sense strand of an RNAi agent. The linker can be any suitable group for coupling the RNAi agent to the ligand. In some embodiments, the linker is a monovalent, bivalent, trivalent, or tetravalent branched linker. In some embodiments, the linker is a phosphorus-containing linker (e.g., phosphate, phosphodiester, phosphorothioate, phosphorodithioate, phosphoroamidate, etc), or other linker. Non-limiting examples of other linkers can include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleotides, ribitol (abasic ribose), and/or PEG groups. In some embodiments, the linker is a phosphorothioate linkage.

(30) The term, nucleoside, as used herein, refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety. The term nucleoside encompasses a natural nucleoside and chemically modified nucleosides (e.g., with modifications in the base and/or sugar moiety).

(31) The term nucleotide, as used herein, refers to a compound comprising a nucleoside linked to a phosphate group. As used herein, linked nucleosides may or may not be linked by phosphate linkages and thus includes, but is not limited to linked nucleotides. As used herein, linked nucleosides are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are present between those that are linked). The term nucleotide encompasses a natural nucleotide and chemically modified nucleotides (e.g., with modifications in the base, sugar moiety, and/or phosphate group).

(32) The term nucleobase, as used herein, refers to nitrogen-containing compounds that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the compound is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified. As used herein a naturally occurring nucleobase is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). The term nucleobase encompasses 5-methylated bases (e.g., 5-methyl cytosine or 5-methyl guanine).

(33) The term modified internucleoside linkage refers to a linkage between two nucleosides (e.g., in an oligonucleotide or in a strand of an RNAi agent) that is not the natural phosphodiester linkage. Non-limiting examples of modified internucleoside linkages include phosphorothioates, phosphorodiamidates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.

(34) The term nucleoside modification or modified nucleoside means a nucleoside that has one or more modifications to the nucleoside, including modifications to the nucleobase moiety and/or the sugar moiety. Any of the modified chemistries or formats of nucleosides described herein can be combined with each other. Non-limiting examples of modified nucleosides includes 2-fluoro (2-F), 2-O-methyl (2-O-Me), 2-O-methoxyethyl (2-MOE), 2-O-aminopropyl (2-O-AP), 2-O-dimethylaminoethyl (2-O-DMAOE), 2-O-dimethylaminopropyl (2-O-DMAP), 2-O-dimethylaminoethyloxyethyl (2-O-DMAEOE), or 2-ON-methylacetamido (2-O-NMA), locked nucleic acid (LNA, methylene-bridged nucleic acid), unlocked nucleic acid (UNA), ethylene-bridged nucleic acid (ENA), and (S)-constrained ethyl-bridged nucleic acid (cEt) modified nucleosides. Further non-limiting examples of modified nucleosides include a conformationally restricted nucleoside, an abasic nucleoside, a 2-amino-modified nucleoside, a morpholino nucleoside, a phosphoramidate, a non-natural base comprising nucleoside, a tetrahydropyran modified nucleoside, a 1,5-anhydrohexitol modified nucleoside (HNA), a cyclohexenyl modified nucleoside (CeNA), a nucleoside comprising a phosphorothioate group, a nucleoside comprising a methylphosphonate group, a nucleoside comprising a 5-phosphate, a nucleoside comprising a 5-phosphate mimic, a thermally destabilizing nucleoside, a glycol modified nucleoside (GNA).

(35) The term 2-modified nucleoside refers to a nucleoside having a sugar moiety modified at the 2 position, meaning the sugar moiety comprises at least one 2-substituent group other than H or OH. Non-limiting examples of 2-modified nucleosides include: 2-fluoro (2-F), 2-O-methyl (2-O-Me), 2-O-methoxyethyl (2-MOE), 2-deoxy, 2-O-aminopropyl (2-O-AP), 2-O-dimethylaminoethyl (2-O-DMAOE), 2-O-dimethylaminopropyl (2-O-DMAP), 2-O-dimethylaminoethyloxyethyl (2-O-DMAEOE), or 2-ON-methylacetamido (2-O-NMA) modified nucleosides. In some embodiments, any one of the 2-modified nucleosides described herein are high-affinity modified nucleosides and a modified RNAi agent has increased affinity to target sequences, relative to an unmodified RNAi agent. In some embodiments, at least one modified nucleoside is a 2 modified nucleoside. In some embodiments the 2 modified nucleoside is a 2-O-methyl (2-O-Me) modified nucleoside or a 2-fluoro (2-F) modified nucleoside or combinations thereof.

(36) The term modified oligonucleotide or modified RNAi agent or modified siRNA refers to oligonucleotides, modified RNAi agents, or modified siRNAs that comprise one or more modified nucleosides and/or one or more modified internucleoside linkages. In some embodiments, a modified oligonucleotide or modified RNAi agent or modified siRNA comprises a mix of modified nucleosides and unmodified nucleosides and/or a mix of modified internucleoside linkages and unmodified modified internucleoside linkages. In some embodiments, each nucleoside of a modified, oligonucleotide or modified RNAi agent or modified siRNA is a modified nucleoside, and/or each internucleoside linkage of a modified oligonucleotide or modified RNAi agent or modified siRNA is a modified internucleoside linkage.

(37) As used herein, the term nucleoside overhang or overhang refers to at least one unpaired nucleoside that protrudes from the duplex structure of an RNAi agent. For example, when a 3-end of one strand of an RNAi agent extends beyond the 5-end of the other strand, or vice versa, there is a nucleoside overhang. An RNAi agent can comprise an overhang of at least one nucleoside, at least two nucleosides, at least three nucleosides, at least four nucleosides, or more. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleoside(s) of an overhang can be present on the 5-end, 3-end, or both ends of either an antisense or sense strand of an RNAi agent.

(38) In some embodiments, at least one strand of an RNAi agent comprises a 3 overhang of at least 1 nucleoside. In some embodiments, at least one strand comprises a 3 overhang of at least 2 nucleosides, e.g., 2, 3, 4, 5, etc. nucleosides. In some embodiments, at least one strand of an RNAi agent comprises a 5 overhang of at least 1 nucleoside. In some embodiments, at least one strand comprises a 5 overhang of at least 2 nucleosides, e.g., 2, 3, 4, 5, etc. nucleosides. In some embodiments, both the 3 and the 5 end of one strand of an RNAi agent comprise an overhang of at least 1 nucleoside.

(39) In some embodiments, the antisense strand of an RNAi agent has a 1-10 nucleoside, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside, overhang at the 3-end or the 5-end. In some embodiments, the sense strand of an RNAi agent has a 1-10 nucleoside, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside, overhang at the 3-end or the 5-end. In another embodiment, one or more of the nucleosides in the overhang is replaced with a nucleoside thiophosphate.

(40) In some embodiments, the antisense strand of an RNAi agent has a 1-10 nucleosides, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleoside, overhang at the 3-end or the 5-end. In some embodiments, the antisense strand of an RNAi agent has a 1-3 nucleosides, e.g., a 1, 2, or 3 nucleoside, overhang at the 3-end.

(41) In some embodiments, the nucleosides in an overhang that is immediately adjacent to the duplex region is linked to the adjacent nucleoside in the duplex region via a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage. In some embodiments, the overhang comprises two or more nucleosides and the nucleosides in an overhang are linked via a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage.

(42) The term passenger strand or sense strand, as used herein, refers to a single stranded nucleic acid molecule which is one strand of a double stranded RNAi molecule, and which has a sequence that is at least substantially complementary (e.g., at least 85% complementary) to that of the guide strand/antisense strand. The sense strand need not be fully complementary over the entire length of the antisense strand, but must at least be sufficiently complementary to hybridize with the antisense strand and result in RNA interference.

(43) The term repeat unit refers to a part of a compound whose repetition would produce the complete compound (except for the end-groups, e.g., the RNAi agent and R.sup.4 as disclosed herein) by linking the repeat units together successively along the chain.

(44) The term region of complementarity, as used herein, refers to a nucleobase sequence, e.g., of an RNAi agent, that is sufficiently complementary to a cognate nucleobase sequence, e.g., of a target nucleic acid, such that the two nucleobase sequences are capable of annealing to one another under physiological conditions (e.g., in a cell). In some embodiments, a region of complementarity is fully complementary to a cognate nucleobase sequence of target nucleic acid. However, in some embodiments, a region of complementarity is partially complementary to a cognate nucleobase sequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementarity). In some embodiments, a region of complementarity contains 1, 2, 3, 4, or 5 mismatches compared with a cognate nucleobase sequence of a target nucleic acid.

(45) The term RNAi agent, or RNA interference agent means a composition that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents may operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. An RNAi agent modulates, e.g., inhibits, the expression of a CYP7A1 in a cell, e.g., a cell within a subject, such as a mammalian subject. RNAi agents include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. Any one of the RNAi agents described herein comprises a strand that is at least partially complementary to the mRNA being targeted. In some embodiments, an RNAi agent is single stranded (e.g., it can be an antisense oligonucleotide). In some embodiments, an RNAi agent is double stranded. In some embodiments, the double stranded RNAi agent is a double stranded siRNA.

(46) In some embodiments, an RNAi agent described herein is double-stranded, and comprises an antisense strand and a sense strand, wherein the antisense strand is at least partially complementary to the mRNA being targeted (e.g., CYP7A1 mRNA), and the sense strand is at least partially complementary to the antisense strand. It is not necessary that there be perfect complementarity between the RNAi agent and the target, but the correspondence is preferably sufficient to enable the RNAi agent to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., CYP7A1 mRNA. An RNAi agent described herein may comprise one or more modified nucleosides and/or one or more modified (e.g., non-phosphodiester) internucleoside linkages.

(47) Modification to stabilize one or more 3- or 5-terminus of an RNAi agent, e.g., against exonucleases may also be present in an RNAi agent described herein. Other modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis. Modifications can also include, e.g., the use of modifications at the 2 OH group of the ribose sugar, e.g., the use of deoxyribonucleosides, e.g., deoxythymidine, instead of ribonucleosides, and modifications in the internucleoside linkages, e.g., phosphothioate internucleoside linkages. In some embodiments, the different strands will include different modifications. In some embodiments, an RNAi agent of the disclosure includes a short interfering RNA (siRNA) that interacts with a target RNA sequence, e.g., a CYP7A1 target sequence, to direct the cleavage of the target RNA. In some embodiments, an RNAi agent described herein is a small interfering RNA (siRNA).

(48) The term siRNA, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary (e.g., at least 85% complementary) nucleic acid strands, referred to as having sense and antisense orientations with respect to a target sequence, i.e., a CYP7A1 sequence. Each strand of the siRNA may optionally and independently comprise ribonucleosides (RNA), RNA analog(s) (e.g., chemically modified ribonucleosides), and/or deoxyribonucleosides (DNA). Each strand of an siRNA comprises between 15 and 30 nucleosides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides). In some embodiments, each strand of an siRNA comprises between 18 and 28 nucleosides (e.g., 18-28, 19-25, 19-23, 19-21). In some embodiments, each strand of an siRNA is 19, 20, 21, 22, or 23 nucleosides in length. In some embodiments of the disclosure, an siRNA induces the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

(49) In some embodiments, any one of the CYP7A1 RNAi agents (e.g., siRNAs) disclosed herein comprises a duplex region of 10-30 base pairs in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length). In some embodiments, an RNAi agent of the present disclosure is blunt ended. In some embodiments, an RNAi agent of the present disclosure has overhangs on one or both strands. The overhang may include 1-10 (e.g., 1-10, 1-8, 1-5, 1-3, 1-2) nucleosides, such that the duplex region in the RNAi agent comprises 17-21 nucleosides, or 19 nucleosides. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.

(50) Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3 overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

(51) In one aspect, the disclosure relates to a single stranded RNA generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., CYP7A1 gene. In some embodiments, an RNAi agent may be a single-stranded RNA (ssRNAi) that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded RNAi agents are generally 15-30 nucleosides and may be chemically modified. The design and testing of single-stranded RNAi agents are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference.

(52) The term sequence identity, as used herein, refers to the extent that sequences are identical (independent of chemical modification) on a nucleobase-by-nucleobase basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a percentage of sequence identity may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

(53) The terms silence, reduce, inhibit, down-regulate, or knockdown when referring to expression of a given gene (e.g., CYP7A1), mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with an RNAi agent or conjugate described herein as compared to a control or reference cell, group of cells, tissue, organ or a subject, e.g., a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated. In some embodiments, when a cell, group of cells, tissue, organ, or subject is treated with an RNAi agent or conjugate described herein, expression of a target gene (e.g., CYP7A1) is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to a control, e.g., baseline level of gene expression prior to treatment.

(54) The term subject, as used herein, refers to a mammal. In some embodiments, a subject is non-human primate, or rodent. In some embodiments, a subject is a human. In some embodiments, a subject is a patient, e.g., a human patient that has or is suspected of having a disease. In some embodiments, the subject is a human patient who has or is suspected of having a CYP7A1 disease or CYP7A1-associated disease.

(55) The term specificity means the ability to inhibit the target RNA without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).

(56) The term symptom as used herein, refers to any manifestation or indication of an underly disease. A symptom can be any biochemical, cellular, genetic, histological, and/or physiological observation, measurement, and/or test result in a subject that deviate from those of a control or reference. For example, a symptom may be an elevated level of a liver enzyme, such as aminotransferases, as compared to a normal reference range.

(57) The term target sequence, as used herein, refers to a nucleoside sequence whose expression or activity is to be modulated. In some embodiments, the target sequence is a contiguous portion of the nucleoside sequence of a gene, a cDNA, or an mRNA molecule formed during the transcription of a target gene, e.g., CYP7A1 gene, including a unprocessed pre-mRNA transcript and mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleoside sequence of an mRNA molecule formed during the transcription of the target gene, e.g., CYP7A1 gene. In some embodiment, the target sequence is within the protein coding region of the target gene, e.g., CYP7A1.

(58) The term treat, treatment, as used herein, mean the methods or steps taken to provide relief from or alleviation of the number, severity, and/or frequency of one or more symptoms of a disease (e.g., a CYP7A1 disease or CYP7A1-associated disease) in a subject. As used herein, treat and treatment may include the prevention, management, prophylactic treatment, and/or inhibition of the number, severity, and/or frequency of one or more symptoms of a disease (e.g., a CYP7A1 disease or CYP7A1-associated disease) in a subject.

(59) The term variant means a molecule (e.g., nucleic acid or polypeptide) that differs from a given molecule (e.g., a reference nucleic acid or polypeptide) in sequence (nucleic acid or amino acid respectively) by the addition (e.g., insertion), deletion, or conservative substitution of nucleic acids or amino acids, respectively, but that retains the biological activity of the given molecule. Nucleic acid variants are closely related overall and, in many regions, identical. Changes in the reference nucleic acid sequence of the variant may be silent. That is, they may not alter the amino acid sequence encoded by the nucleic acid. Alternatively, changes in the nucleoside sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleoside changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. The term variant encompasses fragments of a variant unless otherwise defined. A variant may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, or 75% identical to the reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTn with default settings).

(60) Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75.sup.th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5.sup.th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3.sup.rd Edition, Cambridge University Press, Cambridge, 1987.

(61) Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

(62) In a formula, is a single bond were the stereochemistry of the moieties immediately attached thereto is not specified, is absent or a single bond, and or is a single or double bond.

(63) Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of .sup.19F with .sup.18F, or the replacement of .sup.12C with .sup.13C or .sup.14C are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

(64) When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example C.sub.1-6 alkyl is intended to encompass, C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.1-6, C.sub.1-5, C.sub.1-4, C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-5, C.sub.2-4, C.sub.2-3, C.sub.3-6, C.sub.3-5, C.sub.3-4, C.sub.4-6, C.sub.4-5, and C.sub.5-6 alkyl.

(65) The term aliphatic refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term heteroaliphatic refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

(66) The term alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (C.sub.1-10 alkyl). In some embodiments, an alkyl group has 1 to 9 carbon atoms (C.sub.1-9 alkyl). In some embodiments, an alkyl group has 1 to 8 carbon atoms (C.sub.1-8 alkyl). In some embodiments, an alkyl group has 1 to 7 carbon atoms (C.sub.1-7 alkyl). In some embodiments, an alkyl group has 1 to 6 carbon atoms (C.sub.1-6 alkyl). In some embodiments, an alkyl group has 1 to 5 carbon atoms (C.sub.1-5 alkyl). In some embodiments, an alkyl group has 1 to 4 carbon atoms (C.sub.1-4 alkyl). In some embodiments, an alkyl group has 1 to 3 carbon atoms (C.sub.1-3 alkyl). In some embodiments, an alkyl group has 1 to 2 carbon atoms (C.sub.1-2 alkyl). In some embodiments, an alkyl group has 1 carbon atom (C.sub.1 alkyl). In some embodiments, an alkyl group has 2 to 6 carbon atoms (C.sub.2-6 alkyl). Examples of C.sub.1-6 alkyl groups include methyl (C.sub.1), ethyl (C.sub.2), propyl (C.sub.3) (e.g., n-propyl, isopropyl), butyl (C.sub.4) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C.sub.5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C.sub.6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C.sub.7), n-octyl (C.sub.8), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an unsubstituted alkyl) or substituted (a substituted alkyl) with one or more substituents (e.g., halogen, such as F). In certain embodiments, the alkyl group is an unsubstituted C.sub.1-10 alkyl (such as unsubstituted C.sub.1-6 alkyl, e.g., CH.sub.3.Math.(Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is a substituted C.sub.1-10 alkyl (such as substituted C.sub.1-6 alkyl, e.g., CF.sub.3, Bn).

(67) The term haloalkyl is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms (C.sub.1-8 haloalkyl). In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms (C.sub.1-6 haloalkyl). In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms (C.sub.1-4 haloalkyl). In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (C.sub.1-3 haloalkyl). In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (C.sub.1-2 haloalkyl). Examples of haloalkyl groups include CHF.sub.2, CH.sub.2F, CF.sub.3, CH.sub.2CF.sub.3, CF.sub.2CF.sub.3, CF.sub.2CF.sub.2CF.sub.3, CCl.sub.3, CFCl.sub.2, CF.sub.2Cl, and the like.

(68) The term heteroalkyl refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-20 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 18 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-8 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 16 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-16 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 14 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-14 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-12 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-10 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-8 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (heteroC.sub.1-6 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (heteroC.sub.1-4 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (heteroC.sub.1-3 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (heteroC.sub.1-2 alkyl). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (heteroC.sub.1 alkyl). In some embodiments, the heteroalkyl group defined herein is a partially unsaturated group having 1 or more heteroatoms within the parent chain and at least one unsaturated carbon, such as a carbonyl group. For example, a heteroalkyl group may comprise an amide or ester functionality in its parent chain such that one or more carbon atoms are unsaturated carbonyl groups. Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an unsubstituted heteroalkyl) or substituted (a substituted heteroalkyl) with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC.sub.1-20 alkyl. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC.sub.1-10 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC.sub.1-20 alkyl. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC.sub.1-10alkyl.

(69) The term alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (C.sub.2-9 alkenyl). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (C.sub.2-8 alkenyl). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (C.sub.2-7 alkenyl). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (C.sub.2-6 alkenyl). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (C.sub.2-5 alkenyl). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (C.sub.2-4 alkenyl). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (C.sub.2-3 alkenyl). In some embodiments, an alkenyl group has 2 carbon atoms (C.sub.2 alkenyl). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C.sub.2-4 alkenyl groups include ethenyl (C.sub.2), 1-propenyl (C.sub.3), 2-propenyl (C.sub.3), 1-butenyl (C.sub.4), 2-butenyl (C.sub.4), butadienyl (C.sub.4), and the like. Examples of C.sub.2-6 alkenyl groups include the aforementioned C.sub.2-4 alkenyl groups as well as pentenyl (C.sub.5), pentadienyl (C.sub.5), hexenyl (C.sub.6), and the like. Additional examples of alkenyl include heptenyl (C.sub.7), octenyl (C.sub.8), octatrienyl (C.sub.8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an unsubstituted alkenyl) or substituted (a substituted alkenyl) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C.sub.2-10 alkenyl. In certain embodiments, the alkenyl group is a substituted C.sub.2-10 alkenyl. In an alkenyl group, a CC double bond for which the stereochemistry is not specified (e.g., CHCHCH.sub.3 or

(70) ##STR00024##
may be an (E)- or (Z)-double bond.

(71) The term heteroalkenyl refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-10 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-9 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-8 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-7 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-6 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (heteroC.sub.2-5 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (heteroC.sub.2-4 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (heteroC.sub.2-3 alkenyl). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (heteroC.sub.2-6 alkenyl). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an unsubstituted heteroalkenyl) or substituted (a substituted heteroalkenyl) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC.sub.2-10 alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC.sub.2-10 alkenyl.

(72) The term alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (C.sub.2-10 alkynyl). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (C.sub.2-9 alkynyl). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (C.sub.2-8 alkynyl). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (C.sub.2-7 alkynyl). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (C.sub.2-6 alkynyl). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (C.sub.2-5 alkynyl). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (C.sub.2-4 alkynyl). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (C.sub.2-3 alkynyl). In some embodiments, an alkynyl group has 2 carbon atoms (C.sub.2 alkynyl). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C.sub.2-4 alkynyl groups include, without limitation, ethynyl (C.sub.2), 1-propynyl (C.sub.3), 2-propynyl (C.sub.3), 1-butynyl (C.sub.4), 2-butynyl (C.sub.4), and the like. Examples of C.sub.2-6 alkenyl groups include the aforementioned C.sub.2-4 alkynyl groups as well as pentynyl (C.sub.5), hexynyl (C.sub.6), and the like. Additional examples of alkynyl include heptynyl (C.sub.7), octynyl (C.sub.8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an unsubstituted alkynyl) or substituted (a substituted alkynyl) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C.sub.2-10 alkynyl. In certain embodiments, the alkynyl group is a substituted C.sub.2-10 alkynyl.

(73) The term heteroalkynyl refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-10 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-9 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-8 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-7 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (heteroC.sub.2-6 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (heteroC.sub.2-5 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and lor 2 heteroatoms within the parent chain (heteroC.sub.2-4 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (heteroC.sub.2-3 alkynyl). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (heteroC.sub.2-6 alkynyl). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an unsubstituted heteroalkynyl) or substituted (a substituted heteroalkynyl) with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC.sub.2-10 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC.sub.2-10 alkynyl.

(74) The term carbocyclyl or carbocyclic refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (C.sub.3-14 carbocyclyl) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (C.sub.3-10 carbocyclyl). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (C.sub.3-8 carbocyclyl). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (C.sub.3-7 carbocyclyl). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (C.sub.3-6 carbocyclyl). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (C.sub.4-6 carbocyclyl). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (C.sub.5-6 carbocyclyl). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (C.sub.5-10 carbocyclyl). Exemplary C.sub.3-6 carbocyclyl groups include, without limitation, cyclopropyl (C.sub.3), cyclopropenyl (C.sub.3), cyclobutyl (C.sub.4), cyclobutenyl (C.sub.4), cyclopentyl (C.sub.5), cyclopentenyl (C.sub.5), cyclohexyl (C.sub.6), cyclohexenyl (C.sub.6), cyclohexadienyl (C.sub.6), and the like. Exemplary C.sub.3-8 carbocyclyl groups include, without limitation, the aforementioned C.sub.3-6 carbocyclyl groups as well as cycloheptyl (C.sub.7), cycloheptenyl (C.sub.7), cycloheptadienyl (C.sub.7), cycloheptatrienyl (C.sub.7), cyclooctyl (C.sub.8), cyclooctenyl (C.sub.8), bicyclo[2.2.1]heptanyl (C.sub.7), bicyclo[2.2.2]octanyl (C), and the like. Exemplary C.sub.3-10 carbocyclyl groups include, without limitation, the aforementioned C.sub.3-8 carbocyclyl groups as well as cyclononyl (C.sub.9), cyclononenyl (C.sub.9), cyclodecyl (C.sub.10), cyclodecenyl (C.sub.10), octahydro-1H-indenyl (C.sub.9), decahydronaphthalenyl (C.sub.10), spiro[4.5]decanyl (C.sub.10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (monocyclic carbocyclyl) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (bicyclic carbocyclyl) or tricyclic system (tricyclic carbocyclyl)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. Carbocyclyl also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an unsubstituted carbocyclyl) or substituted (a substituted carbocyclyl) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C.sub.3-14 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C.sub.3-14 carbocyclyl.

(75) In some embodiments, carbocyclyl is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (C.sub.3-14 cycloalkyl). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (C.sub.3-10 cycloalkyl). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (C.sub.3-8 cycloalkyl). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (C.sub.3-6 cycloalkyl). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (C.sub.4-6 cycloalkyl). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (C.sub.5-6 cycloalkyl). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (C.sub.5-10 cycloalkyl). Examples of C.sub.5-6 cycloalkyl groups include cyclopentyl (C.sub.5) and cyclohexyl (C.sub.5). Examples of C.sub.3-6 cycloalkyl groups include the aforementioned C.sub.5-6 cycloalkyl groups as well as cyclopropyl (C.sub.3) and cyclobutyl (C.sub.4). Examples of C.sub.3-8 cycloalkyl groups include the aforementioned C.sub.3-6 cycloalkyl groups as well as cycloheptyl (C.sub.7) and cyclooctyl (C.sub.8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an unsubstituted cycloalkyl) or substituted (a substituted cycloalkyl) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C.sub.3-14 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C.sub.3-14 cycloalkyl.

(76) The term heterocyclyl or heterocyclic refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (3-14 membered heterocyclyl). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (monocyclic heterocyclyl) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (bicyclic heterocyclyl) or tricyclic system (tricyclic heterocyclyl)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an unsubstituted heterocyclyl) or substituted (a substituted heterocyclyl) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.

(77) In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5-10 membered heterocyclyl). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5-8 membered heterocyclyl). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5-6 membered heterocyclyl). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

(78) Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

(79) The term aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ?t electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (C.sub.6-14 aryl). In some embodiments, an aryl group has 6 ring carbon atoms (C.sub.6 aryl; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (C.sub.10 aryl; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (C.sub.14 aryl; e.g., anthracyl). Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an unsubstituted aryl) or substituted (a substituted aryl) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C.sub.6-14 aryl. In certain embodiments, the aryl group is a substituted C.sub.6-14 aryl.

(80) Aralkyl is a subset of alkyl and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.

(81) The term heteroaryl refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5-14 membered heteroaryl). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

(82) In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5-10 membered heteroaryl). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5-8 membered heteroaryl). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (5-6 membered heteroaryl). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an unsubstituted heteroaryl) or substituted (a substituted heteroaryl) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

(83) Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

(84) Heteroaralkyl is a subset of alkyl and refers to an alkyl group substituted by a heteroaryl group, wherein the point of attachment is on the alkyl moiety.

(85) The term unsaturated or partially unsaturated refers to a moiety that includes at least one double or triple bond.

(86) The term saturated refers to a moiety that does not contain a double or triple bond, i.e., the moiety only contains single bonds.

(87) Affixing the suffix -ene to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

(88) A group is optionally substituted unless expressly provided otherwise. The term optionally substituted refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl group). In general, the term substituted means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a substituted group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term substituted is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety. The disclosure is not intended to be limited in any manner by the exemplary substituents described herein.

(89) Exemplary carbon atom substituents include, but are not limited to, halogen, CN, NO.sub.2, N.sub.3, SO.sub.2H, SO.sub.3H, OH, OR.sup.aa, ON(R.sup.bb).sub.2, N(R.sup.bb).sub.2, N(R.sup.bb).sub.3.sup.+X.sup., N(OR.sup.cc)R.sup.bb, SH, SR.sup.aa, SSR.sup.cc, C(O)R.sup.aa, CO.sub.2H, CHO, C(OR.sup.cc).sub.3, CO.sub.2R.sup.aa, OC(O)R.sup.aa, OCO.sub.2R.sup.aa, C(O)N(R.sup.bb).sub.2, OC(O)N(R.sup.bb).sub.2, NR.sup.bbC(O)R.sup.aa, NR.sup.bbCO.sub.2R.sup.aa, NR.sup.bbC(O)N(R.sup.bb).sub.2, C(NR.sup.bb)R.sup.aa, C(NR.sup.bb)OR.sup.aa, OC(NR.sup.bb)R.sup.aa, OC(NR.sup.bb)OR.sup.aa, C(NR.sup.bb)N(R.sup.bb).sub.2, OC(NR.sup.bb)N(R.sup.bb).sub.2, NR.sup.bbC(NR.sup.bb)N(R.sup.bb).sub.2, C(O)NR.sup.bbSO.sub.2R.sup.aa, NR.sup.bbSO.sub.2R.sup.aa, SO.sub.2N(R.sup.bb).sub.2, SO.sub.2R.sup.aa, SO.sub.2OR.sup.aa, OSO.sub.2R.sup.aa, S(O)R.sup.aa, OS(O)R.sup.aa, Si(R.sup.aa).sub.3, OSi(R.sup.aa).sub.3C(S)N(R.sup.bb).sub.2, C(O)SR.sup.aa, C(S)SR.sup.aa, SC(S)SR.sup.aa, SC(O)SR.sup.aa, OC(O)SR.sup.aa, SC(O)OR.sup.aa, SC(O)R.sup.aa, P(O)(R.sup.aa).sub.2, P(O)(OR.sup.cc).sub.2, OP(O)(R.sup.aa).sub.2, OP(O)(OR.sup.cc).sub.2, P(O)(N(R.sup.bb).sub.2).sub.2, OP(O)(N(R.sup.bb).sub.2).sub.2, NR.sup.bbP(O)(R.sup.aa).sub.2, NR.sup.bbP(O)(OR.sup.cc).sub.2, NR.sup.bbP(O)(N(R.sup.bb).sub.2).sub.2, P(R.sup.cc).sub.2, P(OR.sup.cc).sub.2, P(R.sup.cc).sub.3.sup.+X.sup., P(OR.sup.cc).sub.3.sup.+X.sup., P(R.sup.cc).sub.4, P(OR.sup.cc).sub.4, OP(R.sup.cc).sub.2, OP(R.sup.cc).sub.3.sup.+X.sup., OP(OR.sup.cc).sub.2, OP(OR.sup.cc).sub.3.sup.+X.sup., OP(R.sup.cc).sub.4, OP(OR.sup.cc).sub.4, B(R.sup.aa).sub.2, B(OR.sup.cc).sub.2, BR.sup.aa(OR.sup.cc), C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; wherein X.sup. is a counterion; or two geminal hydrogens on a carbon atom are replaced with the group O, S, NN(R.sup.bb).sub.2, NNR.sup.bbC(O)R.sup.aa, NNR.sup.bbC(O)OR.sup.aa, NNR.sup.bbS(O).sub.2R.sup.aa, NR.sup.bb, or =NOR.sup.cc; each instance of R.sup.aa is, independently, selected from C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two R.sup.aa groups are joined to form a 3-14 membered-heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; each instance of R.sup.bb is, independently, selected from hydrogen, OH, OR.sup.aa, N(R.sup.cc).sub.2, CN, C(O)R.sup.aa, C(O)N(R.sup.cc).sub.2, CO.sub.2R.sup.aa, SO.sub.2R.sup.aa, C(NR.sup.cc)OR.sup.aa, C(NR.sup.cc)N(R.sup.cc).sub.2, SO.sub.2N(R.sup.cc).sub.2, SO.sub.2R.sup.cc, SO.sub.2OR.sup.cc, SOR.sup.aa, C(S)N(R.sup.cc).sub.2, C(O)SR.sup.cc, C(S)SR.sup.cc, P(O)(R.sup.aa).sub.2, P(O)(OR.sup.cc).sub.2, P(O)(N(R.sup.cc).sub.2).sub.2, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two R.sup.bb groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; wherein X.sup. is a counterion; each instance of R.sup.cc is, independently, selected from hydrogen, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two R.sup.cc groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; each instance of R.sup.dd is, independently, selected from halogen, CN, NO.sub.2, N.sub.3, SO.sub.2H, SO.sub.3H, OH, OR.sup.cc, ON(R.sup.ff).sub.2, N(R.sup.ff).sub.2, N(R.sup.ff).sub.3.sup.+X.sup., N(OR.sup.ee)R.sup.ff, SH, SR.sup.ee, SSR.sup.ee, C(O)R.sup.ee, CO.sub.2H, CO.sub.2R.sup.ee, OC(O)R.sup.ee, OCO.sub.2R.sup.ee, C(O)N(R.sup.ff).sub.2, OC(O)N(R.sup.ff).sub.2, NR.sup.ffC(O)R.sup.ee, NR.sup.ffCO.sub.2R.sup.ee, NR.sup.ffC(O)N(R.sup.ff).sub.2, C(NR.sup.ff)OR.sup.ee, OC(NR.sup.ff)R.sup.ee, OC(NR.sup.ff)OR.sup.ee, C(NR.sup.ff)N(R.sup.ff).sub.2, OC(NR.sup.ff)N(R.sup.ff).sub.2, NR.sup.ffC(NR.sup.ff)N(R.sup.ff).sub.2, NR.sup.ffSO.sub.2R.sup.ee, SO.sub.2N(R.sup.ff).sub.2, SO.sub.2R.sup.ee, SO.sub.2OR.sup.ee, OSO.sub.2R.sup.ee, S(O)R.sup.ee, Si(R.sup.ee).sub.3, OSi(R.sup.ee).sub.3, C(S)N(R.sup.ff).sub.2, C(O)SR.sup.ee, C(S)SR.sup.ee, SC(S)SR.sup.ee, P(O)(OR.sup.ee).sub.2, P(O)(R.sup.ee).sub.2, OP(O)(R.sup.ee).sub.2, OP(O)(OR.sup.ee).sub.2, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6 alkyl, heteroC.sub.2-6 alkenyl, heteroC.sub.2-6 alkynyl, C.sub.3-10 carbocyclyl, 3-10 membered heterocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups, or two geminal R.sup.dd substituents can be joined to form O or =S; wherein X.sup. is a counterion; each instance of R.sup.ee is, independently, selected from C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6 alkyl, heteroC.sub.2-6 alkenyl, heteroC.sub.2-6 alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups; each instance of R.sup.ff is, independently, selected from hydrogen, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6 alkyl, heteroC.sub.2-6 alkenyl, heteroC.sub.2-6 alkynyl, C.sub.3-10 carbocyclyl, 3-10 membered heterocyclyl, C.sub.6-10 aryl and 5-10 membered heteroaryl, or two R.sup.ff groups are joined to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl, ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups; and each instance of R.sup.gg is, independently, halogen, CN, NO.sub.2, N.sub.3, SO.sub.2H, SO.sub.3H, OH, OC.sub.1-6 alkyl, ON(C.sub.1-6 alkyl).sub.2, N(C.sub.1-6 alkyl).sub.2, N(C.sub.1-6 alkyl).sub.3.sup.+X.sup., NH(C.sub.1-6 alkyl).sub.2.sup.+X.sup., NH.sub.2(C.sub.1-6 alkyl).sub.3.sup.+X.sup., NH.sub.3.sup.+X.sup., N(OC.sub.1-6 alkyl)(C.sub.1-6 alkyl), N(OH)(C.sub.1-6 alkyl), NH(OH), SH, SC.sub.1-6 alkyl, SS(C.sub.1-6 alkyl), C(O)(C.sub.1-6 alkyl), CO.sub.2H, CO.sub.2(C.sub.1-6 alkyl), OC(O)(C.sub.1-6 alkyl), OCO.sub.2(C.sub.1-6 alkyl), C(O)NH.sub.2, C(O)N(C.sub.1-6 alkyl).sub.2, OC(O)NH(C.sub.1-6 alkyl), NHC(O)(C.sub.1-6 alkyl), N(C.sub.1-6 alkyl)C(O)(C.sub.1-6 alkyl), NHCO.sub.2(C.sub.1-6 alkyl), NHC(O)N(C.sub.1-6 alkyl).sub.2, NHC(O)NH(C.sub.1-6 alkyl), NHC(O)NH.sub.2, C(NH)O(C.sub.1-6 alkyl), OC(NH)(C.sub.1-6 alkyl), OC(NH)OC.sub.1-6 alkyl, C(NH)N(C.sub.1-6 alkyl).sub.2, C(NH)NH(C.sub.1-6 alkyl), C(NH)NH.sub.2, OC(NH)N(C.sub.1-6 alkyl).sub.2, OC(NH)NH(C.sub.1-6 alkyl), OC(NH)NH.sub.2, NHC(NH)N(C.sub.1-6 alkyl).sub.2, NHC(NH)NH.sub.2, NHSO.sub.2(C.sub.1-6 alkyl), SO.sub.2N(C.sub.1-6 alkyl).sub.2, SO.sub.2NH(C.sub.1-6 alkyl), SO.sub.2NH.sub.2, SO.sub.2(C.sub.1-6 alkyl), SO.sub.2O(C.sub.1-6 alkyl), OSO.sub.2(C.sub.1-6 alkyl), SO(C.sub.1-6 alkyl), Si(C.sub.1-6 alkyl).sub.3, OSi(C.sub.1-6 alkyl).sub.3-C(S)N(C.sub.1-6 alkyl).sub.2, C(S)NH(C.sub.1-6 alkyl), C(S)NH.sub.2, C(O)S(C.sub.1-6 alkyl), C(S)SC.sub.1-6 alkyl, SC(S)SC.sub.1-6 alkyl, P(O)(OC.sub.1-6 alkyl).sub.2, P(O)(C.sub.1-6 alkyl).sub.2, OP(O)(C.sub.1-6 alkyl).sub.2, OP(O)(OC.sub.1-6 alkyl).sub.2, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6 alkyl, heteroC.sub.2-6 alkenyl, heteroC.sub.2-6 alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal R.sup.99 substituents can be joined to form O or =S; wherein X.sup. is a counterion.

(90) In certain embodiments, an exemplary carbon atom substituent is halogen (.e.g, F, Cl, Br, I), CN, NO.sub.2, N.sub.3, SO.sub.2H, SO.sub.3H, OH, O(C.sub.1-6 alkyl) (e.g., OMe), NH.sub.2, SH, S(C.sub.1-6 alkyl) (e.g., SMe), C(O)(C.sub.1-6 alkyl) (e.g., C(O)Me), CO.sub.2H, or CHO.

(91) The term halo or halogen refers to fluorine (fluoro, F), chlorine (chloro, C.sub.1), bromine (bromo, Br), or iodine (iodo, I).

(92) The term hydroxyl or hydroxy refers to the group OH. The term substituted hydroxyl or substituted hydroxyl, by extension, refers to a hydroxyl group wherein the oxygen atom directly attached to the parent molecule is substituted with a group other than hydrogen, and includes groups selected from OR.sup.aa, ON(R.sup.bb).sub.2, OC(O)SR.sup.aa, OC(O)R.sup.aa, OCO.sub.2R.sup.aa, OC(O)N(R.sup.bb).sub.2, OC(NR.sup.bb)R.sup.aa, OC(NR.sup.bb)OR.sup.aa, OC(NR.sup.bb)N(R.sup.bb).sub.2, OS(O)R.sup.aa, OSO.sub.2R.sup.aa, OSi(R.sup.aa).sub.3; OP(R.sup.cc).sub.2, OP(R.sup.cc).sub.3.sup.+X.sup., OP(OR.sup.cc).sub.2, OP(OR.sup.cc).sub.3.sup.+X.sup., OP(O)(R.sup.aa).sub.2, OP(O)(OR.sup.cc).sub.2, and OP(O)(N(R.sup.bb).sub.2).sub.2, wherein X.sup., R.sup.aa, R.sup.bb, and R.sup.cc are as defined herein.

(93) The term amino refers to the group NH.sub.2. The term substituted amino, by extension, refers to a monosubstituted amino, a disubstituted amino, or a trisubstituted amino. In certain embodiments, the substituted amino is a monosubstituted amino or a disubstituted amino group.

(94) The term monosubstituted amino refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with one hydrogen and one group other than hydrogen, and includes groups selected from NH(R.sup.bb), NHC(O)R.sup.aa, NHCO.sub.2R.sup.aa, NHC(O)N(R.sup.bb).sub.2, NHC(NR.sup.bb)N(R.sup.bb).sub.2, NHSO.sub.2R.sup.aa, NHP(O)(OR.sup.cc).sub.2, and NHP(O)(N(R.sup.bb).sub.2).sub.2, wherein R.sup.aa, R.sup.bb and R.sup.cc are as defined herein, and wherein R.sup.bb of the group NH(R.sup.bb) is not hydrogen.

(95) The term disubstituted amino refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with two groups other than hydrogen, and includes groups selected from N(R.sup.bb).sub.2, NR.sup.bbC(O)R.sup.aa, NR.sup.bbCO.sub.2R.sup.aa, NR.sup.bbC(O)N(R.sup.bb).sub.2, NR.sup.bbC(NR.sup.bb)N(R.sup.bb).sub.2, NR.sup.bbSO.sub.2R.sup.aa, NR.sup.bbP(O)(OR.sup.cc).sub.2, and NR.sup.bbP(O)(N(R.sup.bb).sub.2).sub.2, wherein R.sup.aa, R.sup.bb, and R.sup.cc are as defined herein, with the proviso that the nitrogen atom directly attached to the parent molecule is not substituted with hydrogen.

(96) The term trisubstituted amino refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with three groups, and includes groups selected from N(R.sup.bb).sub.3 and N(R.sup.bb).sub.3.sup.+X.sup., wherein R.sup.bb and X.sup. are as defined herein.

(97) The term acyl refers to a group having the general formula: C(O)R.sup.X1, C(O)OR.sup.X1, C(O)OC(O)R.sup.X1, C(O)SR.sup.X1, C(O)N(R.sup.X1).sub.2, C(S)R.sup.X1, C(S)N(R.sup.X1).sub.2, C(S)O(R.sup.X1), C(S)S(R.sup.X1), C(NR.sup.X1)R.sup.X1, C(NR.sup.X1)OR.sup.X1, C(NR.sup.X1)SR.sup.X1, or C(NR.sup.X1)N(R.sup.X1).sub.2, wherein R.sup.X1 is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino; or two R.sup.X1 groups taken together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (CHO), carboxylic acids (CO.sub.2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

(98) The term oxo refers to the group O, and the term thiooxo refers to the group S.

(99) Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, OH, OR.sup.aa, N(R.sup.cc).sub.2, CN, C(O)R.sup.aa, C(O)N(R.sup.cc).sub.2, CO.sub.2R.sup.aa, SO.sub.2R.sup.aa, C(NR.sup.bb)R.sup.aa, C(NR.sup.cc)OR.sup.aa, C(NR.sup.cc)N(R.sup.cc).sub.2, SO.sub.2N(R.sup.cc).sub.2, SO.sub.2R.sup.cc, SO.sub.2OR.sup.cc, SOR.sup.aa, C(S)N(R.sup.cc).sub.2, C(O)SR.sup.cc, C(S)SR.sup.cc, P(O)(OR.sup.cc).sub.2, P(O)(R.sup.cc).sub.2, P(O)(N(R.sup.cc).sub.2).sub.2, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10alkyl, heteroC.sub.2-10alkenyl, heteroC.sub.2-10alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two R.sup.cc groups attached to an N atom are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups, and wherein R.sup.aa, R.sup.bb, R.sup.cc and R.sup.dd are as defined herein.

(100) In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to herein as an amino protecting group). Nitrogen protecting groups include, but are not limited to, OH, OR.sup.aa, N(R.sup.cc).sub.2, C(O)R.sup.aa, C(O)N(R.sup.cc).sub.2, CO.sub.2R.sup.aa, SO.sub.2R.sup.aa, C(NR.sup.cc)R.sup.aa, C(NR.sup.cc)OR.sup.aa, C(NR.sup.cc)N(R.sup.cc).sub.2, SO.sub.2N(R.sup.cc).sub.2, SO.sub.2R.sup.cc, SO.sub.2OR.sup.cc, SOR.sup.aa, C(S)N(R.sup.cc).sub.2, C(O)SR.sup.cc, C(S)SR.sup.cc, C.sub.1-10 alkyl (e.g., aralkyl, heteroaralkyl), C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups, and wherein R.sup.aa, R.sup.bb, R.sup.cc and R.sup.dd are as defined herein. Nitrogen protecting groups are well known in the art and include 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, incorporated herein by reference.

(101) For example, nitrogen protecting groups such as amide groups (e.g., C(O)R.sup.aa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N-dithiobenzyloxyacylamino)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 and o-(benzoyloxymethyl)benzamide.

(102) Nitrogen protecting groups such as carbamate groups (e.g., C(O)OR.sup.aa) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl 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 or 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-nitrobenzyl 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, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl 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, isobornyl 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, and 2,4,6-trimethylbenzyl carbamate.

(103) Nitrogen protecting groups such as sulfonamide groups (e.g., S(O).sub.2R.sup.aa) include, but are not limited to, 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.

(104) Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N-p-toluenesulfonylaminoacyl derivative, N-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 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-pyrrolin-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(pentaacylchromium- or tungsten)acyl]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, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys). In certain embodiments, a nitrogen protecting group is benzyl (Bn), tert-butyloxycarbonyl (BOC), carbobenzyloxy (Cbz), 9-flurenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethyloxycarbonyl (Troc), triphenylmethyl (Tr), tosyl (Ts), brosyl (Bs), nosyl (Ns), mesyl (Ms), triflyl (Tf), or dansyl (Ds).

(105) In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an hydroxyl protecting group). Oxygen protecting groups include, but are not limited to, R.sup.aa, N(R.sup.bb).sub.2, C(O)SR.sup.aa, C(O)R.sup.aa, CO.sub.2R.sup.aa, C(O)N(R.sup.bb).sub.2, C(NR.sup.bb)R.sup.aa, C(NR.sup.bb)OR.sup.aa, C(NR.sup.bb)N(R.sup.bb).sub.2, S(O)R.sup.aa, SO.sub.2R.sup.aa, Si(R.sup.aa).sub.3, P(R.sup.cc).sub.2, P(R.sup.cc).sub.3.sup.+X.sup., P(OR.sup.cc).sub.2, P(OR.sup.cc).sub.3.sup.+X.sup., P(O)(R.sup.aa).sub.2, P(O)(OR.sup.cc).sub.2, and P(O)(N(R.sup.bb).sub.2).sub.2, wherein X.sup., R.sup.aa, R.sup.bb, and R.sup.cc are as defined herein. Oxygen protecting groups are well known in the art and include 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, incorporated herein by reference.

(106) Exemplary oxygen protecting groups include, but are not limited to, 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 (Bn), 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), t-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), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, 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-(methoxyacyl)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). In certain embodiments, an oxygen protecting group is silyl. In certain embodiments, an oxygen protecting group is t-butyldiphenylsilyl (TBDPS), t-butyldimethylsilyl (TBDMS), triisoproylsilyl (TIPS), triphenylsilyl (TPS), triethylsilyl (TES), trimethylsilyl (TMS), triisopropylsiloxymethyl (TOM), acetyl (Ac), benzoyl (Bz), allyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethyl carbonate, methoxymethyl (MOM), 1-ethoxyethyl (EE), 2-methyoxy-2-propyl (MOP), 2,2,2-trichloroethoxyethyl, 2-methoxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), p-methoxyphenyl (PMP), triphenylmethyl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMTr), allyl, p-methoxybenzyl (PMB), t-butyl, benzyl (Bn), allyl, or pivaloyl (Piv).

(107) In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups include, but are not limited to, R.sup.aa, N(R.sup.bb).sub.2, C(O)SR.sup.aa, C(O)R.sup.aa, CO.sub.2R.sup.aa, C(O)N(R.sup.bb).sub.2, C(NR.sup.bb)R.sup.aa, C(NR.sup.bb)OR.sup.aa, C(NR.sup.bb)N(R.sup.bb).sub.2, S(O)R.sup.aa, SO.sub.2R.sup.aa, Si(R.sup.aa).sub.3, P(R.sup.cc).sub.2, P(R.sup.cc).sub.3.sup.+X.sup., P(OR.sup.cc).sub.2, P(OR.sup.cc).sub.3.sup.+X.sup., P(O)(R.sup.aa).sub.2, P(O)(OR.sup.cc).sub.2, and P(O)(N(R.sup.bb).sub.2).sub.2, wherein R.sup.aa, R.sup.bb, and R.sup.cc are as defined herein. Sulfur protecting groups are well known in the art and include 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, incorporated herein by reference. In certain embodiments, a sulfur protecting group is acetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl.

(108) A counterion or anionic counterion is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. An anionic counterion may be monovalent (i.e., including one formal negative charge). An anionic counterion may also be multivalent (i.e., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F.sup., Cl.sup., Br.sup., I.sup.), NO.sub.3.sup., ClO.sub.4.sup., OH.sup., H.sub.2PO.sub.4.sup., HCO.sub.3.sup., HSO.sub.4.sup., sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF.sub.4.sup., PF.sub.4.sup., PF.sub.6.sup., AsF.sub.6.sup., SbF.sub.6.sup., B[3,5-(CF.sub.3).sub.2C.sub.6H.sub.3].sub.4].sup., B(C.sub.6F.sub.5).sub.4.sup., BPh.sub.4.sup., Al(OC(CF.sub.3).sub.3).sub.4.sup., and carborane anions (e.g., CB.sub.11H.sub.12.sup. or (HCB.sub.11Me.sub.5Br.sub.6).sup.). Exemplary counterions which may be multivalent include CO.sub.3.sup.2, HPO.sub.4.sup.2, P.sub.4.sup.3, B.sub.4O.sub.7.sup.2, SO.sub.4.sup.2, S.sub.2O.sub.3.sup.2, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.

(109) As used herein, the term salt refers to any and all salts, and encompasses pharmaceutically acceptable salts.

(110) The term pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and/or 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, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts 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, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include 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. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N.sup.+(C.sub.1-4 alkyl).sub.4.sup. salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

(111) It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed isomers. Isomers that differ in the arrangement of their atoms in space are termed stereoisomers.

(112) Stereoisomers that are not mirror images of one another are termed diastereomers and those that are non-superimposable mirror images of each other are termed enantiomers. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or ()-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a racemic mixture.

(113) The term Cytochrome P450 family 7 subfamily A member 1, used interchangeably with the term CYP7A1, refers to the well-known gene and polypeptide, also known in the art as Cytochrome P450 family 7 subfamily A member 1.

(114) The term CYP7A1 includes human (Homo sapiens) CYP7A1, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 1581, NCBI Accession Nos. NP_000771.2 and NM_000780.4, UniProt ID: P22680; mouse (Mus musculus) CYP7A1, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 13122, NCBI Accession Nos. NP_031850.2 and NM_007824.3; UniProt ID: Q64505; rat (Rattus norvegicus) CYP7A1, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 25428, NCBI Accession No. NP_037074.1 and NM_012942.2, UniProt ID: P18125; and chimpanzee (Pan troglodytes) CYP7A1, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 464191, NCBI Accession No. XM_519773.3, UniProt ID: H2QW73. The term CYP7A1 also includes cynomolgus monkey (Macaca fascicularis) CYP7A1, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI: 102132452, NCBI Accession No. XP_005563427.1 and XM_005563370.2, UniProt ID: G7PBW5. Additional examples of CYP7A1 mRNA sequences are readily available using, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site. Exemplary CYP7A1 nucleotide and amino acid sequences may also be found in Table 1, SEQ ID NOs: 1-8.

(115) The term CYP7A1, as used herein, also refers to naturally occurring DNA sequence variations of the CYP7A1 gene. Numerous sequence variations within the CYP7A1 gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov/snp).

(116) Further information on CYP7A1 can be found, for example, at ncbi.nlm.nih.gov/gene/1581. The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

(117) Table 1 below summarizes exemplary amino acid sequences of CYP7A1 proteins and DNA sequence of CYP7A1 genes of various mammals.

(118) TABLE-US-00007 TABLE1 ExemplaryAminoAcidandDNASequencesofCYP7A1 SEQ ID Sequence NO Human AGTGGCATCCTTCCCTTTCTAATCAGAGATTTTCTTCCTCAGAGATTTTGGCCTAGATTT 1 CYP7A1 GCAAAATGATGACCACATCTTTGATTTGGGGGATTGCTATAGCAGCATGCTGTTGTCTAT Nucleic GGCTTATTCTTGGAATTAGGAGAAGGCAAACGGGTGAACCACCTCTAGAGAATGGATTAA Acid TTCCATACCTGGGCTGTGCTCTGCAATTTGGTGCCAATCCTCTTGAGTTCCTCAGAGCAA (NM_000780.4) ATCAAAGGAAACATGGTCATGTTTTTACCTGCAAACTAATGGGAAAATATGTCCATTTCA TCACAAATCCCTTGTCATACCATAAGGTGTTGTGCCACGGAAAATATTTTGATTGGAAAA AATTTCACTTTGCTACTTCTGCGAAGGCATTTGGGCACAGAAGCATTGACCCGATGGATG GAAATACCACTGAAAACATAAACGACACTTTCATCAAAACCCTGCAGGGCCATGCCTTGA ATTCCCTCACGGAAAGCATGATGGAAAACCTCCAACGTATCATGAGACCTCCAGTCTCCT CTAACTCAAAGACCGCTGCCTGGGTGACAGAAGGGATGTATTCTTTCTGCTACCGAGTGA TGTTTGAAGCTGGGTATTTAACTATCTTTGGCAGAGATCTTACAAGGCGGGACACACAGA AAGCACATATTCTAAACAATCTTGACAACTTCAAGCAATTCGACAAAGTCTTTCCAGCCC TGGTAGCAGGCCTCCCCATTCACATGTTCAGGACTGCGCACAATGCCCGGGAGAAACTGG CAGAGAGCTTGAGGCACGAGAACCTCCAAAAGAGGGAAAGCATCTCAGAACTGATCAGCC TGCGCATGTTTCTCAATGACACTTTGTCCACCTTTGATGATCTGGAGAAGGCCAAGACAC ACCTCGTGGTCCTCTGGGCATCGCAAGCAAACACCATTCCAGCGACTTTCTGGAGTTTAT TTCAAATGATTAGGAACCCAGAAGCAATGAAAGCAGCTACTGAAGAAGTGAAAAGAACAT TAGAGAATGCTGGTCAAAAAGTCAGCTTGGAAGGCAATCCTATTTGTTTGAGTCAAGCAG AACTGAATGACCTGCCAGTATTAGATAGTATAATCAAGGAATCGCTGAGGCTTTCCAGTG CCTCCCTCAACATCCGGACAGCTAAGGAGGATTTCACTTTGCACCTTGAGGACGGTTCCT ACAACATCCGAAAAGATGACATCATAGCTCTTTACCCACAGTTAATGCACTTAGATCCAG AAATCTACCCAGACCCTTTGACTTTTAAATATGATAGGTATCTTGATGAAAACGGGAAGA CAAAGACTACCTTCTATTGTAATGGACTCAAGITAAAGTATTACTACATGCCCTTTGGAT CGGGAGCTACAATATGTCCTGGAAGATTGTTCGCTATCCACGAAATCAAGCAATTTTTGA TTCTGATGCTTTCTTATTTTGAATTGGAGCTTATAGAGGGCCAAGCTAAATGTCCACCTT TGGACCAGTCCCGGGCAGGCTTGGGCATTTTGCCGCCATTGAATGATATTGAATTTAAAT ATAAATTCAAGCATTTGTGAATACATGGCTGGAATAAGAGGACACTAGATGATATTACAG GACTGCAGAACACCCTCACCACACAGTCCCTTTGGACAAATGCATTTAGTGGTGGTAGAA ATGATTCACCAGGTCCAATGTTGTTCACCAGTGCTTGCTTGTGAATCTTAACATTTTGGT GACAGTTTCCAGATGCTATCACAGACTCTGCTAGTGAAAAGAACTAGTTTCTAGGAGCAC AATAATTTGTTTTCATTTGTATAAGTCCATGAATGTTCATATAGCCAGGGATTGAAGTTT ATTATTTTCAAAGGAAAACACCTTTATTTTATTTTTTTTCAAAATGAAGATACACATTAC AGCCAGGTGTGGTAGCAGGCACCTGTAGTCTTAGCTACTCGAGAGGCCAAAGAAGGAGGA TGGCTTGAGCCCAGGAGTTCAAGACCAGCCTGGACAGCTTAGTGAGATCCCGTCTCCGAA GAAAAGATATGTATTCTAATTGGCAGATTGTTTTTTCCTAAGGAAACTGCTTTATTTTTA TAAAACTGCCTGACAATTATGAAAAAATGTTCAAATTCACGTTCTAGTGAAACTGCATTA TTTGTTGACTAGATGGTGGGGTTCTTCGGGTGTGATCATATATCATAAAGGATATTTCAA ATGATTATGATTAGTTATGTCTTTTAATAAAAAGGAAATATTTTTCAACTTCTTCTATAT CCAAAATTCAGGGCTTTAAACATGATTATCTTGATTTCCCAAAAACACTAAAGGTGGTTT TATTTTCCCTTCATGTTTTAACTTATTGTTGCTGAAAACTCTATGTCCGGCTTTAACTAT CTTCTCTATATTTTTATTTCATTCACATTAATGAGAAGAGTTTTCTCAGAGATTAAAAAA GGTAGTTTTTCTGTCATTGTTAAATACACATTATCACTGAAAAAATGTAGCTTTTATGTG ATATGTTTTAAAGTTAAAACTGGATGGAAATAGCCATTTGGAAGCTTTGGTTATGAAACA TGTGGAGTGTATTAAGTGCAGCTTGACATTATGTTTTATTTAAATGCTTTTTATCGCTAA ATGACTTGCAGATGAAAAAAACTAAGGTGACTCGAGTGTTTAAATGCTGTGTACAACAAT GCTTTGATAAAATATTTTAAGTATGAGTTATCAGCTCTATGTCAATTGATATTTCTGTGT AGTATTTATATTTAAATTATATTTACCTTTTTGCTTATTTTACAAATATTAAGAAAATAT TCTAACATTTGATAATTTTGAAATGATTCATCTTTCAGAAATAAAAGTATGAATCTA Human MMTTSLIWGIAIAACCCLWLILGIRRRQTGEPPLENGLIPYLGCALQFGANPLEFLRANQ 2 CYP7A1 RKHGHVFTCKLMGKYVHFITNPLSYHKVLCHGKYFDWKKFHFATSAKAFGHRSIDPMDGN Protein TTENINDTFIKTLQGHALNSLTESMMENLORIMRPPVSSNSKTAAWVTEGMYSFCYRVMF (NP_000771.2) EAGYLTIFGRDLTRRDTQKAHILNNLDNFKQFDKVFPALVAGLPIHMERTAHNAREKLAE SLRHENLQKRESISELISLRMFLNDTLSTEDDLEKAKTHLVVLWASQANTIPATFWSLFQ MIRNPEAMKAATEEVKRTLENAGQKVSLEGNPICLSQAELNDLPVLDSIIKESLRLSSAS LNIRTAKEDFTLHLEDGSYNIRKDDIIALYPQLMHLDPEIYPDPLTFKYDRYLDENGKTK TTFYCNGLKLKYYYMPFGSGATICPGRLFAIHEIKOFLILMLSYFELELIEGQAKCPPLD QSRAGLGILPPLNDIEFKYKFKHL Mouse GGACCGTCTTGCTTTGCTAAGCACAGATTCTCCCCTTGGGACGTTTTCCTGCTTTTGCAA 3 CYP7A1 AATGATGAGCATTTCTTTGATCTGGGGGATTGCTGTGGTAGTGAGCTGTTGCATATGGTT Nucleic TATCATTGGAATAAGGAGAAGGAAAGTAGGTGAACCTCCTTTGGACAACGGGTTGATTCC Acid ATACCTGGGCTGTGCTCTGAAGTTCGGATCCAATCCTCTTGAATTCCTAAGAGCAAAGCA (NM_007824.3) AAGGAAACATGGCCATGTTTTTACCTGCAAACTGATGGGGAAATATGTCCACTTCATCAC AAACTCCCTGTCATACCACAAAGTCTTATGTCACGGAAAATATTTTGACTGGAAAAAATT TCATTACACTACTTCTGCGAAGGCATTTGGACACAGAAGCATAGACCCAAGTGATGGAAA TACCACGGAAAACATAAACAAGACTTTTAACAAAACCCTCCAGGGAGATGCTCTGTGTTC ACTCTCTGAAGCCATGATGCAAAACCTCCAATCTGTCATGAGACCTCCGGGCCTTCCTAA ATCAAAGAGCGCTGTCTGGGTCACGGAAGGGATGTATGCCTTCTGCTACCGAGTGATGTT TGAAGCCGGATATCTAACGCTGTTTGGCAAAGATATTTCAAAGACAGACTCACAAAGAGC ATTTATTCAAAACAACCTTGACAGCTTCAAACAATTTGACCAAGTATTTCCGGCACTAGT GGCGGGCGTCCCTATTCACTTGTTCAAGACCGCACATAAAGCCCGGGAAAGGCTGGCTGA GAGCTTGAAGCACAAGAACCTGTACATGAGGGACCAGGTCTCTGAACTGATCCGTCTACG CATGTTTCTCAACGATACACTCTCCACCTTTGATGACATGGAGAAGGCTAAGACGCACCT CGTGATCCTCTGGGCATCTCAAGCAAACACCATTCCTGCAACCTTCTGGAGCTTATTTCA AATGATCAGGAGCCCTGAAGCAATGAAAGCAGCCTCTGAAGAAGTGAATGGAGCATTACA GAGTGCTGGCCAAGAGCTCAGCTCTGGAGGGAATGCCATTTACTTGGATCAAGAGCAACT AAACAACCTGCCAGTACTAGATAGCATCATCAAGGAGGCTCTGCGGCTCTCCAGTGCATC CTTGAATATCCGGACAGCTAAGGAGGACTTCACTCTACACCTTGAGGATGGTTCCTATAA CATTCGAAAAGATGACATCATAGCTCTTTACCCACAGTTAATGCACTTGGATCCTGAAAT CTACCCAGACCCTTTGACTTTTAAATACGACCGGTACCTTGATGAAAGTGGGAAAGCAAA GACCACCTTCTATAGAAATGGAAACAAGCTGAAGTATTTCTACATGCCCTTTGGATCAGG AGCTACAATATGTCCTGGAAGACTATTTGCTGTCCAAGAAATCAAGCAATTTTTGATTCT GATGCTGTCTTACTTTGAACTGGAGCTTGTAGAGAGCCACACCAAGTGTCCCCCTCTAGA TCAGTCCCGGGCAGGCTTGGGAATTTTGCCACCACTAAATGATATTGAGTTTAAATATAA ACTGAAACACTGATATGTGGTTGGAAGAAGAGGACACTGGATGATGTAACGACTGCTGAG CGTTATCAGTAAACAGGCCTTTGGGACTAGTGCTCACTGAAGCCCCCTAGTAGCTGTATT AGTGAGAAGAACTCTGTTCTTACTGCTCACGTTCCTGGGGATTCGTGTAGCTGGGGCCTG AGTTTCATCACTTTCAGAGCAACGTCTTTTGTTTTTATTTTCAAAATGAAGATATTCCAA TTGGCAGGGTTCTTTCCTAAGGAAATTGCTTTATATTTTTATGAAAACTACCGATTAATT ATGGAAGTACTTCAAATTCACGTTTTAGTGAAATTATTAATTTTTCACTAGTAAGGTTCT TCATGTGTGAGTATATTATAAAAATGTTTTAGCTGATCATATCATGCTTTGCATAAAGGG AAAGGAAATTATTGTTCAGCTTTTTTTTTATGGTGGTGAGAGCTTGAAAATGATCTTACT ATTCTAGAACTACTAGGGAAGTTTCGACATGCTCTCGCTATATTTTAATTTATTGTTGCT GGAGATTTTTATTCCAGTTTTGAACTACTTTATCTTTCCTTCTTTTTGACACACATACCA ATAAGAAGAGCATTTTTCAGAAATTATAAAGGCACCTCCAAGAACCACACCATGAGTCTT TTAAGCCTTTAATTCCAGCTCCCATGAGGCAAAGCCAGGCAAATCTCTATGAGTCTGAGG TTATTCTGGTCTACATCAGCTCTAGACAAGCCAGGACTACAGAAAGGACCTTGTCTAAAA AGAAAAAAAGTTAATTTCTATGTCATATTTGATTATGAATCAACATGAAATATAAATTTA AAATCAGGACTCAGAGAAATGATCAATTAAAAAACTTAGCTATGAAGTATGTGGAGTTCT TTAAGTACAAGTTGACATTATATGTTCTTTTTTTATGGTTTGTTGTAGAAAGGCACGGAG AAAGAAAGAAGGTAGAAAGAGAGAGACACCGGCCATGGCCACGTGGAGAGAGGGGGGAAG GGAAAGAGAGAAGGAGGCCTAGAGAGTAAGAAAGGTGAGGGCTTAAAGAGCTATATGCTC TTTAAAAATGTTTTATGTTTTTATCTCTAAATGCCTTAAAGATGAAGAACAATAATGAAA GGCTGAGTAATAGTGTTTAAATACTGAGTGCAATAGTGCTTTAGTAATATACTTTAAAGA GAGTTATTAGCTATGTCATTTTTACTGAAAATATATTTATATATAAATTATATTTATCTT TTTCTTATACCATATATATAAAAATATTGTAACATTTAGTAATTTTAAAATTACACACCT TTCAGAAATTAACATATGAATGTTCGTGTTTTAAACTTTGAACAGAACATTTAAATTATT CATCTACTGGTAAGCTACAATAATTTTTCTCATATTTATTTAAAATATTCATATTTTCAA GAAATCCAAAAATATCCAAAGTAATCACTCAGTCAAATAGATCCCTAAGATGACAGTAAA TTCAATACCATGCTTTTGTCTGCGAGGGCTGGAGCAGGGTCCAGTGGATGCTGTGCAGGT CCTCCAGCAGAGAGCTAAATCCACAGACTCCTCAAAATCTTAAAACCTTACCACTGAATA CTGGCACAGAGTTATTACCTAGGTACGCTATGCTTCCTTCCTTTTTCATTTTGACAGAGG CCCATAGCATCCCAGGCTGGAACATTCCGCGTAGCTTAAGCAAGCTTGGAACTCACTGTG CTTCCTGCTTTGCCTTGTTATAGGAAGTAATTTTCAAGACCAAGAAAACTTAATTGTTTA GATGAGCTATAAGAAAGACACAATTGTGTTGTATACTAATCTGTACTAAGCTAAATTTGT TTTTAAAACAAGTTTTATGACTCCCTGAACTGAATGTATCCACATACCTTCCCATGGCTT TTTAAAAGCATATTTTTCATACAGAATGATGGGTCTCCTGGTGGTGCCTTCTTCTCATAT TTTTATTCTTGTGAAGTGATGGCTAGCAAACAATAGCCTGTCAACCAAAGTTACTCTTCC CGTTTCTGTGCAGCCTAAGTGTTAAAAGTAATTTTTACATTTTTAAATGCTTAGGGGAAA AAAACCACATGTCTATTTTGTGTCATATACGAAGTATATGAAATACAGACTAGCAAACCC GTGACTAAAACTTTTATTGGGATATCAGAATCTGCCTAATTCTTTGCGTTATGTCTACAA TTCCATTTTTTGACATGTGTATGTAGTGTGCATTTTTGTGGAAGCATGTGTGAGTGTGTG TGCACATGTGGGTAAACATGTGTGCATGTGTGTAGAGGCTGGAGGTGATGTTGAGTGTTT TCCTCCACTGTTCTCCCTTGAGGGTCTCTCCCTTGAGCCAGAGTCCAATGCTTAGGCTAG CATAGCCAACTTGCCGCAGTGCCCTTGAATTGCAGGAGGTTGCCACATCCTCCTGGCATT TACCCTGGTGTGAGATGAGCTTCAGAAGGGCAAGTGCTTTGTCTGCTAGTCTCAGTGTCT ACAGCTCTTGCTGTGTAATTGTAAAGGCAGGGTAGTGTGACATAGACAGGAGGACACAGA GCTTACCATTTCAAAAATCAGTTTCTTCAGAGT Mouse MMSISLIWGIAVVVSCCIWFIIGIRRRKVGEPPLDNGLIPYLGCALKFGSNPLEFLRAKQ 4 CYP7A1 RKHGHVFTCKLMGKYVHFITNSLSYHKVLCHGKYFDWKKFHYTTSAKAFGHRSIDPSDGN Protein TTENINKTENKTLCGDALCSLSEAMMCNLQSVMRPPGLPKSKSAVWVTEGMYAFCYRVMF (NP_031850.2) EAGYLTLFGKDISKTDSQRAFIQNNLDSFKQFDQVFPALVAGVPIHLFKTAHKARERLAE SLKHKNLYMRDQVSELIRLRMFLNDTLSTFDDMEKAKTHLVILWASQANTIPATFWSLFQ MIRSPEAMKAASEEVNGALQSAGQELSSGGNAIYLDQEQLNNLPVLDSIIKEALRLSSAS LNIRTAKEDFTLHLEDGSYNIRKDDIIALYPQLMHLDPEIYPDPLTFKYDRYLDESGKAK TTFYRNGNKLKYFYMPFGSGATICPGRLFAVQEIKCFLILMLSYFELELVESHTKCPPLD QSRAGLGILPPLNDIEFKYKLKH Rat GTCTCCCCTTTGGAAATTTTCCTGCTTTTGCAAAATGATGACTATTTCTTTGATTTGGGG 5 CYP7A1 AATTGCCGTGTTGGTGAGCTGTTGCATATGGTTTATTGTTGGAATAAGGAGAAGGAAAGC Nucleic TGGTGAACCTCCTTTGGAGAACGGGTTGATTCCGTACCTGGGCTGTGCTCTGAAATTTGG Acid ATCTAATCCTCTTGAGTTCCTAAGAGCTAATCAAAGGAAGCATGGTCACGTTTTTACCTG (NM_012942.2) CAAACTGATGGGGAAATATGTCCATTTCATCACAAACTCCCTGTCATACCACAAAGTCTT ATGTCATGGAAAATATTTTGACTGGAAAAAATTTCATTACACTACTTCTGCGAAGGCATT TGGACACAGAAGCATTGACCCAAATGATGGAAATACCACGGAAAATATAAACAACACTTT TACCAAAACCCTCCAGGGAGATGCTCTGTGTTCACTTTCTGAAGCCATGATGCAAAACCT CCAATCTGTCATGAGACCTCCTGGCCTTCCTAAATCAAAGAGCAATGCCTGGGTCACGGA AGGGATGTATGCCTTCTGTTACCGAGTGATGTTTGAAGCTGGCTATCTAACACTGTTTGG CAGAGATATTTCAAAGACAGACACACAAAAAGCACTTATTCTAAACAACCTTGACAACTT CAAACAATTTGACCAAGTCTTTCCGGCACTGGTGGCAGGCCTTCCTATTCACTTGTTCAA GACCGCACATAAAGCTCGGGAAAAGCTGGCTGAGGGATTGAAGCACAAGAACCTGTGTGT GAGGGACCAGGTCTCTGAACTGATCCGTCTACGTATGTTTCTCAATGACACGCTCTCCAC CTTTGACGACATGGAGAAGGCCAAGACGCACCTCGCTATTCTCTGGGCATCTCAAGCAAA CACCATTCCTGCAACCTTTTGGAGCTTATTTCAAATGATCAGGAGTCCTGAAGCAATGAA AGCAGCCTCTGAAGAAGTGAGTGGAGCTTTACAGAGTGCTGGCCAAGAGCTCAGCTCTGG AGGGAGTGCCATTTACTTGGATCAAGTGCAACTGAATGACCTGCCGGTACTAGACAGCAT CATCAAGGAGGCTCTGAGGCTTTCCAGTGCATCCTTGAATATCCGCACAGCTAAGGAGGA CTTCACTCTCCATCTTGAGGACGGTTCCTATAACATCCGAAAAGATGACATGATAGCTCT TTATCCACAGTTAATGCACTTGGATCCTGAAATCTACCCAGACCCTTTGACTTTCAAATA TGACCGGTACCTTGATGAAAGCGGGAAAGCAAAGACCACCTTCTACAGTAATGGAAACAA GCTGAAGTGTTTCTACATGCCCTTCGGATCAGGCGCGACAATATGTCCTGGAAGACTCTT TGCCGTCCAAGAAATCAAGCAGTTTTTGATCCTGATGCTCTCCTGCTTTGAACTGGAGTT TGTGGAGAGCCAAGTCAAGTGTCCCCCTCTAGACCAGTCCCGGGCAGGCTTGGGAATTTT GCCACCACTACATGATATTGAGTTTAAATATAAACTGAAACACTGATACGTGGTTGGAAG AAGCGAACACTGGATGATGTCACTTGGCGGCTGAGAGTCATCACTAAACAGGCCTTCGGG ACCAATGCTCACTGATGCGCCCTAGCGACTGGATTAGTGGGAAGAACTTTGTTCTCGCTG CCCACATTCCTGGGTGTTCACATAGCTGGGGCCAGAGCTTCATCACTTTCAGAAAGCAAT GTCTTTTGTATTTATTTTCAAAATGAAGATATTCCAATTGGCAGGATATTTTTCCTAAGG AAATTGCTTTATATTTTTATGAAAACTACCAATTAATTATGAAAGGGCTTGAAATTCACG TTTTAGTGAAATTACTGATTTTTCACTAGTAAGGTTCTTCAGGTGTGAAACTGTATTATA AAAATGTTGTAATGGGTCACACTGTGCTTTGCATAAAGGTAAAGGAAACTATGTTTCAGC CTTTTCTGTGTCTATGAGCTTCGAAAATAATCTTACTGTTCTAGAAACACTGGGGAGGTT TCGACATGCTCTCGCTATATTTTATTTTACTGTTGCTAGAAATTTTCATTCCAGTTTTCA ACTACCTTATCTTTCCCCCATTTTGACATGCATGCCAATGAGAAGAGTATTTTTTAGGAA TTAACAAGGCACCTCCCAGAACCCTACCCTGAGACTTTTAAGCCTTTAATCCCAGCACTC GAGAAGTAGAGCCAGGCAGATCTCTGAGTCTGAGGTTATTCTGGTCTACATCAGCTCCAG ACAAGCCAGGACTACAGAATGGGATCTTGTCTAAAAAATACAGCTAATCTTTATGTCATA ACTGATTATGAATCAACCTAAAAGATAAATTTTCAATCAGGACTCAGAGAAAATGAGCAA TTAAAAAACTTAGCTCTGAGGTATGTGGAATTCATTAAGTACAAGTTGACATTACATGTT CTTTAAAAATAGTTTATGTTTTATCTCTAAATGCCCTGCAGATGAAGAATAATAATGAAA AGTTGAATAATACTGTTTAAACACTAAGTGCAATAATGCTTTGGTAATGTACTTTAAGAG AATCATTAGCCGTGCCAGTTTTACTAAAATATATTTATATGTAAATTATATTTATCTTTT TCTTATACCATAAATATAAAAATATTGCAACATTTAGTAATTTTAAAATTATATACCTTT CAGAAAATGATGTATGAATGTTTGTATGTTTTTTAACTTTGAACAGAACATTTAAATTAT TCATCTACGGTGATTTTTATCTTATTTATTTCTTTTTGTCTCATTCATATCTTGAAGAAA TCCAAAAATATCTGAAGGAATCGCTCACTCAAATGTCTCCCTATGGTTACAGAAAAATTC AATACCATGTTTTTGTCCTCGGGGACTGAAGCAGGGTGTCGTGGGTGCCGAGCAGAGGCT CCTGCTGCAGCGAGCTTTATCCACGGGACTCCTTAAACTTTTAAAATCTTATCACTATTA TCATGCATTTATTACCTAAGTAGGATATTTCCCTTTCCTTTTTCATTTCAGCCGAGTCCC TTAGCAACCCAGGCTGACTGGGACCCTCCATGTAGCTTAAGCTGTGAACTCACTGTACTT CCTGTTTTCACTTATTTTAGGAAGTAATTTTCCCTATCAGAAATTTTAATTGTTTAGATG ATGTATAAGAGTAACACAATTCTGTTATATACTAATCTGTAGTAAACTAAATTTGTTCTT AGAACAAGTTTGATGACTCTCAAATTGAATGTATCCATACATCTTTCCATGGCTTCTTGA ATGCCCATTTCTCATACACAGAATGATGGGTTTCACGGTGATGTCTTCCTTTCATGTCTT TATTCTTGTGCGGTGATGGTTGGCAAATGATACCCATGGAGCAAGGTTACTCTTCCTATT TCTGTGCAGCCTAAGTGTTAAGAATAATTTTTAAATACTTGGAGGGAAGGCACATTTTGT GTCATATGTGAAGTGACATGTGACACACAGACTAGCAAATCCTTGAGTAAAATTTTATTG GGAT Rat MMTISLIWGIAVLVSCCIWFIVGIRRRKAGEPPLENGLIPYLGCALKFGSNPLEFLRANQ 6 CYP7A1 RKHGHVFTCKLMGKYVHFITNSLSYHKVLCHGKYFDWKKFHYTTSAKAFGHRSIDPNDGN Protein TTENINNTFTKTLQGDALCSLSEAMMCNLQSVMRPPGLPKSKSNAWVTEGMYAFCYRVMF (NP_037074.1) EAGYLTLFGRDISKTDTQKALILNNLDNFKQFDQVFPALVAGLPIHLFKTAHKAREKLAE GLKHKNLCVRDQVSELIRLRMFLNDTLSTFDDMEKAKTHLAILWASQANTIPATFWSLFQ MIRSPEAMKAASEEVSGALQSAGQELSSGGSAIYLDQVQLNDLPVLDSIIKEALRLSSAS LNIRTAKEDFTLHLEDGSYNIRKDDMIALYPQLMHLDPEIYPDPLTFKYDRYLDESGKAK TTFYSNGNKLKCFYMPFGSGATICPGRLFAVQEIKCFLILMLSCFELEFVESQVKCPPLD QSRAGLGILPPLHDIEFKYKLKH Cyno GTCAACAAGCTCAAGTTAATGGATCTGGGTACTATGTATATAAAAAAACCTAGCTTGAGT 7 CYP7A1 CTCTTTTCAGTGACATCTTTGCCTTTCTAATCAGAGATTTTCTTCCTCAGAGATTTTGGC Nucleic CTAGATTTGCAAAATGATGACCATATCTTTGATTTGGGGGATTGCTATAGCAGCATGCTG Acid TTGTCTATGGCTTATTCTTGGAATAAGGAGAAGGCAAACGGGTGAACCACCTCTAGAGAA (XM_ TGGGTTGATTCCATACCTGGGCTGTGCGCTGCAATTTGGTGCCAATCCTCTTGAGTTCCT 005563370.2) CAGAGCAAATCAAAGGAAACATGGTCATGTTTTTACCTGCAAACTAATGGGAAAATATGT CCATTTCATCACAAATCCCTTGTCATACCATAAGGTGTTGTGCCATGGAAAATACTTTGA TTGGAAAAAATTTCACTTTGCTACTTCTGCAAAGGCTTTTGGGCACAGAAGCATTGACCC AAAGGATGGAAATACCACTGAAAACATAAACAACACTTTCATCAAAACCCTGCAGGGCAA TGCCTTGAATTCCCTCACGGAAAGCATGATGGAAAACCTCCAACGTATCATGAGACCTCC AGTCTTCTCTAACTCAAAGACCGCTGCCTGGGTGACAGAAGGGATGTATTCCTTCTGCTA CCGAGTGATGTTTGAAGCTGGGTATTTAACTATCTTTGGCAGGGATCTTACAAGGCAAGA CACACAGAAAGCACATATTCTAAACAATCTTGACAACTTCAAGCAGTTCGACAAAGTCTT TCCAGCCCTGGTAGCAGGCCTCCCCATTCACATGTTCAGGACCGCGCACAGTGCCCGGGA GAAACTGGCAGAGAGCTTGAGGCACGAGAACCTCCAAAAGAGGGAAAGCGTCTCAGAACT GATCAGACTGCGCATGTTTCTCAATGACACTTTGTCCACCTTTGATGACCTGGAGAAGGC CAAGACGCACCTCGTGGTCCTCTGGGCATCGCAAGCAAACACCATTCCAGCAACTTTCTG GAGTTTATTTCAAATGATTAGGAACCCGGAAGCAATGAAAGCAGCTACTGAAGAAGTGAA AAGAACATTAGAGAATGCTGGTCAAAAAGTCAGCTTGGAAGGCAATCCCATTTGTTTGAG TCAAACACAACTGAATGACCTGCCAGTATTAGAAAGCATAATCAAGGAATCACTGAGGCT TTCCAGTGCCTCCCTCAACATTCGGACAGCTAAGGAGGATTTCACTTTGCACCTTGAGGA CGGTTCCTACAACATCCGAAAAGATGACATCATAGCTCTTTACCCACAGTTAATGCACTT AGATCCAGAAATCTACCCAGACCCTTTGATTTTTAAATATGATAGGTATCTTGATGAAAA CGGGAAGACAAAGACTACCTTCTACTGTAATGGACTCAAATTAAAGTATTACTACATGCC CTTTGGATCAGGAGCTACAATATGCCCTGGAAGAGTGTTTGCTATCCACGAAATCAAGCA ATTTTTGGTTTTGATGCTTTCTTATTTTGAACTGGAGCTTGTCGAGGGCCAAGATAAATG TCCGCCTTTGGACCAGTCCCGGGCAGGCTTGGGCATTTTGCCGCCATTATATGACATTGA GTTTAAATATAAATTCAAGCATTTGTGAATACGTGGCTGAAATAAGAGGACACTAGATGA TATTACAGGACTGCAGAACACCATCACCACACAGTCCCTTTGGATAAATGCATTTAGTGG TGGTAGAAATGATTCACCAGGTCCAATGTTGTTCACCAATGCTTGCTTGTGAATCTTAAT ATTTTGGTGACAGTTTCCAGATGGTGTCGCAGACTGTGCTAGTGAAAAGAACTAGTTTCT AGGAGCACGATAATTTGTTTTCATCTGTATGAGTCCATGAATGTTCATATAGCCAGGGGT TGAAGTTTATTTTTTTCAGAGGAAAACACCTTTTTTTTTTTTCCCCCCCCCAAAATGAAG ATACACATTCCAGCCAGGTATTGTAGCAGGCACCTGTAGTCTTAGCTACTCGAGAGGCCA AAGAAGGAGGATGGCTTGAGCCCAGGAGTTCAAGACTAGCCTGGACAGCTTAGTGAGACC CCGTCTCTAAAGAAAAGATACGTATTCTAATTGGCAGATTGTTTTTTCCTAAGGAAACTG CTTTATTTTTATAAAACTGCCCAACAATTATGAAACATGTTCAAATTCACGTTCTAGTGA AACTGCATTATTTTTTTACTAGATGTTGGGGTTCTTCAGGTGTGATCATATATCATAAAG GATATTTCAAATGATTATGATTAGTTATGTCTTTTAATAAAAAGGAAATACTTTTCAACT TTTTATATATCCAAAATTCAGGGCTTTAAAAATGATTATTTTGATTTCCCAAAAACACTA AAGGTGGTTTTAATATTTTCCCTTTATGTTTTAACTTATTGTTGCTGAAAACTCTATGTC CAGCTTTAACTATCTTCTCTGTATTTTTATTTCATTTCATATTAATGAGAAGAGTTTTTT TCAGAGATTAAAAAAGGCAGTTTTTCTGTTATTGTTAAATACACACTATCACTGAACAAA TGTAGCTTTTATGTGATCTGTTTTAAAGTTAAAACCGGATGGAAATAGCCGTTTGAAAGC TTTGGTTATGAAACATGCGGAGTGTATTAAGTACGGCTTGACATTATGTTTTATTTAAAT GCTTTTTATCGCTAAATGACTTGCAGATGAAAAAAAACTAAGGTGGCCTGAGAGTTTAAA TGCTGTGTACAACAATGCTTTGATAATATATTTTAAGTATGAGTTAT CAGCTATATGTCAATTGATATTTCTGTGCAGTATTTATATGTAAATTATATTTACCTTTT TGCTTATTTTATAAATATTAAGAAAGTATTCTAACATTTCATAATTTTGAAATGATTCAT CTTTCAGAAATAAAAGTATGAATCTA Cyno MMTISLIWGIAIAACCCLWLILGIRRRQTGEPPLENGLIPYLGCALQFGANPLEFLRANQ 8 CYP7A1 RKHGHVFTCKLMGKYVHFITNPLSYHKVLCHGKYFDWKKFHFATSAKAFGHRSIDPKDGN Protein TTENINNTFIKTLQGNALNSLTESMMENLCRIMRPPVFSNSKTAAWVTEGMYSFCYRVMF (XP_ EAGYLTIFGRDLTRQDTQKAHILNNLDNFKQFDKVFPALVAGLPIHMERTAHSAREKLAE 005563427.1) SLRHENLQKRESVSELIRLRMFLNDTLSTFDDLEKAKTHLVVLWASQANTIPATFWSLFQ MIRNPEAMKAATEEVKRTLENAGQKVSLEGNPICLSQTQLNDLPVLESIIKESLRLSSAS LNIRTAKEDFTLHLEDGSYNIRKDDIIALYPQLMHLDPEIYPDPLIFKYDRYLDENGKTK TTFYCNGLKLKYYYMPFGSGATICPGRVFAIHEIKCFLVLMLSYFELELVEGQDKCPPLD QSRAGLGILPPLYDIEFKYKFKHL

(119) As used herein, the term CYP7A1 disease or CYP7A1-associated disease, is a disease or disorder that is caused by, or associated with, CYP7A1 expression and/or activity. The term CYP7A1-associated disease includes a disease, disorder or condition that would benefit from a decrease in CYP7A1 gene expression, replication, or protein activity. In some embodiments, a subject having a CYP7A1 disease or a CYP7A1-associated disease may benefit from the reduction in expression of CYP7A1 gene. In some embodiments, a CYP7A1 disease or CYP7A1-associated disease is a disease that is characterized by accumulation of toxic bile acids. A CYP7A1 disease or CYP7A1-associated disease may be a liver disease. In some embodiments, a CYP7A1-associated disease is a cholestatic liver disease. Cholestasis is resulted from disrupted bile flow from the liver to the intestine tract, leading to accumulation of toxic bile acids and other metabolites in the liver, decreased bile acids in the intestine and increased bile acids in the systemic circulation. The accumulation of toxic bile acids in the hepatobiliary system damages bile duct epithelial cells and hepatocytes, causing liver injury and inflammation. Chronic cholestasis leads to fibrosis, cirrhosis and eventually liver failure or hepatocellular or cholangiocellular carcinomas. Non-limiting examples of CYP7A1-associated diseases include, for example, cholestatic liver diseases such as primary sclerosing cholangitis (PSC), familial intrahepatic cholestasis (PFIC, including Type 1, PFIC1, Type 2, PFIC2, and Type 3, PFIC3) and primary biliary cholangitis (PBC), Alagille syndrome, biliary atresia, and other liver diseases such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD).

(120) Further details regarding signs and symptoms of the various diseases or conditions are provided herein and are well known in the art.

(121) Compositions

(122) CYP7A1 RNAi Agents

(123) Some aspects of the present disclosure provide RNAi agents that target CYP7A1 (referred to herein as CYP7A1 RNAi agent) In some embodiments, a CYP7A1 RNAi agent disclosed herein targets a CYP7A1 sequence (e.g., gene sequence, pre-mRNA sequence, or mRNA sequence). In some embodiments, a CYP7A1 RNAi agent disclosed herein is a double stranded siRNA comprising a sense strand and an antisense strand. In some embodiments, the antisense strand of a CYP7A1 RNAi agent disclosed herein comprises a region of complementary to a CYP7A1 RNA (e.g., pre-mRNA or mRNA) sequence. In some embodiments, a CYP7A1 RNAi agent disclosed herein are useful for reducing levels of CYP7A1 RNA and/or protein.

(124) In some embodiments, a CYP7A1 RNAi agent disclosed herein target a CYP7A1 sequence at or near a position corresponding to a position provided in Table 2. In some embodiments, a CYP7A1 RNAi agent disclosed herein is a siRNA oligonucleotide comprising an antisense strand comprising a region of complementary to a CYP7A1 sequence at or near a position in a CYP7A1 sequence corresponding to a position provided in Table 2. Near a position, as used herein, means within 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) nucleosides upstream (5 end) or downstream (3 end) of the position.

(125) As used herein, the position of a target sequence is represented by the position of the first nucleoside of the target sequence in a reference sequence. For example, for the CYP7A1 target sequences provided in Table 2, the reference sequence used is human CYP7A1 mRNA transcript and its corresponding cDNA sequence as set forth in NM_000780.4 (the reference sequence). The position for each target sequence provided in Table 2 refers to the position of the first nucleoside of a target sequence in SEQ ID NO: 1 (NM_000780.4). For example, the first C of the target sequence as set forth in SEQ ID NO: 9 is the 34.sup.th nucleoside of SEQ ID NO: 1 (NM_000780.4). For the purposes of the present disclosure, when referring to RNAi agents that target a CYP7A1 sequence at or near a position (e.g., position 34), such RNAi agents encompasses RNAi agents that target a CYP7A1 sequence at or near the position in the reference sequence (SEQ ID NO: 1; NM_000780.4), and RNAi agents that target at or near a position in another CYP7A1 sequence (e.g., gene sequence, pre-mRNA sequence, or a variant or a homologue of human CYP7A1 mRNA, e.g., a splicing variant or a mRNA from a closely related species, such as a cynomolgus monkey) that corresponds to the position in the reference sequence. To identify a position in another CYP7A1 sequence that corresponds to a certain position in the reference sequence (SEQ ID NO: 1; NM_000780.4), such other CYP7A1 sequences can be aligned to the reference sequence and the position of a nucleoside corresponding the nucleoside in the reference sequence (SEQ ID NO: 1; NM_000780.4) can be determined. For example, the nucleosides at positions 34-52 of SEQ ID NO: 1 (NM_000780.4; upper sequence) are aligned to the nucleosides at positions 102-120 of the cynomolgus CYP7A1 sequence SEQ ID NO: 7 (XM_005563370.2; lower sequence) as follows:

(126) TABLE-US-00008 CTTCCTCAGAGATTTTGGC ||||||||||||||||||| CTTCCTCAGAGATTTTGGC

(127) In the example above, when referring to RNAi agents that target a CYP7A1 sequence at or near position 34, the present disclosure encompasses RNAi agents that target at or near position 34 of NM_000780.4 (SEQ ID NO: 1) and RNAi agents that target at or near position 102 of XM_005563370.2 (SEQ ID NO: 7). In the present disclosure, unless otherwise indicated, the position of a nucleoside on another sequence is designated as the position of the corresponding nucleoside on the reference sequence. For example, in the exemplary alignment above, the first C of XM_005563370.2 (SEQ ID NO: 7) is designated as position 34 although it is the 102th nucleoside in XM_005563370.2 (SEQ ID NO: 7). In determining the position of a nucleoside of another sequence in relation to a reference sequence, the other sequence and the reference sequence are optimally aligned over the window of comparison, which comprises sufficient number of nucleosides for the alignment. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7:0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389, 1997.

(128) One or more insertions in another CYP7A1 sequence (e.g., gene sequence, pre-mRNA sequence, or a variant or a homologue of human CYP7A1 mRNA, e.g., a splicing variant or a mRNA from a closely related species, such as a cynomolgus monkey) relative to the reference sequence (SEQ ID NO: 1; NM_000780.4) are represented as position N-M, wherein N represents the corresponding position immediately before the insertions in the reference sequence, and M represents the position of the nucleoside within the one or more insertions. For example, in the following alignment between positions 2336-2349 of the sequence set forth in SEQ ID NO: 1 (NM_000780.4; upper sequence) and positions 2406-2422 of the sequence set forth in SEQ ID NO: 7 (XM_005563370.2; lower sequence),

(129) TABLE-US-00009 GGTTT---TATTTTCCC |||||||||||||| GGTTTTAATATTTTCCC

(130) The insertions TAA in SEQ ID NO: 7 (XM_005563370.2) follows the position 2340 (bolded T in the alignment above) in SEQ ID NO: 1 (NM_000780.4). Accordingly, the positions of insertions TAA are designated as 2340-1, 2340-2, and 2340-3, respectively.

(131) One or more deletions or mismatches in another CYP7A1 sequence (e.g., gene sequence, pre-mRNA sequence, or a variant or a homologue of human CYP7A1 mRNA, e.g., a splicing variant or a mRNA from a closely related species, such as a cynomolgus monkey) relative to the reference sequence (SEQ ID NO: 1; NM_000780.4) may result in the lack of a corresponding position. In these instances, as long as the deletions/mismatches (e.g., no more than 5, 4, 3, 2, 1 mismatches) are within a stretch of sequences of sufficient length (e.g., at least 20, 25, 30, 35, or 40 nucleosides) that aligned between the two sequences, the mismatched positions are represented as the position in the reference sequence that directly aligned even if the nucleoside in the reference is different. The position for the deletion in the other sequence is skipped.

(132) For example, the first 13 nucleosides (positions 1-13) of the SEQ ID NO: 1 (NM_000780.4; upper sequence) are aligned to the nucleosides at positions 69-82 of the cynomolgus CYP7A1 sequence SEQ ID NO: 7 (XM_005563370.2; lower sequence) as follows:

(133) TABLE-US-00010 AGTGGCATCCTTC ||||||||||. AGTGACATCTTTG

(134) The bolded G at position 5 of SEQ ID NO: 1 (NM_000780.4) aligns with the bolded A at position 73 of SEQ ID NO: 7 (XM_005563370.2). When referring to RNAi agents that target a CYP7A1 sequence at or near position 5, the present disclosure encompasses RNAi agents that target at or near position 5 of NM_000780.4 (SEQ ID NO: 1) and RNAi agents that target at or near position 73 of XM_005563370.2 (SEQ ID NO: 7).

(135) In some embodiments, the antisense strand of a CYP7A1 RNAi agent disclosed herein comprises a core stretch of sequence that is fully, substantially, or at least partially complementary to a CYP7A1 target sequence disclosed in Table 2. CYP7A1 target sequences disclosed in Table 2 are 19 nucleosides in length. The core stretch of sequence that is fully, substantially, or at least partially complementary to a CYP7A1 target sequence disclosed in Table 2 may be, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleosides in length.

(136) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-30 (e.g., 8-30, 8-25, 8-20, 8-15, 8-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) nucleosides in length to a CYP7A1 sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, and 7 (e.g., SEQ ID NO: 1). In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length to a CYP7A1 sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, and 7 (e.g., SEQ ID NO: 1). In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of at least 15 (e.g., 15, 16, 17, 18, 19, 20, or 21) nucleosides in length to a CYP7A1 target sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, and 7 (e.g., SEQ ID NO: 1).

(137) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence of nucleotides 113-133, 221-241, 249-269, 290-321, 475496, 504-524, 593-620, 671-691, 779-799, 839-862, 1003-1029, 1037-1057, 1082-1102, 1189-1209, 1207-1235, 1225-1246, 1235-1255, 1289-1316, 1384-1404, 1415-1435, 1431-1451, or 1559-1579 of SEQ ID NO: 1.

(138) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence of nucleotides 113-133, 221-241, 249-269, 290-310, 301-321, 475-495, 476-496, 504-524, 593-613, 600-620, 671-691, 779-799, 839-859, 842-862, 1003-1023, 1009-1029, 1937-1057, 1082-1102, 1189-1209, 1207-1227, 1215-1235, 1225-1245, 1226-1246, 1235-1255, 1289-1309, 1296-1316, 1384-1404, 1415-1435, 1431-1451, or 1559-1579 of SEQ ID NO: 1.

(139) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence of nucleotides 113-133, 475-495, 1226-1246, or 1235-1255 of SEQ ID NO: 1.

(140) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence of nucleotides 113-133 of SEQ ID NO: 1.

(141) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence of nucleotides 475-495 of SEQ ID NO: 1.

(142) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence of nucleotides 1226-1246 of SEQ ID NO: 1.

(143) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence of nucleotides 1235-1255 of SEQ ID NO: 1.

(144) In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8-21 (e.g., 8-21, 8-15, 8-10, 10-21, 10-15, 15-21, or 19-21) nucleosides in length to a CYP7A1 target sequence as set forth in any one of SEQ ID NOs: 9-776. In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementary of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleosides in length to a CYP7A1 target sequence as set forth in any one of SEQ ID NOs: 9-776. For the purposes of the present disclosure, a region of complementary need not be 100% complementary to that of its target to be specifically hybridizable or specific for a CYP7A1 sequence. In some embodiments, the region of complementarity is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target region in a CYP7A1 sequence. In some embodiments, the target region is a region of consecutive nucleosides in the CYP7A1 sequence. In some embodiments, the region of complementarity comprises a nucleoside sequence that contains no more than 1, 2, 3, 4, or 5 base mismatches compared to the complementary portion of a CYP7A1 sequence. In some embodiments, the region of complementarity comprises a nucleoside sequence that has up to 3 mismatches over 15 nucleosides, or up to 2 mismatches over 10 nucleosides. In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the sense strand is complementary or substantially complementary to the antisense strand.

(145) In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length. In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the antisense strand is 19, 20, 21, 22, or 23 nucleosides in length. In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleosides in length. In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the sense strand is 19, 20, 21, 22, or 23 nucleosides in length. In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the antisense strand is 19, 20, 21, 22, or 23 nucleosides in length and the sense strand is 19, 20, or 21 nucleosides in length. In some embodiments, in any one of the CYP7A1 RNAis agent disclosed herein, the antisense strand and the sense strand are of the same length, e.g., 19, 20, 21, 22, or 23 nucleosides in length. In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the antisense strand and the sense strand are of different lengths. In some embodiments, in any one of the CYP7A1 RNAi agents disclosed herein, the antisense strand is 23 nucleosides in length and the sense strand is 21 nucleosides in length.

(146) In some embodiments, a CYP7A1 RNAi agent disclosed herein may be blunt-ended on both ends. In some embodiments, a CYP7A1 RNAi agent disclosed herein may be blunt-ended on one end and has an overhang (5 or 3 overhang) on the other end. In some embodiments, a CYP7A1 RNAi agent disclosed herein has an overhang (5 or 3 overhang) on both ends. In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a duplex region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length. In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a duplex region of 19, 20, or 21 base pairs in length. In some embodiments, a CYP7A1 RNAi agent comprises an overhang (5 or 3 overhang) of at least 1 nucleoside on at least one strand. In some embodiments, a CYP7A1 RNAi agent comprises a 3 overhang of at least 1 nucleoside on at least one strand. In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a duplex region of 19, 20, 21 base pairs in length and comprises an overhang (5 or 3 overhang) of 1, 2, or 3 nucleosides in length on the sense strand. In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a duplex region of 19, 20, 21 base pairs in length and comprises an overhang (5 or 3 overhang) of 1, 2, or 3 nucleosides in length on the antisense strand. In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a duplex region of 19, 20, 21 base pairs in length and comprises a 3 overhang of 1, 2, or 3 nucleosides in length on the antisense strand. In some embodiments, a CYP7A1 RNAi agent disclosed herein comprises a duplex region of 19, 20, 21 base pairs in length and comprises a 3 overhang of 2 nucleosides on the antisense strand.

(147) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that is 18-25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and comprises a region of complementarity to a target sequence as set forth in any one of SEQ ID NOs: 9-776, wherein the region of complementarity is at least 15 nucleosides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleosides) in length. In some embodiments, the antisense strand is 23 nucleosides in length and comprises a region of complementarity to a target sequence as set forth in any one of SEQ ID NOs: 9-776, wherein the region of complementarity is 19, 20, 21, 22, or 23 nucleosides in length. In some embodiments, the region of complementarity is fully complementarity with all or a portion of its target sequence. In some embodiments, the region of complementarity includes 1, 2, 3 or more mismatches.

(148) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that is 18-25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleobases of any one of SEQ ID NOs: 777-1190, and a sense strand that is 18-25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleobases of any one of SEQ ID NOs: 9-776.

(149) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand comprising nucleobases 2-21 (counting 5.fwdarw.3) of any one of SEQ ID NOs: 777-1190 and a sense strand comprising the nucleobase sequence of any one of SEQ ID NOs: 393-776.

(150) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 777-1190, and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 393-776. In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand form a duplex region, and wherein the antisense strand is fully complementary to the sense strand within the duplex region. In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand form a duplex region, wherein the antisense strand is complementary to the sense strand within the duplex region, and wherein there are 1, 2, 3, 4, or 5 mismatches between the sense strand and antisense strand.

(151) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises nucleobases 2-21 (counting 5.fwdarw.3) of a nucleobase sequence selected from (5.fwdarw.3):

(152) TABLE-US-00011 (i) (SEQIDNO:1161) UUUGCUCUGAGGAACUCAAGAAG (ii) (SEQIDNO:1162) UUGUCAUUGAGAAACAUGCGCAG (iii) (SEQIDNO:1165) UUAACUGUGGGUAAAGAGCUAAG (iv) (SEQIDNO:1166) UAUACUGGCAGGUCAUUCAGUAG (v) (SEQIDNO:1169) UGAUUUGUGAUGAAAUGGACAAG (vi) (SEQIDNO:1173) UCUAAGUGCAUUAACUGUGGGAG (vii) (SEQIDNO:1174) UUUAACUGUGGGUAAAGAGCUAG (viii) (SEQIDNO:1180) UCAAGAAUAAGCCAUAGACAAAG (ix) (SEQIDNO:1181) UUUCCGUGAGGGAAUUCAAGGAG (x) (SEQIDNO:1184) UGUAUGACAAGGGAUUUGUGAAG (xi) (SEQIDNO:1188) UAAAUACCCAGCUUCAAACAUAG; and (xii) (SEQIDNO:1190) UAUGAUACGUUGGAGGUUUUCAG.

(153) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises a nucleobase sequence selected from (5.fwdarw.3):

(154) TABLE-US-00012 (i) (SEQIDNO:1161) UUUGCUCUGAGGAACUCAAGAAG (ii) (SEQIDNO:1162) UUGUCAUUGAGAAACAUGCGCAG (iii) (SEQIDNO:1165) UUAACUGUGGGUAAAGAGCUAAG (iv) (SEQIDNO:1166) UAUACUGGCAGGUCAUUCAGUAG (v) (SEQIDNO:1169) UGAUUUGUGAUGAAAUGGACAAG (vi) (SEQIDNO:1173) UCUAAGUGCAUUAACUGUGGGAG (vii) (SEQIDNO:1174) UUUAACUGUGGGUAAAGAGCUAG (viii) (SEQIDNO:1180) UCAAGAAUAAGCCAUAGACAAAG (ix) (SEQIDNO:1181) UUUCCGUGAGGGAAUUCAAGGAG (x) (SEQIDNO:1184) UGUAUGACAAGGGAUUUGUGAAG (xi) (SEQIDNO:1188) UAAAUACCCAGCUUCAAACAUAG; and (xii) (SEQIDNO:1190) UAUGAUACGUUGGAGGUUUUCAG.

(155) In some embodiments, a CYP7A1 RNAi agent described herein comprises a sense strand that comprises a nucleobase sequence selected from (5.fwdarw.3):

(156) TABLE-US-00013 (i) (SEQIDNO:419) UCUUGAGUUCCUCAGAGCAAA; (ii) (SEQIDNO:517) GCGCAUGUUUCUCAAUGACAA; (iii) (SEQIDNO:597) UAGCUCUUUACCCACAGUUAA; (iv) (SEQIDNO:566) ACUGAAUGACCUGCCAGUAUA; (v) (SEQIDNO:439) UGUCCAUUUCAUCACAAAUCA; (vi) (SEQIDNO:604) CCCACAGUUAAUGCACUUAGA; (vii) (SEQIDNO:598) AGCUCUUUACCCACAGUUAAA; (viii) (SEQIDNO:404) UUGUCUAUGGCUUAUUCUUGA; (ix) (SEQIDNO:464) CCUUGAAUUCCCUCACGGAAA; (x) (SEQIDNO:443) UCACAAAUCCCUUGUCAUACA; (xi) (SEQIDNO:493) AUGUUUGAAGCUGGGUAUUUA; and (xii) (SEQIDNO:473) GAAAACCUCCAACGUAUCAUA.

(157) In some embodiments, a CYP7A1 RNAi agent described herein comprises nucleobase sequences (e.g., nucleobase sequences of the antisense strand and the sense strand) of the siRNAs listed in Table 5A, Table 7A, and Table 9.

(158) In some embodiments, a CYP7A1 RNAi agent described herein comprises nucleobase sequences (e.g., nucleobase sequences of the antisense strand and the sense strand) of the siRNAs selected from siRNA1-siRNA384, siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272. The siRNA numbers correspond to the siRNA numbers in Table 5A and Table 7A.

(159) In some embodiments, a CYP7A1 RNAi agent described herein comprises nucleobase sequences (e.g., nucleobase sequences of the antisense strand and the sense strand) of the siRNA molecule selected from siRNA27, siRNA125, siRNA205, siRNA174, siRNA47, siRNA212, siRNA206, siRNA12, siRNA72, siRNA51, sRNA101, and siRNA81. The siRNA numbers correspond to the siRNA numbers in Table 7A.

(160) In some embodiments, a CYP7A1 RNAi agent described herein comprises nucleobase sequences (e.g., nucleobase sequences of the antisense strand and the sense strand) of the siRNA molecule selected from siRNA72, siRNA206, siRNA12, and siRNA212. The siRNA numbers correspond to the siRNA numbers in Table 7A.

(161) It is to be understood that, for the purposes of the present disclosure, a CYP7A1 RNAi agent comprising nucleobase sequences (e.g., nucleobase sequences of the antisense strand and the sense strand) of the siRNAs listed in Table 5A and Table 7A encompasses CYP7A1 RNAi agents comprising such nucleobase sequences and comprising no chemical modifications (e.g., modified nucleosides and/or modified internucleoside linkages), and CYP7A1 RNAi agents comprising such nucleobase sequences and comprising chemical modifications (e.g., one or more modified nucleosides and/or one or more modified internucleoside linkages; e.g., those provided in Table 5B, Table 7B, and Table 9), and encompasses such modified or unmodified RNAi agents unconjugated or conjugated to a targeting moiety.

(162) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand and sense strand, each comprising a structure as provided in Table 5B, Table 7B, or Table 9. In some embodiments, the CYP7A1 RNAi agent comprises an antisense strand comprising a structure as set forth in any one of SEQ ID NOs: 1605-2054. In some embodiments, the CYP7A1 RNAi agent comprises a sense strand comprising a structure as set forth in any one of SEQ ID NOs: 1191-1604. In some embodiments, the CYP7A1 RNAi agent comprises an antisense strand comprising a structure as set forth in any one of SEQ ID NOs: 1605-2054 and a sense strand comprising a structure as set forth in any one of SEQ ID NOs: 1191-1604. In some embodiments, the CYP7A1 RNAi agent is selected from any one of the siRNAs listed in Tables 5B, 7B, and 9.

(163) In some embodiments, an RNAi agent described herein (e.g., the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9) comprises a sense strand of 21 nucleosides in length and an antisense strand of 23 nucleosides in length, resulting in an siRNA having a 2 nucleoside overhang at the 3 end of the antisense strand. In some embodiments, the 2 nucleoside overhang at the 3 end of the antisense strand is UU (e.g., the siRNAs listed in Tables 5A and 5B). In some embodiments, the 2 nucleoside overhang at the 3 end of the antisense strand is AG (e.g., the siRNAs listed in Tables 7A and 7B, and 9). It is to be understood that the present disclosure contemplates and encompasses any one of the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9 with the 2 nucleoside overhang at the 3 end of the antisense strand being replaced with an overhang of a different nucleobase sequence (e.g., any known overhangs such as UG, TG or TT) with the same or different chemical modifications (e.g., in the sugar moieties and/or the internucleoside linkages).

(164) In some embodiments, an RNAi agent described herein has a U as the 5 terminal nucleoside of the antisense strand (i.e., position 1 counting from 5 to 3) (e.g., the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9). It is to be understood that the present disclosure contemplates and encompasses any one of the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9 with the 5 terminal U of the antisense strand replaced with a different nucleobase (e.g., A, T, G, or C) with the same or different chemical modification (e.g., in the sugar moiety and/or the internucleoside linkage).

(165) In some embodiments, any one or more of the uracil bases (U's) in any one of the target sequences provided herein (e.g., the target sequences provided in Table 2) may independently and optionally be thymine bases (T's).

(166) In some embodiments, any one or more of the uracil bases (U's) in any one of the siRNAs provided herein (e.g., the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9) may independently and optionally be thymine bases (T's).

(167) In some embodiments, any one or more of the purine bases (G's or A's) in any one of the siRNAs provided herein (e.g., the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9) may independently and optionally be a base that can base pair with a pyrimidine base, e.g., inosine bases (I's).

(168) In some embodiments, any one or more of the pyrimidine bases (C's or U's) in any one of the siRNAs provided herein (e.g., the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9) may independently and optionally be a base that can base pair with a purine base, e.g., inosine bases (I's).

(169) In some embodiments, any one of the siRNAs provided herein (e.g., the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9) may comprise: (i) a sense strand that comprises a 5-OH or 5-O-methyl (5-O-Me); (ii) a sense strand that comprises a 3-OH; (iii) an antisense strand that comprises a 5-OH, 5-phosphate, or 5-vinylphosphonate; and/or (iv) an antisense strand that comprises a 3-OH.

(170) In some embodiments, any one of the siRNAs provided herein (e.g., the siRNAs listed in Tables 5A, 5B, 7A, 7B, and 9) may comprise: (i) a sense strand that comprises a 5-OH, or 5-O-methyl (5-O-Me); (ii) a sense strand that is conjugated at the 3 end to a targeting moiety (e.g., via a phosphorothioate linkage); (iii) an antisense strand that comprises a 5-OH, 5-phosphate, or 5-vinylphosphonate; and/or (iv) an antisense strand that comprises a 3-OH.

(171) TABLE-US-00014 TABLE2 CYP7A119-mermRNATargetSequences(taken fromhumanCYP7A1mRNAtranscriptandcDNA sequencesetforthinGenBankAccessionNo. NM_000780.4(theRNAversionofSEQIDNO:1, withallT'sinSEQIDNO:1replacedbyU's)) SEQID NO. Position Sequence 9 34 CUUCCUCAGAGAUUUUGGC 10 40 CAGAGAUUUUGGCCUAGAU 11 41 AGAGAUUUUGGCCUAGAUU 12 42 GAGAUUUUGGCCUAGAUUU 13 43 AGAUUUUGGCCUAGAUUUG 14 45 AUUUUGGCCUAGAUUUGCA 15 105 GCAUGCUGUUGUCUAUGGC 16 110 CUGUUGUCUAUGGCUUAUU 17 111 UGUUGUCUAUGGCUUAUUC 18 112 GUUGUCUAUGGCUUAUUCU 19 113 UUGUCUAUGGCUUAUUCUU 20 115 GUCUAUGGCUUAUUCUUGG 21 116 UCUAUGGCUUAUUCUUGGA 22 117 CUAUGGCUUAUUCUUGGAA 23 118 UAUGGCUUAUUCUUGGAAU 24 139 GGAGAAGGCAAACGGGUGA 25 141 AGAAGGCAAACGGGUGAAC 26 142 GAAGGCAAACGGGUGAACC 27 143 AAGGCAAACGGGUGAACCA 28 181 UUCCAUACCUGGGCUGUGC 29 182 UCCAUACCUGGGCUGUGCU 30 183 CCAUACCUGGGCUGUGCUC 31 201 CUGCAAUUUGGUGCCAAUC 32 203 GCAAUUUGGUGCCAAUCCU 33 210 GGUGCCAAUCCUCUUGAGU 34 214 CCAAUCCUCUUGAGUUCCU 35 223 UUGAGUUCCUCAGAGCAAA 36 225 GAGUUCCUCAGAGCAAAUC 37 229 UCCUCAGAGCAAAUCAAAG 38 230 CCUCAGAGCAAAUCAAAGG 39 236 AGCAAAUCAAAGGAAACAU 40 238 CAAAUCAAAGGAAACAUGG 41 242 UCAAAGGAAACAUGGUCAU 42 243 CAAAGGAAACAUGGUCAUG 43 244 AAAGGAAACAUGGUCAUGU 44 245 AAGGAAACAUGGUCAUGUU 45 246 AGGAAACAUGGUCAUGUUU 46 251 ACAUGGUCAUGUUUUUACC 47 253 AUGGUCAUGUUUUUACCUG 48 254 UGGUCAUGUUUUUACCUGC 49 262 UUUUUACCUGCAAACUAAU 50 264 UUUACCUGCAAACUAAUGG 51 266 UACCUGCAAACUAAUGGGA 52 267 ACCUGCAAACUAAUGGGAA 53 268 CCUGCAAACUAAUGGGAAA 54 291 GUCCAUUUCAUCACAAAUC 55 292 UCCAUUUCAUCACAAAUCC 56 294 CAUUUCAUCACAAAUCCCU 57 300 AUCACAAAUCCCUUGUCAU 58 302 CACAAAUCCCUUGUCAUAC 59 303 ACAAAUCCCUUGUCAUACC 60 304 CAAAUCCCUUGUCAUACCA 61 305 AAAUCCCUUGUCAUACCAU 62 309 CCCUUGUCAUACCAUAAGG 63 310 CCUUGUCAUACCAUAAGGU 64 311 CUUGUCAUACCAUAAGGUG 65 312 UUGUCAUACCAUAAGGUGU 66 313 UGUCAUACCAUAAGGUGUU 67 315 UCAUACCAUAAGGUGUUGU 68 319 ACCAUAAGGUGUUGUGCCA 69 360 AAAUUUCACUUUGCUACUU 70 363 UUUCACUUUGCUACUUCUG 71 372 GCUACUUCUGCGAAGGCAU 72 375 ACUUCUGCGAAGGCAUUUG 73 377 UUCUGCGAAGGCAUUUGGG 74 390 UUUGGGCACAGAAGCAUUG 75 392 UGGGCACAGAAGCAUUGAC 76 394 GGCACAGAAGCAUUGACCC 77 474 GCCUUGAAUUCCCUCACGG 78 475 CCUUGAAUUCCCUCACGGA 79 476 CUUGAAUUCCCUCACGGAA 80 477 UUGAAUUCCCUCACGGAAA 81 478 UGAAUUCCCUCACGGAAAG 82 482 UUCCCUCACGGAAAGCAUG 83 484 CCCUCACGGAAAGCAUGAU 84 486 CUCACGGAAAGCAUGAUGG 85 488 CACGGAAAGCAUGAUGGAA 86 501 AUGGAAAACCUCCAACGUA 87 503 GGAAAACCUCCAACGUAUC 88 505 AAAACCUCCAACGUAUCAU 89 506 AAACCUCCAACGUAUCAUG 90 507 AACCUCCAACGUAUCAUGA 91 509 CCUCCAACGUAUCAUGAGA 92 510 CUCCAACGUAUCAUGAGAC 93 511 UCCAACGUAUCAUGAGACC 94 512 CCAACGUAUCAUGAGACCU 95 513 CAACGUAUCAUGAGACCUC 96 514 AACGUAUCAUGAGACCUCC 97 539 CUCUAACUCAAAGACCGCU 98 552 ACCGCUGCCUGGGUGACAG 99 562 GGGUGACAGAAGGGAUGUA 100 563 GGUGACAGAAGGGAUGUAU 101 564 GUGACAGAAGGGAUGUAUU 102 585 UUCUGCUACCGAGUGAUGU 103 587 CUGCUACCGAGUGAUGUUU 104 588 UGCUACCGAGUGAUGUUUG 105 589 GCUACCGAGUGAUGUUUGA 106 593 CCGAGUGAUGUUUGAAGCU 107 594 CGAGUGAUGUUUGAAGCUG 108 595 GAGUGAUGUUUGAAGCUGG 109 602 GUUUGAAGCUGGGUAUUUA 110 605 UGAAGCUGGGUAUUUAACU 111 606 GAAGCUGGGUAUUUAACUA 112 607 AAGCUGGGUAUUUAACUAU 113 608 AGCUGGGUAUUUAACUAUC 114 609 GCUGGGUAUUUAACUAUCU 115 610 CUGGGUAUUUAACUAUCUU 116 612 GGGUAUUUAACUAUCUUUG 117 613 GGUAUUUAACUAUCUUUGG 118 653 CACACAGAAAGCACAUAUU 119 663 GCACAUAUUCUAAACAAUC 120 665 ACAUAUUCUAAACAAUCUU 121 666 CAUAUUCUAAACAAUCUUG 122 667 AUAUUCUAAACAAUCUUGA 123 669 AUUCUAAACAAUCUUGACA 124 670 UUCUAAACAAUCUUGACAA 125 671 UCUAAACAAUCUUGACAAC 126 673 UAAACAAUCUUGACAACUU 127 679 AUCUUGACAACUUCAAGCA 128 712 UUCCAGCCCUGGUAGCAGG 129 771 GAGAAACUGGCAGAGAGCU 130 781 CAGAGAGCUUGAGGCACGA 131 788 CUUGAGGCACGAGAACCUC 132 841 UGCGCAUGUUUCUCAAUGA 133 844 GCAUGUUUCUCAAUGACAC 134 846 AUGUUUCUCAAUGACACUU 135 850 UUCUCAAUGACACUUUGUC 136 851 UCUCAAUGACACUUUGUCC 137 852 CUCAAUGACACUUUGUCCA 138 861 ACUUUGUCCACCUUUGAUG 139 862 CUUUGUCCACCUUUGAUGA 140 901 ACCUCGUGGUCCUCUGGGC 141 903 CUCGUGGUCCUCUGGGCAU 142 904 UCGUGGUCCUCUGGGCAUC 143 905 CGUGGUCCUCUGGGCAUCG 144 906 GUGGUCCUCUGGGCAUCGC 145 912 CUCUGGGCAUCGCAAGCAA 146 915 UGGGCAUCGCAAGCAAACA 147 916 GGGCAUCGCAAGCAAACAC 148 917 GGCAUCGCAAGCAAACACC 149 920 AUCGCAAGCAAACACCAUU 150 921 UCGCAAGCAAACACCAUUC 151 950 CUGGAGUUUAUUUCAAAUG 152 954 AGUUUAUUUCAAAUGAUUA 153 955 GUUUAUUUCAAAUGAUUAG 154 956 UUUAUUUCAAAUGAUUAGG 155 957 UUAUUUCAAAUGAUUAGGA 156 980 AGAAGCAAUGAAAGCAGCU 157 982 AAGCAAUGAAAGCAGCUAC 158 983 AGCAAUGAAAGCAGCUACU 159 988 UGAAAGCAGCUACUGAAGA 160 989 GAAAGCAGCUACUGAAGAA 161 992 AGCAGCUACUGAAGAAGUG 162 993 GCAGCUACUGAAGAAGUGA 163 994 CAGCUACUGAAGAAGUGAA 164 995 AGCUACUGAAGAAGUGAAA 165 996 GCUACUGAAGAAGUGAAAA 166 1005 GAAGUGAAAAGAACAUUAG 167 1006 AAGUGAAAAGAACAUUAGA 168 1007 AGUGAAAAGAACAUUAGAG 169 1011 AAAAGAACAUUAGAGAAUG 170 1012 AAAGAACAUUAGAGAAUGC 171 1013 AAGAACAUUAGAGAAUGCU 172 1018 CAUUAGAGAAUGCUGGUCA 173 1019 AUUAGAGAAUGCUGGUCAA 174 1020 UUAGAGAAUGCUGGUCAAA 175 1021 UAGAGAAUGCUGGUCAAAA 176 1026 AAUGCUGGUCAAAAAGUCA 177 1027 AUGCUGGUCAAAAAGUCAG 178 1029 GCUGGUCAAAAAGUCAGCU 179 1036 AAAAAGUCAGCUUGGAAGG 180 1039 AAGUCAGCUUGGAAGGCAA 181 1042 UCAGCUUGGAAGGCAAUCC 182 1084 UGAAUGACCUGCCAGUAUU 183 1087 AUGACCUGCCAGUAUUAGA 184 1134 UCCAGUGCCUCCCUCAACA 185 1151 CAUCCGGACAGCUAAGGAG 186 1152 AUCCGGACAGCUAAGGAGG 187 1155 CGGACAGCUAAGGAGGAUU 188 1158 ACAGCUAAGGAGGAUUUCA 189 1160 AGCUAAGGAGGAUUUCACU 190 1162 CUAAGGAGGAUUUCACUUU 191 1165 AGGAGGAUUUCACUUUGCA 192 1168 AGGAUUUCACUUUGCACCU 193 1169 GGAUUUCACUUUGCACCUU 194 1174 UCACUUUGCACCUUGAGGA 195 1177 CUUUGCACCUUGAGGACGG 196 1178 UUUGCACCUUGAGGACGGU 197 1189 AGGACGGUUCCUACAACAU 198 1191 GACGGUUCCUACAACAUCC 199 1194 GGUUCCUACAACAUCCGAA 200 1196 UUCCUACAACAUCCGAAAA 201 1200 UACAACAUCCGAAAAGAUG 202 1201 ACAACAUCCGAAAAGAUGA 203 1203 AACAUCCGAAAAGAUGACA 204 1204 ACAUCCGAAAAGAUGACAU 205 1205 CAUCCGAAAAGAUGACAUC 206 1207 UCCGAAAAGAUGACAUCAU 207 1209 CGAAAAGAUGACAUCAUAG 208 1212 AAAGAUGACAUCAUAGCUC 209 1214 AGAUGACAUCAUAGCUCUU 210 1215 GAUGACAUCAUAGCUCUUU 211 1217 UGACAUCAUAGCUCUUUAC 212 1226 AGCUCUUUACCCACAGUUA 213 1227 GCUCUUUACCCACAGUUAA 214 1228 CUCUUUACCCACAGUUAAU 215 1229 UCUUUACCCACAGUUAAUG 216 1230 CUUUACCCACAGUUAAUGC 217 1231 UUUACCCACAGUUAAUGCA 218 1233 UACCCACAGUUAAUGCACU 219 1236 CCACAGUUAAUGCACUUAG 220 1237 CACAGUUAAUGCACUUAGA 221 1238 ACAGUUAAUGCACUUAGAU 222 1239 CAGUUAAUGCACUUAGAUC 223 1240 AGUUAAUGCACUUAGAUCC 224 1241 GUUAAUGCACUUAGAUCCA 225 1242 UUAAUGCACUUAGAUCCAG 226 1248 CACUUAGAUCCAGAAAUCU 227 1249 ACUUAGAUCCAGAAAUCUA 228 1261 AAAUCUACCCAGACCCUUU 229 1283 UUUUAAAUAUGAUAGGUAU 230 1285 UUAAAUAUGAUAGGUAUCU 231 1286 UAAAUAUGAUAGGUAUCUU 232 1288 AAUAUGAUAGGUAUCUUGA 233 1290 UAUGAUAGGUAUCUUGAUG 234 1291 AUGAUAGGUAUCUUGAUGA 235 1292 UGAUAGGUAUCUUGAUGAA 236 1293 GAUAGGUAUCUUGAUGAAA 237 1296 AGGUAUCUUGAUGAAAACG 238 1297 GGUAUCUUGAUGAAAACGG 239 1298 GUAUCUUGAUGAAAACGGG 240 1299 UAUCUUGAUGAAAACGGGA 241 1300 AUCUUGAUGAAAACGGGAA 242 1301 UCUUGAUGAAAACGGGAAG 243 1302 CUUGAUGAAAACGGGAAGA 244 1303 UUGAUGAAAACGGGAAGAC 245 1306 AUGAAAACGGGAAGACAAA 246 1307 UGAAAACGGGAAGACAAAG 247 1311 AACGGGAAGACAAAGACUA 248 1313 CGGGAAGACAAAGACUACC 249 1314 GGGAAGACAAAGACUACCU 250 1353 UUAAAGUAUUACUACAUGC 251 1354 UAAAGUAUUACUACAUGCC 252 1358 GUAUUACUACAUGCCCUUU 253 1360 AUUACUACAUGCCCUUUGG 254 1361 UUACUACAUGCCCUUUGGA 255 1363 ACUACAUGCCCUUUGGAUC 256 1364 CUACAUGCCCUUUGGAUCG 257 1382 GGGAGCUACAAUAUGUCCU 258 1386 GCUACAAUAUGUCCUGGAA 259 1388 UACAAUAUGUCCUGGAAGA 260 1389 ACAAUAUGUCCUGGAAGAU 261 1413 GCUAUCCACGAAAUCAAGC 262 1415 UAUCCACGAAAUCAAGCAA 263 1416 AUCCACGAAAUCAAGCAAU 264 1417 UCCACGAAAUCAAGCAAUU 265 1424 AAUCAAGCAAUUUUUGAUU 266 1425 AUCAAGCAAUUUUUGAUUC 267 1431 CAAUUUUUGAUUCUGAUGC 268 1433 AUUUUUGAUUCUGAUGCUU 269 1506 CAGUCCCGGGCAGGCUUGG 270 1507 AGUCCCGGGCAGGCUUGGG 271 1510 CCCGGGCAGGCUUGGGCAU 272 1511 CCGGGCAGGCUUGGGCAUU 273 1513 GGGCAGGCUUGGGCAUUUU 274 1518 GGCUUGGGCAUUUUGCCGC 275 1555 UUAAAUAUAAAUUCAAGCA 276 1556 UAAAUAUAAAUUCAAGCAU 277 1557 AAAUAUAAAUUCAAGCAUU 278 1558 AAUAUAAAUUCAAGCAUUU 279 1559 AUAUAAAUUCAAGCAUUUG 280 1561 AUAAAUUCAAGCAUUUGUG 281 1593 AAUAAGAGGACACUAGAUG 282 1594 AUAAGAGGACACUAGAUGA 283 1597 AGAGGACACUAGAUGAUAU 284 1599 AGGACACUAGAUGAUAUUA 285 1601 GACACUAGAUGAUAUUACA 286 1605 CUAGAUGAUAUUACAGGAC 287 1606 UAGAUGAUAUUACAGGACU 288 1609 AUGAUAUUACAGGACUGCA 289 1610 UGAUAUUACAGGACUGCAG 290 1614 AUUACAGGACUGCAGAACA 291 1638 ACCACACAGUCCCUUUGGA 292 1660 AUGCAUUUAGUGGUGGUAG 293 1661 UGCAUUUAGUGGUGGUAGA 294 1662 GCAUUUAGUGGUGGUAGAA 295 1663 CAUUUAGUGGUGGUAGAAA 296 1669 GUGGUGGUAGAAAUGAUUC 297 1671 GGUGGUAGAAAUGAUUCAC 298 1673 UGGUAGAAAUGAUUCACCA 299 1676 UAGAAAUGAUUCACCAGGU 300 1683 GAUUCACCAGGUCCAAUGU 301 1686 UCACCAGGUCCAAUGUUGU 302 1689 CCAGGUCCAAUGUUGUUCA 303 1690 CAGGUCCAAUGUUGUUCAC 304 1692 GGUCCAAUGUUGUUCACCA 305 1712 UGCUUGCUUGUGAAUCUUA 306 1713 GCUUGCUUGUGAAUCUUAA 307 1769 UGCUAGUGAAAAGAACUAG 308 1770 GCUAGUGAAAAGAACUAGU 309 1824 AGUCCAUGAAUGUUCAUAU 310 1825 GUCCAUGAAUGUUCAUAUA 311 1826 UCCAUGAAUGUUCAUAUAG 312 1827 CCAUGAAUGUUCAUAUAGC 313 1828 CAUGAAUGUUCAUAUAGCC 314 1829 AUGAAUGUUCAUAUAGCCA 315 1830 UGAAUGUUCAUAUAGCCAG 316 1895 UUUUUCAAAAUGAAGAUAC 317 2052 UAUUCUAAUUGGCAGAUUG 318 2053 AUUCUAAUUGGCAGAUUGU 319 2075 UUCCUAAGGAAACUGCUUU 320 2127 AUGUUCAAAUUCACGUUCU 321 2128 UGUUCAAAUUCACGUUCUA 322 2132 CAAAUUCACGUUCUAGUGA 323 2133 AAAUUCACGUUCUAGUGAA 324 2134 AAUUCACGUUCUAGUGAAA 325 2135 AUUCACGUUCUAGUGAAAC 326 2136 UUCACGUUCUAGUGAAACU 327 2137 UCACGUUCUAGUGAAACUG 328 2139 ACGUUCUAGUGAAACUGCA 329 2140 CGUUCUAGUGAAACUGCAU 330 2144 CUAGUGAAACUGCAUUAUU 331 2189 GGUGUGAUCAUAUAUCAUA 332 2190 GUGUGAUCAUAUAUCAUAA 333 2191 UGUGAUCAUAUAUCAUAAA 334 2192 GUGAUCAUAUAUCAUAAAG 335 2193 UGAUCAUAUAUCAUAAAGG 336 2194 GAUCAUAUAUCAUAAAGGA 337 2195 AUCAUAUAUCAUAAAGGAU 338 2199 UAUAUCAUAAAGGAUAUUU 339 2209 AGGAUAUUUCAAAUGAUUA 340 2210 GGAUAUUUCAAAUGAUUAU 341 2211 GAUAUUUCAAAUGAUUAUG 342 2215 UUUCAAAUGAUUAUGAUUA 343 2216 UUCAAAUGAUUAUGAUUAG 344 2217 UCAAAUGAUUAUGAUUAGU 345 2218 CAAAUGAUUAUGAUUAGUU 346 2220 AAUGAUUAUGAUUAGUUAU 347 2221 AUGAUUAUGAUUAGUUAUG 348 2223 GAUUAUGAUUAGUUAUGUC 349 2224 AUUAUGAUUAGUUAUGUCU 350 2225 UUAUGAUUAGUUAUGUCUU 351 2313 GAUUUCCCAAAAACACUAA 352 2316 UUCCCAAAAACACUAAAGG 353 2317 UCCCAAAAACACUAAAGGU 354 2319 CCAAAAACACUAAAGGUGG 355 2320 CAAAAACACUAAAGGUGGU 356 2321 AAAAACACUAAAGGUGGUU 357 2353 AUGUUUUAACUUAUUGUUG 358 2355 GUUUUAACUUAUUGUUGCU 359 2356 UUUUAACUUAUUGUUGCUG 360 2362 CUUAUUGUUGCUGAAAACU 361 2364 UAUUGUUGCUGAAAACUCU 362 2365 AUUGUUGCUGAAAACUCUA 363 2366 UUGUUGCUGAAAACUCUAU 364 2370 UGCUGAAAACUCUAUGUCC 365 2503 AAAUGUAGCUUUUAUGUGA 366 2562 AAGCUUUGGUUAUGAAACA 367 2563 AGCUUUGGUUAUGAAACAU 368 2620 UUAAAUGCUUUUUAUCGCU 369 2621 UAAAUGCUUUUUAUCGCUA 370 2622 AAAUGCUUUUUAUCGCUAA 371 2623 AAUGCUUUUUAUCGCUAAA 372 2624 AUGCUUUUUAUCGCUAAAU 373 2625 UGCUUUUUAUCGCUAAAUG 374 2629 UUUUAUCGCUAAAUGACUU 375 2630 UUUAUCGCUAAAUGACUUG 376 2632 UAUCGCUAAAUGACUUGCA 377 2633 AUCGCUAAAUGACUUGCAG 378 2635 CGCUAAAUGACUUGCAGAU 379 2639 AAAUGACUUGCAGAUGAAA 380 2679 UUUAAAUGCUGUGUACAAC 381 2682 AAAUGCUGUGUACAACAAU 382 2683 AAUGCUGUGUACAACAAUG 383 2687 CUGUGUACAACAAUGCUUU 384 2690 UGUACAACAAUGCUUUGAU 385 2691 GUACAACAAUGCUUUGAUA 386 2692 UACAACAAUGCUUUGAUAA 387 2833 UAAUUUUGAAAUGAUUCAU 388 2834 AAUUUUGAAAUGAUUCAUC 389 2835 AUUUUGAAAUGAUUCAUCU 390 2838 UUGAAAUGAUUCAUCUUUC 391 2839 UGAAAUGAUUCAUCUUUCA 392 2858 GAAAUAAAAGUAUGAAUCU Each uracil base (U) in any one of the target sequences provided in Table 2 may independently and optionally be replaced with a thymine base (T).
Modified RNAi Agents (e.g., Modified CYP7A1 RNAi Agents)

(172) Some aspects of the present disclosure provide modified RNAi agents (e.g., modified CYP7A1 RNAi agents). In some embodiments, a modified RNAi agent (e.g., modified CYP7A1 RNAi agent) comprise modified nucleosides and/or modified internucleoside linkages.

(173) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) 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, 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, or more) modified nucleosides and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modified internucleoside linkages. In some embodiments, a modified nucleoside comprises a modification at the 2 position of the sugar (referred to herein as a 2-modified nucleoside). In some embodiments, all modified nucleosides in an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) are 2-modified nucleosides. In some embodiments, the 2-modified nucleoside is selected from 2-fluoro (2-F), 2-O-methyl (2-O-Me), 2-O-methoxyethyl (2-MOE), 2-O-aminopropyl (2-O-AP), 2-O-dimethylaminoethyl (2-O-DMAOE), 2-O-dimethylaminopropyl (2-O-DMAP), 2-O-dimethylaminoethyloxyethyl (2-O-DMAEOE), or 2-ON-methylacetamido (2-O-NMA) modified nucleoside, and combinations thereof. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a mix of two or more different 2-modified nucleosides (e.g., any combination of 2-F modified nucleosides, 2-O-Me modified nucleosides and 2-MOE modified nucleosides).

(174) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) modified internucleoside linkages. Nonlimiting examples of internucleoside linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3-amino phosphoramidate and aminoalkylphosphoramidates, mesyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) phosphorothioate internucleoside linkages.

(175) Table 3 lists various exemplary nucleosides with 3-phosphate or 3-phosphorothioate and the structures thereof.

(176) TABLE-US-00015 TABLE 3 Exemplary nucleosides with 3-phosphate or 3-phosphorothioate and structures Abbreviation Nucleosides or linkage Structure A Adenosine-3-phosphate fA 2-fluoroadenosine-3- phosphate fAs 2-fluoroadenosine-3- phosphorothioate C Cytidine-3-phosphate fC 2-fluorocytidine-3- phosphate fCs 2-fluorocytidine-3- phosphorothioate 0 G Guanosine-3-phosphate fG 2-fluoroguanosine-3- phosphate fGs 2-fluoroguanosine- 3-phosphorothioate U Uridine-3-phosphate fU 2-fluorouridine-3- phosphate fUs 2-fluorouridine-3- phosphorothioate mA 2-O-methyladenosine-3- phosphate mAs 2-O-methyladenosine-3- phosphorothioate mC 2-O-methylcytidine- 3-phosphate mCs 2-O-methylcytidine-3- phosphorothioate 0 mG 2-O-methylguanosine- 3-phosphate mGs 2-O-methylguanosine- 3-phosphorothioate mU 2'-O-methyluridine-3- phosphate mUs 2-O-methyluridine-3- phosphorothioate 2-O-methoxyethyl- adenosine-3-phosphate 2-O-methoxyethyl- adenosine-3-phos- phorothioate 2-O-methoxyethyl- cytidine-3-phosphate 2-O-methoxyethyl- cytidine-3- phosphorothioate 2-O-methoxyethyl- guanosine-3-phosphate 2-O-methoxyethyl- guanosine-3-phos- phorothioate 0 2-O-methoxyethyl- luridine-3-phosphate 2-O-methoxyethyl- uridine-3-phosphoro- thioate s Phosphorothioate linkage

(177) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises an antisense strand comprising one or more modified nucleosides (e.g., one or more 2-modified nucleosides). In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises an antisense strand comprising a mix of two or more different 2-modified nucleosides (e.g., any combination of 2-F modified nucleosides, 2-O-Me modified nucleosides, and 2-MOE modified nucleosides). In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand comprising one or more modified nucleosides (e.g., one or more 2-modified nucleosides). In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand comprising a mix of two or more different 2-modified nucleosides (e.g., any combination of 2-F modified nucleosides, 2-O-Me modified nucleosides, and 2-MOE modified nucleosides).

(178) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises an antisense strand and a sense strand, wherein each nucleoside of the antisense strand is a modified nucleoside (e.g., 2-modified nucleoside) and each nucleoside of the sense strand is a modified nucleoside (e.g., 2-modified nucleoside). In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises an antisense strand and a sense strand, wherein each nucleoside of the antisense strand is a 2-modified nucleoside selected from a 2-F modified nucleoside, a 2-O-Me modified nucleoside, and a 2-MOE modified nucleoside, and each nucleoside of the sense strand is a 2-modified nucleoside selected from a 2-F modified nucleoside, a 2-O-Me modified nucleoside, and a 2-MOE modified nucleoside. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises an antisense strand and a sense strand, wherein each nucleoside of the antisense strand is a 2-modified nucleoside selected from a 2-F modified nucleoside and a 2-O-Me modified nucleoside, and each nucleoside of the sense strand is a 2-modified nucleoside selected from a 2-F modified nucleoside and a 2-O-Me modified nucleoside.

(179) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at two or more (e.g., 2, 3, 4, 5, or 6) of positions 8, 9, 10, 11, 12, and 13 (counting 5.fwdarw.3) of the sense strand are nucleosides with the same 2 chemistry in the sugar moiety, e.g., unmodified (i.e., 2-hydroxy nucleoside), 2-F modified nucleoside, 2-O-Me modified nucleoside, 2-MOE modified nucleoside, or 2-deoxy nucleoside. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein each of the nucleosides at positions 8-12, 8-13, 9-12, or 9-13 (counting 5.fwdarw.3) of the sense strand are nucleosides with the same 2 chemistry in the sugar moiety, e.g., unmodified (i.e., 2-hydroxy nucleoside), 2-F modified nucleoside, 2-O-Me modified nucleoside, 2-MOE modified nucleoside, or 2-deoxy nucleoside. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein each of the nucleosides at positions 8-12, 8-13, 9-12, or 9-13 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein each of the nucleosides at positions 8-12, 8-13, 9-12, or 9-13 (counting 5-43) of the sense strand are unmodified nucleosides (i.e., 2-hydroxy nucleosides). The ranges referred to includes the nucleosides at both ends of the range. For example, nucleosides at positions 9-12 includes nucleosides at positions 9, 10, 11, and 12.

(180) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at two or more (e.g., 2, 3, 4, 5, or 6) of positions 8, 9, 10, 11, 12, and 13 (counting 5.fwdarw.3) of the sense strand are consecutive nucleosides with the same 2 chemistry in the sugar moiety, e.g., unmodified nucleosides (i.e., 2-hydroxy nucleosides), 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides and the nucleosides immediately upstream and downstream to the two or more consecutive nucleosides with the same 2 chemistry in the sugar moiety are nucleosides with different 2 chemistry in the sugar moiety.

(181) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, and 12 (counting 5.fwdarw.3) of the sense strand are nucleosides with the same 2 chemistry in the sugar moiety, e.g., 2-F modified nucleoside, 2-O-Me modified nucleoside, 2-MOE modified nucleoside, or 2-deoxy nucleoside and the nucleosides at positions 8 or 13 (counting 5.fwdarw.3) are nucleosides with different 2 chemistry in the sugar moiety including, e.g., unmodified nucleosides, i.e., 2-hydroxy nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, and 12 (counting 5.fwdarw.3) of the sense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides, and the nucleosides at positions 8 and 13 (counting 5.fwdarw.3) have a different 2 chemistry in the sugar moiety, e.g., 2-F modified nucleoside, 2-O-Me modified nucleoside, 2-MOE modified nucleoside, or 2-deoxy nucleoside.

(182) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 53) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 8 and 13 (counting 5.fwdarw.3) are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, one or more nucleosides of the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides. In some embodiments, all nucleosides of the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides.

(183) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more positions 1, 2, 3, 4, 5, 6, 7, 8 and 13, 14, 15, 16, 17, 18, 19, 20, 21 (counting 5.fwdarw.3) of the sense strand are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more positions 1, 2, 3, 4, 5, 6, 7, 8 and 13, 14, 15, 16, 17, 18, 19, 20, 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides.

(184) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8 and 13, 14, 15, 16, 17, 18, 19, 20, 21 (counting 5.fwdarw.3) of the sense strand are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8 and 13, 14, 15, 16, 17, 18, 19, 20, 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides.

(185) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more of positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides positions 2 and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides positions at 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, and one or more of positions 5, 6, and 8 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more of positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides and the nucleosides at other positions are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more of positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides and all nucleosides that are not 2-F modified nucleosides are 2-O-Me modified nucleosides.

(186) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more positions 1, 4, 5, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more positions 1, 4, 5, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides.

(187) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 4, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 4, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides.

(188) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, the nucleosides at positions 1, 4, 6, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, one or more nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides. In some embodiments, all nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides.

(189) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, the nucleosides at positions 1, 4, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, one or more nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides. In some embodiments, all nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides.

(190) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, the nucleosides at positions 1, 4, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, one or more nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides. In some embodiments, all nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides.

(191) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, the nucleosides at positions 1, 4, 5, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides, 2-MOE modified nucleosides, 2-deoxy nucleosides, or 2-hydroxy nucleosides. In some embodiments, one or more nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides. In some embodiments, all nucleosides of the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides.

(192) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises one or more (e.g., 1, 2, or 3) phosphorothioate internucleoside linkages in at least one strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises one or more (e.g., 1, 2, or 3) phosphorothioate internucleoside linkages in the sense strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises one or more (e.g., 1, 2, 3, 4, or 5) phosphorothioate internucleoside linkages in the antisense strand.

(193) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises two phosphorothioate internucleoside linkages in the sense strand and four phosphorothioate internucleoside linkages in the antisense strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises two phosphorothioate internucleoside linkages in the sense strand (e.g., the first two internucleoside linkages from 5.fwdarw.3) and four phosphorothioate internucleoside linkages in the antisense strand (e.g., the first two internucleoside linkages and the last two internucleoside linkages from 5.fwdarw.3). It is to be understood that, in any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein, the rest of internucleoside linkages, unless otherwise specified, are all phosphodiester internucleoside linkages.

(194) In some embodiments, provided is a population of two or more of the same modified RNAi agents as described herein, wherein all of the phosphorothioate internucleoside linkages are stereorandom. In some embodiments, provided is a chirally enriched population of such modified RNAi agents as described herein, wherein the chirally enriched population is enriched for modified RNAi agents comprising at least one particular phosphorothioate internucleoside linkage having a particular stereochemical configuration. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents comprising at least one particular phosphorothioate internucleoside linkage having the (Sp) or (Rp) configuration. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents comprising at least one particular phosphorothioate internucleoside linkage having the (Sp) configuration. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents comprising at least one particular phosphorothioate internucleoside linkage having the (Rp) configuration. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents having a particular, independently selected stereochemical configuration at each phosphorothioate internucleoside linkage. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents having the (Rp) configuration at one particular phosphorothioate internucleoside linkage and the (Sp) configuration at each of the remaining phosphorothioate internucleoside linkages. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents having the (Sp) configuration at one particular phosphorothioate internucleoside linkage and the (Rp) configuration at each of the remaining phosphorothioate internucleoside linkages. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents having at least 3 contiguous phosphorothioate internucleoside linkages in the (Sp), (Sp), and (Rp) configurations, in the 5 to 3 direction. In some embodiments, the chirally enriched population of modified RNAi agents is enriched for modified RNAi agents having at least 4 contiguous phosphorothioate internucleoside linkages in the (Sp), (Sp), (Sp), and (Rp) configurations, in the 5 to 3 direction.

(195) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages. An example of such a modified RNAi agent is provided in FIG. 1MOD 1 (M1).

(196) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages. An example of such a modified RNAi agent is provided in FIG. 1MOD 2 (M2).

(197) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages. An example of such a modified RNAi agent is provided in FIG. 1MOD 3 (M3).

(198) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages. An example of such a modified RNAi agent is provided in FIG. 1MOD 4 (M4).

(199) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that is 18-25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleobases of any one of SEQ ID NOs: 777-1190, and a sense strand that is 18-25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleobases of any one of SEQ ID NOs: 9-776, wherein: (i) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages; (ii) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages; (iii) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages; or (iv) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(200) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 777-1190 and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 393-776, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(201) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 777-1190, and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 393-776, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(202) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 777-1190, and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 393-776, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(203) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 777-1190, and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 393-776, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(204) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 1161-1190 and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 404, 419, 430, 439, 443, 464, 465, 473, 492, 493, 510, 514, 516, 517, 550, 553, 564, 566, 582, 591, 595, 597, 598, 604, 618, 623, 642, 648, 652, and 664, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(205) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 1161, 1162, 1165, 1166, 1169, 1173, 1174, 1180, 1181, 1184, 1188, and 1190 and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 404, 419, 439, 443, 464, 473, 493, 517, 566, 597, 598, 604, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(206) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 1161, 1162, 1165, 1166, 1169, 1173, 1174, 1180, 1181, 1184, 1188, and 1190 and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 404, 419, 439, 443, 464, 473, 493, 517, 566, 597, 598, 604, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(207) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 1161, 1162, 1165, 1166, 1169, 1173, 1174, 1180, 1181, 1184, 1188, and 1190 and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 404, 419, 439, 443, 464, 473, 493, 517, 566, 597, 598, 604, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(208) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that comprises the nucleobase sequence of any one of SEQ ID NOs: 1161, 1162, 1165, 1166, 1169, 1173, 1174, 1180, 1181, 1184, 1188, and 1190 and further comprises a sense strand that is substantially complementary to the antisense strand and comprises the nucleobase sequence of any one of SEQ ID NOs: 404, 419, 439, 443, 464, 473, 493, 517, 566, 597, 598, 604, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(209) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 419 and a structure of (5.fwdarw.3) [mUs][mCs][mU][mU][mG][mA][mG][mU][fU][fC][fC][fU][mC][mA][mG][mA][mG][mC][mA][mA][mAs] (SEQ ID NO: 1219), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1161 and a structure of (5.fwdarw.3):

(210) TABLE-US-00016 (i) (SEQIDNO:1989) [[mUs]][[fUs]][[fU]][[mG]][[fC]][[fU]][[fC]][[fU]][[mG]][[fA]][[mG]] [[mG]][[mA]][[fA]][[mC]][[mU]][[mC]][[mA]][[mA]][[mG]][[mAs]][[mAs]] [[mG]]; (ii) (SEQIDNO:1990) [[mUs]][[fUs]][[fU]][[mG]][[mC]][[mU]][[fC]][[mU]][[mG]][[fA]][[mG]] [[mG]][[mA]][[fA]][[mC]][[mU]][[mC]][[mA]][[mA]][[mG]][[mAs]][[mAs]] [[mG]]; (iii) (SEQIDNO:1991) [[mUs]][[fUs]][[fU]][[mG]][[fC]][[mU]][[fC]][[fU]][[mG]][[fA]][[mG]] [[mG]][[mA]][[fA]][[mC]][[mU]][[mC]][[mA]][[mA]][[mG]][[mAs]][[mAs]] [[mG]]; or (iv) (SEQIDNO:1992) [[mUs]][[fUs]][[fU]][[mG]][[fC]][[fU]][[fC]][[mU]][[mG]][[fA]][[mG]] [[mG]][[mA]][[fA]][[mC]][[mU]][[mC]][[mA]][[mA]][[mG]][[mAs]][[mAs]][[mG]]
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(211) ##STR00054##
wherein X is SH, i.e.,

(212) ##STR00055##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (Ib-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(213) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 517 and a structure of (5.fwdarw.3) [mGs][mCs][mG][mC][mA][mU][mG][mU][fU][fU][fC][fU][mC][mA][mA][mU][mG][mA][mC][mA][mAs] (SEQ ID NO: 1329), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1162 and a structure of (5.fwdarw.3):

(214) TABLE-US-00017 (i) (SEQIDNO:1993) [[mUs]][[fUs]][[fG]][[mU]][[fC]][[fA]][[fU]][[fU]][[mG]][[fA]][[mG]] [[mA]][[mA]][[fA]][[mC]][[mA]][[mU]][[mG]][[mC]][[mG]][[mCs]][[mAs]] [[mG]]; (ii) (SEQIDNO:1994) [[mUs]][[fUs]][[fG]][[mU]][[mC]][[mA]][[fU]][[mU]][[mG]][[fA]][[mG]] [[mA]][[mA]][[fA]][[mC]][[mA]][[mU]][[mG]][[mC]][[mG]][[mCs]][[mAs]] [[mG]]; (iii) (SEQIDNO:1995) [[mUs]][[fUs]][[fG]][[mU]][[fC]][[mA]][[fU]][[fU]][[mG]][[fA]][[mG]] [[mA]][[mA]][[fA]][[mC]][[mA]][[mU]][[mG]][[mC]][[mG]][[mCs]][[mAs]] [[mG]]; or (iv) (SEQIDNO:1996) [[mUs]][[fUs]][[fG]][[mU]][[fC]][[fA]][[fU]][[mU]][[mG]][[fA]][[mG]] [[mA]][[mA]][[fA]][[mC]][[mA]][[mU]][[mG]][[mC]][[mG]][[mCs]][[mAs]] [[mG]];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(215) ##STR00056##
wherein X is SH, i.e.,

(216) ##STR00057##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(217) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 597 and a structure of (5.fwdarw.3) [mUs][mAs][mG][mC][mU][mC][mU][mU][fU][fA][fC][fC][mC][mA][mC][mA][mG][mU][mU][mA][mAs] (SEQ ID NO: 1417), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1165 and a structure of (5.fwdarw.3):

(218) TABLE-US-00018 (i) (SEQIDNO:1999) [[mUs]][[fUs]][[fA]][[mA]][[fC]][[fU]][[fG]][[fU]][[mG]][[fG]][[mG]] [[mU]][[mA]][[fA]][[mA]][[mG]][[mA]][[mG]][[mC]][[mU]][[mAs]][[mAs]] [[mG]]; (ii) (SEQIDNO:2000) [[mUs]][[fUs]][[fA]][[mA]][[mC]][[mU]][[fG]][[mU]][[mG]][[fG]][[mG]] [[mU]][[mA]][[fA]][[mA]][[mG]][[mA]][[mG]][[mC]][[mU]][[mAs]][[mAs]] [[mG]]; (iii) (SEQIDNO:2001) [[mUs]][[fUs]][[fA]][[mA]][[fC]][[mU]][[fG]][[fU]][[mG]][[fG]][[mG]] [[mU]][[mA]][[fA]][[mA]][[mG]][[mA]][[mG]][[mC]][[mU]][[mAs]][[mAs]] [[mG]]; or (iv) (SEQIDNO:2002) [[mUs]][[fUs]][[fA]][[mA]][[fC]][[fU]][[fG]][[mU]][[mG]][[fG]][[mG]] [[mU]][[mA]][[fA]][[mA]][[mG]][[mA]][[mG]][[mC]][[mU]][[mAs]][[mAs]] [[mG]];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(219) ##STR00058##
wherein X is SH, i.e.,

(220) ##STR00059##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II, (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(221) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 566 and a structure of (5 .fwdarw.3) [mAs][mCs][mU][mG][mA][mA][mU][mG][fA][fC][fC][fU][mG][mC][mC][mA][mG][mU][mA][mU][mAs] (SEQ ID NO: 1382), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1166 and a structure of (5.fwdarw.3):

(222) TABLE-US-00019 (i) (SEQIDNO:2003) [mUs][fAs][fU][mA][fC][fU][fG][fG][mC][fA][mG][mG] [mU][fC][mA][mU][mU][mC][mA][mG][mUs][mAs][mG]; (ii) (SEQIDNO:2004) [mUs][fAs][fU][mA][mC][mU][fG][mG][mC][fA][mG][mG] [mU][fC][mA][mU][mU][mC][mA][mG][mUs][mAs][mG]; (iii) (SEQIDNO:2005) [mUs][fAs][fU][mA][fC][mU][fG][fG][mC][fA][mG][mG] [mU][fC][mA][mU][mU][mC][mA][mG][mUs][mAs][mG]; or (iv) (SEQIDNO:2006) [mUs][fAs][fU][mA][fC][fU][fG][mG][mC][fA][mG][mG] [mU][fC][mA][mU][mU][mC][mA][mG][mUs][mAs][mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(223) ##STR00060##
wherein X is SH, i.e.,

(224) ##STR00061##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(225) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 439 and a structure of (5.fwdarw.3) [mUs][mGs][mU][mC][mC][mA][mU][mU][fU][fC][fA][fU][mC][mA][mC][mA][mA][mA][mU][mC][mAs] (SEQ ID NO: 1241), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1169 and a structure of (5.fwdarw.3):

(226) TABLE-US-00020 (i) (SEQIDNO:2009) [mUs][fGs][fA][mU][fU][fU][fG][fU][mG][fA][mU][mG] [mA][fA][mA][mU][mG][mG][mA][mC][mAs][mAs][mG]; (ii) (SEQIDNO:2010) [mUs][fGs][fA][mU][mU][mU][fG][mU][mG][fA][mU][mG] [mA][fA][mA][mU][mG][mG][mA][mC][mAs][mAs][mG]; (iii) (SEQIDNO:2011) [mUs][fGs][fA][mU][fU][mU][fG][fU][mG][fA][mU][mG] [mA][fA][mA][mU][mG][mG][mA][mC][mAs][mAs][mG]; or (iv) (SEQIDNO:2012) [mUs][fGs][fA][mU][fU][fU][fG][mU][mG][fA][mU][mG] [mA][fA][mA][mU][mG][mG][mA][mC][mAs][mAs][mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(227) ##STR00062##
wherein X is SH, i.e.,

(228) ##STR00063##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (1-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(229) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 604 and a structure of (5.fwdarw.3) [mCs][mCs][mC][mA][mC][mA][mG][mU][fU][fA][fA][fU][mG][mC][mA][mC][mU][mU][mA][mG][mAs] (SEQ ID NO: 1425), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1173 and a structure of (5.fwdarw.3):

(230) TABLE-US-00021 (i) (SEQIDNO:2017) [mUs][fCs][fU][mA][fA][fG][fU][fG][mC][fA][mU][mU] [mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG]; (ii) (SEQIDNO:2018) [mUs][fCs][fU][mA][mA][mG][fU][mG][mC][fA][mU][mU] [mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG]; (iii) (SEQIDNO:2016) [mUs][fCs][fU][mA][fA][mG][fU][fG][mC][fA][mU][mU] [mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG]; or (iv) (SEQIDNO:2019) [mUs][fCs][fU][mA][fA][fG][fU][mG][mC][fA][mU][mU] [mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(231) ##STR00064##
wherein X is SH, i.e.,

(232) ##STR00065##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(233) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 598 and a structure of (5.fwdarw.3) [mAs][mGs][mC][mU][mC][mU][mU][mU][fA][fC][fC][fC][mA][mC][mA][mG][mU][mU][mA][mA][mAs] (SEQ ID NO: 1418), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1174 and a structure of (5.fwdarw.3):

(234) TABLE-US-00022 (i) (SEQIDNO:2021) [mUs][fUs][fU][mA][fA][fC][fU][fG][mU][fG][mG][mG] [mU][fA][mA][mA][mG][mA][mG][mC][mUs][mAs][mG]; (ii) (SEQIDNO:2018) [mUs][fCs][fU][mA][mA][mG][fU][mG][mC][fA][mU][mU] [mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG]; (iii) (SEQIDNO:2020) [mUs][fUs][fU][mA][fA][mC][fU][fG][mU][fG][mG][mG] [mU][fA][mA][mA][mG][mA][mG][mC][mUs][mAs][mG]; or (iv) (SEQIDNO:2023) [mUs][fUs][fU][mA][fA][fC][fU][mG][mU][fG][mG][mG] [mU][fA][mA][mA][mG][mA][mG][mC][mUs][mAs][mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(235) ##STR00066##
wherein X is SH, i.e.,

(236) ##STR00067##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(237) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 404 and a structure of (5.fwdarw.3) [mUs][mUs][mG][mU][mC][mU][mA][mU][fG][fG][fC][fU][mU][mA][mU][mU][mC][mU][mU][mG][mAs] (SEQ ID NO: 1202), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1180 and a structure of (5.fwdarw.3):

(238) TABLE-US-00023 (i) (SEQIDNO:2029) [mUs][fCs][fA][mA][fG][fA][fA][fU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG]; (ii) (SEQIDNO:2030) [mUs][fCs][fA][mA][mG][mA][fA][mU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG]; (iii) (SEQIDNO:2031) [mUs][fCs][fA][mA][fG][mA][fA][fU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG]; or (iv) (SEQIDNO:2032) [mUs][fCs][fA][mA][fG][fA][fA][mU][mA][fA][mG][mC] [mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(239) ##STR00068##
wherein X is SH, i.e.,

(240) ##STR00069##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (1-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(241) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 464 and a structure of (5.fwdarw.3) [mCs][mCs][mU][mU][mG][mA][mA][mU][fU][fC][fC][fC][mU][mC][mA][mC][mG][mG][mA][mA][mAs] (SEQ ID NO: 1267), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1181 and a structure of (5.fwdarw.3):

(242) TABLE-US-00024 (i) (SEQIDNO:2034) [mUs][fUs][fU][mC][fC][fG][fU][fG][mA][fG][mG][mG] [mA][fA][mU][mU][mC][mA][mA][mG][mGs][mAs][mG]; (ii) (SEQIDNO:2035) [mUs][fUs][fU][mC][mC][mG][fU][mG][mA][fG][mG][mG] [mA][fA][mU][mU][mC][mA][mA][mG][mGs][mAs][mG]; iii) (SEQIDNO:2036) [mUs][fUs][fU][mC][fC][mG][fU][fG][mA][fG][mG][mG] [mA][fA][mU][mU][mC][mA][mA][mG][mGs][mAs][mG]; or (iv) (SEQIDNO:2033) [mUs][fUs][fU][mC][fC][fG][fU][mG][mA][fG][mG][mG] [mA][fA][mU][mU][mC][mA][mA][mG][mGs][mAs][mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(243) ##STR00070##
wherein X is SH, i.e.,

(244) ##STR00071##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(245) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 443 and a structure of (5.fwdarw.3) [mUs][mCs][mA][mC][mA][mA][mA][mU][fC][fC][fC][fU][mU][mG][mU][mC][mA][mU][mA][mC][mAs] (SEQ ID NO: 1246), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1184 and a structure of (5.fwdarw.3):

(246) TABLE-US-00025 (i) (SEQIDNO:2039) [mUs][fGs][fU][mA][fU][fG][fA][fC][mA][fA][mG] [mG][mG][fA][mU][mU][mU][mG][mU][mG][mAs][mAs] [mG]; (ii) (SEQIDNO:2040) [mUs][fGs][fU][mA][mU][mG][fA][mC][mA][fA][mG] [mG][mG][fA][mU][mU][mU][mG][mU][mG][mAs][mAs] [mG]; (iii) (SEQIDNO:2041) [mUs][fGs][fU][mA][fU][mG][fA][fC][mA][fA][mG] [mG][mG][fA][mU][mU][mU][mG][mU][mG][mAs][mAs] [mG]; or (iv) (SEQIDNO:2042) [mUs][fGs][fU][mA][fU][fG][fA][mC][mA][fA][mG] [mG][mG][fA][mU][mU][mU][mG][mU][mG][mAs][mAs] [mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(247) ##STR00072##
wherein X is SH, i.e.,

(248) ##STR00073##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(249) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 493 and a structure of (5.fwdarw.3) [mAs][mUs][mG][mU][mU][mU][mG][mA][fA][fG][fC][fU][mG][mG][mG][mU][mA][mU][mU][mU][mAs] (SEQ ID NO: 1301), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1188 and a structure of (5.fwdarw.3):

(250) TABLE-US-00026 (i) (SEQIDNO:2046) [mUs][fAs][fA][mA][fU][fA][fC][fC][mC][fA][mG] [mC][mU][fU][mC][mA][mA][mA][mC][mA][mUs][mAs] [mG]; (ii) (SEQIDNO:2047) [mUs][fAs][fA][mA][mU][mA][fC][mC][mC][fA][mG] [mC][mU][fU][mC][mA][mA][mA][mC][mA][mUs][mAs] [mG]; (iii) (SEQIDNO:2048) [mUs][fAs][fA][mA][fU][mA][fC][fC][mC][fA][mG] [mC][mU][fU][mC][mA][mA][mA][mC][mA][mUs][mAs] [mG]; or (iv) (SEQIDNO:2049) [mUs][fAs][fA][mA][fU][fA][fC][mC][mC][fA][mG] [mC][mU][fU][mC][mA][mA][mA][mC][mA][mUs][mAs] [mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(251) ##STR00074##
wherein X is SH, i.e.,

(252) ##STR00075##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(253) In some embodiments, a CYP7A1 RNAi agent described herein comprises: a sense strand comprising the nucleobase sequence of SEQ ID NO: 473 and a structure of (5.fwdarw.3) [mGs][mAs][mA][mA][mA][mC][mC][mU][fC][fC][fA][fA][mC][mG][mU][mA][mU][mC][mA][mU][mAs] (SEQ ID NO: 1279), and an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1190 and a structure of (5.fwdarw.3):

(254) TABLE-US-00027 (i) (SEQIDNO:2051) [mUs][fAs][fU][mG][fA][fU][fA][fC][mG][fU][mU] [mG][mG][fA][mG][mG][mU][mU][mU][mU][mCs][mAs] [mG]; (ii) (SEQIDNO:2052) [mUs][fAs][fU][mG][mA][mU][fA][mC][mG][fU][mU] [mG][mG][fA][mG][mG][mU][mU][mU][mU][mCs][mAs] [mG]; (iii) (SEQIDNO:2053) [mUs][fAs][fU][mG][fA][mU][fA][fC][mG][fU][mU] [mG][mG][fA][mG][mG][mU][mU][mU][mU][mCs][mAs] [mG]; or (iv) (SEQIDNO:2054) [mUs][fAs][fU][mG][fA][fU][fA][mC][mG][fU][mU] [mG][mG][fA][mG][mG][mU][mU][mU][mU][mCs][mAs] [mG];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage. It is to be understood that the s of the 3-terminal [mAs] of the sense strand indicates a phosphorothioate linkage between the 3-terminal nucleoside of the sense strand and a conjugated moiety (e.g., a targeting moiety described herein). In some embodiments, the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(255) ##STR00076##
wherein X is SH, i.e.,

(256) ##STR00077##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).

(257) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8, 14, 15, 16, 17, 18, 20, and 21 (counting 5.fwdarw.3) of the sense strand are nucleosides with a first 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides, and the nucleosides at positions 9, 10, 11, 12, 13, and 19 (counting 5.fwdarw.3) of the sense strand are nucleosides with a second 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides, wherein the first 2 chemistry in the sugar moiety and the second 2 chemistry in the sugar moiety are different. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8, 14, 15, 16, 17, 18, 20, and 21 (counting 5.fwdarw.3) of the sense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides, and the nucleosides at positions 9, 10, 11, 12, 13, and 19 (counting 5.fwdarw.3) of the sense strand are nucleosides with a different 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12, 13, and 19 (counting 5.fwdarw.3) of the sense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides, and the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8, 14, 15, 16, 17, 18, 20, and 21 (counting 5.fwdarw.3) of the sense strand are nucleosides with a different 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides.

(258) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8, 14, 15, 16, 17, 18, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides and the nucleosides at positions 9, 10, 11, 12, 13, and 19 (counting 5.fwdarw.3) of the sense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides.

(259) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 4, 11, 12, 16, 18, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are nucleosides with a first 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides, and the nucleosides at positions 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 17, 19, and 20 (counting 5.fwdarw.3) of the antisense strand are nucleosides with a second 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides, wherein the first 2 chemistry in the sugar moiety and the second 2 chemistry in the sugar moiety are different. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 4, 11, 12, 16, 18, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides, and the nucleosides at positions 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 17, 19, and 20 (counting 5.fwdarw.3) of the antisense strand are nucleosides with a different 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleoside. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 17, 19, and 20 (counting 5.fwdarw.3) of the antisense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides, and the nucleosides at positions 1, 4, 11, 12, 16, 18, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are nucleosides with a different 2 chemistry in the sugar moiety, e.g., 2-F modified nucleosides, 2-O-Me modified nucleosides, 2-MOE modified nucleosides, or 2-deoxy nucleosides.

(260) In some embodiment, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 4, 11, 12, 16, 18, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are the 2-O-Me modified nucleosides, and the nucleosides at positions 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 17, 19, and 20 (counting 5.fwdarw.3) of the antisense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides.

(261) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand that is 18-25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleobases of any one of SEQ ID NOs: 777-1160, and a sense strand that is 18-25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and comprises at least 15 (e.g., 16, 17, 18, 19, 20, 21, 22, or 23) consecutive nucleobases of any one of SEQ ID NOs: 9-776, wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8, 14, 15, 16, 17, 18, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides and the nucleosides at positions 9, 10, 11, 12, 13, and 19 (counting 5.fwdarw.3) of the sense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides, and wherein the nucleosides at positions 1, 4, 11, 12, 16, 18, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are the 2-O-Me modified nucleosides, and the nucleosides at positions 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 17, 19, and 20 (counting 5.fwdarw.3) of the antisense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides.

(262) In some embodiments, a CYP7A1 RNAi agent described herein comprises an antisense strand comprising the nucleobase sequence of any one of SEQ ID NOs: 777-1160, and a sense strand comprising the nucleobase sequence of any one of SEQ ID NOs: 9-776, wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides and the nucleosides at positions 9, 10, 11, and 12 (counting 5.fwdarw.3) of the sense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides, and wherein the nucleosides at positions 1, 4, 11, 12, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are the 2-O-Me modified nucleosides, and the nucleosides at positions 2, 3, 5, 6, 7, 8, 9, 10, 13, 14, 15, 16, and 17 (counting 5.fwdarw.3) of the antisense strand are unmodified nucleosides, i.e., 2-hydroxy nucleosides.

(263) Additional Modified CYP7A1 RNAi Agents

(264) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprising nucleobase sequences (e.g., nucleobase sequences of the antisense strand and the sense strand) of the siRNAs listed in Table 5A and Table 7A can include any of the chemical modifications (e.g., one or more modified nucleosides and/or one or more modified internucleoside linkages) described below. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein can include any of the chemical modifications as disclosed in Hu et al. Therapeutic siRNA: State of the Art. Sig Transduct Target Ther 5, 101 (2020), the entire contents of which is herein incorporated by reference.

(265) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises any of the chemical modifications and/or modification patterns as disclosed in U.S. Pat. Nos. 10,233,448, 11,504,391, 9,290,760, 9,796,974, 9,399,775 9,796,974, 11,015,198, 10,676,742, 11,661,604, 11,655,473, and WO2022204430. The disclosures in these references related to chemical modifications of oligonucleotides (e.g., RNAi agents) are incorporated herein by reference.

(266) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises one or more chemical modifications and/or modification patterns as described in U.S. Pat. Nos. 10,233,448, 11,504,391, 9,290,760, 9,796,974, 9,399,775, 9,796,974, and 11,015,198. For example, in some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein one or both of the sense strand and the antisense strand comprise chemical modifications on nucleosides of a portion or the entire strand that form an alternating motif. The term alternating motif refers to a type of modification pattern formed by one or more repeats of a sequence (e.g., a sequence of two, three, four, five, six, or more nucleosides), wherein each repeat of the sequence comprises a chemical modification on each nucleoside forming a pattern (e.g., an alternating pattern). For example, if A, B and C each represent one type of modified nucleoside, the alternating motif can be ABABABABABAB . . . , AABBAABBAABB . . . , AABAABAABAAB . . . , AAABAAABAAAB . . . , AAABBBAAABBB . . . , or ABCABCABCABC . . . , etc.

(267) The type of modifications contained in an alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on a nucleoside, an alternating motif, e.g., modifications on every other nucleoside, may be the same throughout the entire alternating motif (e.g., ABABABAB . . . throughout), or may be different in one portion of the motif than another portion (e.g., ABABABABACACACAC). In some embodiments, each of the sense strand or antisense strand can comprise several possibilities of alternating patterns such as ABABAB . . . , ACACAC . . . , BDBDBD . . . , or CDCDCD . . . , etc., within an alternating motif.

(268) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein may comprise, on one or both of the sense strand and the antisense strand, one or more alternating motifs. In some embodiments, the type of modifications contained in one or more alternating motif of the sense strand may be the same or different from the one or more alternating motifs of the antisense strand.

(269) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein comprises a sense strand and an antisense strand, wherein the sense strand comprises a different alternating motif relative to the alternating motif on the antisense strand.

(270) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, each of which comprises an alternating motif composed of the same types of modifications on nucleosides, but each nucleoside on the antisense strand comprises a different modification than the nucleoside it base pairs with on the sense strand. In some embodiments, in an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein, the sense strand when paired with the antisense strand to form a duplex region, the alternating motif in the sense strand may start with ABABAB from 5.fwdarw.3 of the sense strand and the alternating motif in the antisense strand may start with ABABAB from 5.fwdarw.3 of the antisense strand within the duplex region. In some embodiments, the alternating motif in the sense strand may start with AABBAABB from 5 .fwdarw.3 of the sense strand and the alternating motif in the antisense strand may start with AABBAABB from 5.fwdarw.3 of the antisense strand within the duplex region. In some embodiments, in an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein, the sense strand when paired with the antisense strand to form a duplex region, the alternating motif in the sense strand may start with ABABAB from 5.fwdarw.3 of the sense strand and the alternating motif in the antisense strand may start with BABABA from 5.fwdarw.3 of the antisense strand within the duplex region. In some embodiments, the alternating motif in the sense strand may start with AABBAABB from 5.fwdarw.3 of the sense strand and the alternating motif in the antisense strand may start with BBAABBAA from 5.fwdarw.3 of the antisense strand within the duplex region.

(271) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand or the antisense strand comprises at least two different types of modified nucleosides (e.g., 2-O-Me and 2-F modified nucleosides). In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein each of the sense strand and the antisense strand comprises at least two different types of modified nucleosides (e.g., 2-O-Me and 2-F modified nucleosides) on each strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein each nucleoside of the sense strand and the antisense strand is a modified nucleoside, and wherein each of the sense strand and the antisense strand comprises an alternating motif of 2-F modified nucleosides and 2-O-Me modified nucleosides.

(272) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand comprises an alternating motif of 2-O-Me modified nucleosides and 2-F modified nucleosides and the antisense strand comprises an alternating motif of 2-O-Me modified nucleosides and 2-F modified nucleosides, wherein a 2-O-Me modified nucleoside on the sense strand base pairs with a 2-F modified nucleoside on the antisense strand and vice versa. In some embodiments, position 1 (counting 5.fwdarw.3) of the sense strand is a 2-F modified nucleoside, and the corresponding position of the antisense strand is a 2-O-Me modified nucleoside. In some embodiments, position 1 (counting 5.fwdarw.3) of the sense strand is a 2-O-Me modified nucleoside, and the corresponding position of the antisense strand is a 2-F modified nucleoside. In some embodiments, an RNAi agent (e.g., CYP7A1 RNA agent) further comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the first two internucleoside linkages (from 5.fwdarw.3) and the last two internucleoside linkages (from 5.fwdarw.3) of the sense strand are internucleoside linkages and/or (e.g., and) the last two internucleoside linkages (from 5.fwdarw.3) of the antisense strand are internucleoside linkages. In some embodiments, the sense strand and the antisense strand are both 19 nucleosides in length and for a duplex of 19 base pairs in length.

(273) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein one or both of the sense strand and the antisense strand comprises one or more motifs of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications). In some embodiments, at least one of the one or more motifs of three nucleosides is located on the sense and/or antisense strand at positions corresponding to or near the site on a target mRNA where cleavage occurs (these positions on the sense and/or antisense strand are collectively referred to herein as the cleavage site). In some embodiments, at least one of the one or more motifs of three nucleosides is located at or near the cleavage site on the antisense strand (e.g., positions 10, 11, and 12 (counting 5.fwdarw.3) of the antisense strand). For an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein comprising a duplex region of 19-23 nucleosides in length, the positions on the antisense strand corresponding to the cleavage site is typically around positions 10, 11, and 12 (counting 5.fwdarw.3). Thus, the three identical sugar modifications (e.g., identical 2-modifications) may occur at positions 9, 10, 11; positions 10, 11, 12; positions 11, 12, 13; positions 12, 13, 14; or positions 13, 14, 15 of the antisense strand (counting 5.fwdarw.3 or counting from the first paired nucleoside within the duplex region from the 5-end of the antisense strand). The positions on the antisense strand corresponding to the cleavage site may also change according to the length of the duplex region of the RNAi agent from the 5-end.

(274) In some embodiments, when the sense strand and antisense strand of an RNAi agent comprise one or more motifs of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at or near the cleavage site on at least one strand of an RNAi agent, gene silencing activity of the RNAi agent is observed.

(275) In some embodiments, the sense strand of an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein comprises at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications), wherein at least one of the motifs occurs at or near the cleavage site on the sense strand.

(276) In some embodiments, the antisense strand of the RNAi agent comprises at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications), wherein at least one of the motifs occurs at or near the cleavage site on the antisense strand.

(277) In some embodiments, the rest of the nucleosides in the sense strand and antisense strand of the RNAi agent are modified nucleosides. In some embodiments, the rest of the nucleosides in the sense strand and antisense strand of the RNAi agent are modified nucleosides with a different modification than the modification on the motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications).

(278) In some embodiments, the sense strand of an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein comprises at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at or near the cleavage site on the sense strand and the antisense strand comprises at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at or near the cleavage site on the antisense strand. When the sense strand and the antisense strand form a duplex region, the sense strand and the antisense strand can be so aligned that one motif of the three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) on the sense strand and one motif of the three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) on the antisense strand have at least one nucleoside overlap, i.e., at least one of the three consecutive nucleosides of the motif on the sense strand forms a base pair with at least one of the three consecutive nucleosides of the motif on the antisense strand. Alternatively, at least two nucleosides may overlap, or all three nucleosides may overlap. In some embodiments, the overlapping nucleoside on the sense strand comprises a different sugar modification than the nucleoside it overlaps with on the antisense strand.

(279) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein is an RNAi agent comprising a duplex region of 19 nucleosides in length, wherein the duplex region is formed by a sense strand and an antisense strand, wherein the nucleosides at three consecutive positions of the sense strand are 2-F modified nucleosides and the nucleosides at three consecutive positions of the antisense strand are 2-O-Me modified nucleosides. In some embodiments, the nucleosides at positions 7, 8, and 9 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 11, 12, and 13 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides.

(280) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) is an RNAi agent comprising a duplex region of 20 nucleosides in length, wherein the duplex region is formed by a sense strand and an antisense strand, wherein the nucleosides at three consecutive positions of the sense strand are 2-F modified nucleosides and the nucleosides at three consecutive positions of the antisense strand are 2-O-Me modified nucleosides. In some embodiments, the nucleosides at positions 8, 9, and 10 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 11, 12, and 13 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides.

(281) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein is an RNAi agent comprising a duplex region of 21 nucleosides in length, wherein the duplex region is formed by a sense strand and an antisense strand, wherein nucleosides at three consecutive positions of the sense strand are 2-F modified nucleosides and the nucleosides at three consecutive positions of the antisense strand are 2-O-Me modified nucleosides. In some embodiments, the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 11, 12, and 13 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides.

(282) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein comprises a sense strand and an antisense strand wherein the sense strand is 21 nucleosides in length and the antisense strand is 23 nucleosides in length, wherein the sense strand comprises at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., 2-F modified nucleosides) and the antisense strand comprises at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., 2-O-Me modified nucleosides). In some embodiments, the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 11, 12, and 13 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides. In some embodiments, the rest of the nucleosides in the sense strand and the antisense strand of the RNAi agent are modified nucleosides (e.g., 2 modified nucleosides). In some embodiments, the rest of the nucleosides in the sense strand and antisense strand are a mix of 2-F modified nucleosides and 2-O-Me modified nucleosides.

(283) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein one or both of the sense strand and the antisense strand comprise the at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at or near the cleavage site and comprise one or more alternating motifs. In some embodiments, the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 11, 12, and 13 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, and the rest of the nucleosides in the sense strand and the antisense strand comprise alternating motifs of 2-O-Me modified nucleosides and 2-F modified nucleosides.

(284) In some embodiments, when the motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) is present on any of the strands, the modification of the nucleoside next to the motif is a different modification than the modification of the motif. For example, in some embodiments, the portion of the sequence containing the motif is . . . N.sub.aYYYN.sub.b . . . , wherein Y represents the modification of the motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications), and N.sub.a and N.sub.b represent a modification to the nucleoside next to the motif YYY that is different than the modification of Y, and where N.sub.a and N.sub.b can be the same or different modifications.

(285) In some embodiments, an RNAi agent comprising a sense strand and an antisense strand each or both comprising one or more motifs of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) and alternating motifs described herein comprise two blunt ends, one blunt end and one end with an overhang, or two ends each with overhangs. In some embodiments, one end of the RNAi agent is blunt, while the other end comprises a two nucleoside overhang (e.g., formed by a sense strand of 21 nucleosides in length and an antisense strand of 23 nucleosides in length). In some embodiments, the two nucleoside overhang is at the 3-end of the antisense strand.

(286) In some embodiments, a two nucleoside overhang is at the 3-end of the antisense strand, and the antisense strand comprises two phosphorothioate internucleoside linkages between the terminal three nucleosides, wherein two of the three nucleosides are the overhang nucleosides, and the third nucleoside is a paired nucleoside next to the overhang nucleoside. In some embodiments, the RNAi agent further comprises two phosphorothioate internucleoside linkages of the sense strand (e.g., first two internucleoside linkages from 5.fwdarw.3) and two phosphorothioate internucleoside linkages in the antisense strand (e.g., first two internucleoside linkages from 5.fwdarw.3).

(287) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 4, 6, 8, 10, 14, 16, 18, 20, and 22 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 5, 7, 9, 11, 12, 13, 15, 17, 19, 21, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, and wherein the last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(288) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent) described herein comprises a sense strand and an antisense strand, wherein one or both of the sense strand and the antisense strand comprise more than one (e.g., 2, 3, or more) motifs of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications). The first motif may occur at or near the cleavage site on the sense strand or antisense strand and the other motif(s) may occur at another portion of the strand that is separated from (e.g., by at least one or more nucleosides) the first motif or may be adjacent to the first motif. When the other motif(s) are adjacent to the first motif, the chemistries are different from that of the first motif. When the motifs are separated by one or more nucleosides, the chemistries can be the same or different. In some embodiments, when two other motifs are present, each motif may occur at one end relative to the first motif which is at or near cleavage site or on either side of the first motif.

(289) Like the sense strand, in some embodiments, the antisense strand of the RNAi agent may contain more than one motif (e.g., 2, 3, or more) of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications), with at least one of the motifs occurring at or near the cleavage site on the strand. In some embodiments, the antisense strand comprises one or more motifs of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) in an alignment with the motifs of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) that are present on the sense strand.

(290) In some embodiments, a motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two nucleosides at the 3-end, 5-end, or both ends of the strand.

(291) In some embodiments, a motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleosides within the duplex region at the 3-end, 5-end, or both ends of the strand.

(292) When the sense strand and the antisense strand of the RNAi agent each comprise at least one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications), the motifs may fall on the same end of the duplex region, and have an overlap of one, two, or three nucleosides.

(293) When the sense strand and the antisense strand of the RNAi agent each comprise at least two motifs of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications), the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, comprising an overlap of one, two, or three nucleosides; two modifications each from one strand fall on the other end of the duplex region, comprising an overlap of one, two or three nucleosides; two modifications one strand fall on each side of the lead motif, comprising an overlap of one, two or three nucleosides in the duplex region.

(294) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at the 5 end or 3 end of the sense strand and/or the antisense strand. In some embodiments, the antisense strand comprises a motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at the 3 end of the strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand and antisense strand each comprise a motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at or near the cleavage site, wherein the RNAi agents further comprise a motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications) at the 3 end of the sense strand, and wherein the sense strand and antisense strand each comprise an alternating motif comprising chemical modifications in an alternating pattern. In some embodiments, the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 11, 12, and 13 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides and the rest of the nucleosides in the sense strand and the antisense strand comprise one or more alternating motifs.

(295) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, 19, and 21 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting 5 3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 5, 7, 9, 11, 12, 13, 15, 17, 19, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(296) In some embodiments, the sense strand comprises modified nucleosides that form a modification pattern represented by formula (IV):

(297) TABLE-US-00028 (IV) 5n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.j-N.sub.a-n.sub.q3 wherein: i and j are each independently 0 or 1; p and q are each independently 0-6; each N.sub.a independently represents an oligonucleotide sequence comprising 0-25 modified nucleosides, each sequence comprising at least two differently modified nucleosides; each N.sub.b independently represents an oligonucleotide sequence comprising 0-10 modified nucleosides; each n.sub.p and n.sub.q independently represent an overhang nucleoside; wherein N.sub.b and Y do not have the same modification; and XXX, YYY, and ZZZ each independently represent one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications). Preferably YYY is all 2-F modified nucleosides.

(298) In some embodiments, the N.sub.a or N.sub.b comprises an alternating pattern of modified nucleosides.

(299) In some embodiments, the YYY motif occurs at or near the cleavage site on the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleosides in length, the YYY motif can occur at or the near the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) on the sense strand, (counting 5.fwdarw.3) or counting from the first paired nucleoside within the duplex region, from the 5-end.

(300) In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

(301) TABLE-US-00029 (IVb) 5n.sub.p-N.sub.a-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3; (IVc) 5n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.a-n.sub.q3; or (IVd) 5n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3.

(302) When the sense strand is represented by formula (IVb), N.sub.b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Each N.sub.a independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(303) When the sense strand is represented as formula (IVc), N.sub.b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Each N.sub.a can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(304) When the sense strand is represented as formula (IVd), each N.sub.b independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Preferably, N.sub.b is 0, 1, 2, 3, 4, 5, or 6. Each N.sub.a can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(305) Each of X, Y and Z may be the same or different from each other.

(306) In some embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

(307) TABLE-US-00030 (IVa) 5n.sub.p-N.sub.a-YYY-N.sub.a-n.sub.q3.

(308) When the sense strand is represented by formula (IVa), each N.sub.a independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(309) In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (V):

(310) TABLE-US-00031 (V) 5n.sub.q-N.sub.a-(ZZZ).sub.k-N.sub.b-YYY-N.sub.b-(XXX).sub.l-N.sub.a- n.sub.p3 wherein: k and l are each independently 0 or 1; p and q are each independently 0-6; each N.sub.a independently represents an oligonucleotide sequence comprising 0-25 modified nucleosides, each sequence comprising at least two differently modified nucleosides; each N.sub.b independently represents an oligonucleotide sequence comprising 0-10 modified nucleosides; each n.sub.p and n.sub.q independently represent an overhang nucleoside; wherein N.sub.b and Y do not have the same modification; and XXX, YYY, and ZZZ each independently represent one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications).

(311) In some embodiments, the N.sub.a or N.sub.b comprises an alternating pattern of modified nucleosides.

(312) The YYY motif occurs at or near the cleavage site on the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleosides in length, the YYY motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleoside, from the 5-end; or optionally, the count starting at the first paired nucleoside within the duplex region, from the 5-end. Preferably, the YYY motif occurs at positions 11, 12, 13.

(313) In some embodiments, YYY motif is all 2-O-Me modified nucleosides.

(314) In some embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

(315) The antisense strand can therefore be represented by the following formulas:

(316) TABLE-US-00032 (Vb) 5n.sub.q-N.sub.a-ZZZ-N.sub.b-YYY-N.sub.a-np3; (IIc) 5n.sub.q-N.sub.a-YYY-N.sub.b-XXX-n.sub.p3; or (Vd) 5n.sub.q-N.sub.a-ZZZ-N.sub.b-YYY-N.sub.b-XXX-N.sub.a-n.sub.p3.

(317) When the antisense strand is represented by formula (IIb), N.sub.b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Each N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(318) When the antisense strand is represented as formula (Vc), N.sub.b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Each N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(319) When the antisense strand is represented as formula (Vd), each N.sub.b independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Each N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides. Preferably, N.sub.b is 0, 1, 2, 3, 4, 5, or 6.

(320) In some embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

(321) TABLE-US-00033 (IVa) 5n.sub.p-N.sub.a-YYY-N.sub.a-n.sub.q3.

(322) When the antisense strand is represented as formula (Va), each N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides. Each of X, Y and Z may be the same or different from each other.

(323) Each nucleoside of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, GNA, cEt, HNA, CeNA, 2-methoxyethyl, 2-O-Me, 2-O-allyl, 2-C-allyl, 2-hydroxyl, or 2-F. For example, each nucleoside of the sense strand and antisense strand is independently modified with 2-O-Me or 2-F. Each X, Y, Z, X, Y, and Z, in particular, may represent a 2-O-Me modified nucleoside or a 2-F modified nucleoside.

(324) In some embodiments, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10, and 11 positions of the strand when the duplex region is 21 nucleosides, the count starting from the first nucleoside from the 5-end, or optionally, the count starting at the first paired nucleoside within the duplex region, from the 5-end; and Y represents a 2-F modified nucleoside. The sense strand may additionally contain XXX motif or ZZZ motifs at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2-O-Me modified nucleoside or a 2-F modified nucleoside.

(325) In some embodiments the antisense strand may contain YYY motif occurring at positions 11, 12, 13 of the strand, the count starting from the first nucleoside from the 5-end, or optionally, the count starting at the first paired nucleoside within the duplex region, from the 5-end; and Y represents 2-O-Me modification. The antisense strand may additionally contain XXX motif or ZZZ motifs at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2-O-Me modified nucleoside or a 2-F modified nucleoside.

(326) The sense strand represented by any one of the above formulas (IVa), (IVb), (IVc), and (IVd) forms a duplex with an antisense strand being represented by any one of formulas (Va), (Vb), (Vc), and (Vd), respectively.

(327) Accordingly, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein may comprise a sense strand and an antisense strand, each strand comprising 14 to 30 nucleosides, the RNAi agent represented by formula (VI):

(328) TABLE-US-00034 (VI) sense: 5n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.j-N.sub.a-n.sub.q3 antisense: 3n.sub.p.sup.-N.sub.a.sup.-(XXX).sub.k-N.sub.b.sup.-YYY-N.sub.b.sup.-(ZZZ).sub.l-N.sub.a.sup.- n.sub.q.sup.5 wherein: i, j, k, and l are each independently 0 or 1; p, p, q, and q are each independently 0-6; each N.sub.a and N.sub.a independently represents an oligonucleotide sequence comprising 0-25 modified nucleosides, each sequence comprising at least two differently modified nucleosides; each N.sub.b and N.sub.b independently represents an oligonucleotide sequence comprising 0-10 modified nucleosides; wherein each n.sub.p, n.sub.p, n.sub.q, and n.sub.q, each of which may or may not be present, independently represents an overhang nucleoside; and XXX, YYY, ZZZ, XXX, YYY, and ZZZ each independently represent one motif of three consecutive nucleosides with identical sugar modifications (e.g., identical 2-modifications).

(329) In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

(330) Exemplary combinations of the sense strand and antisense strand forming a RNAi agent include the formulas below:

(331) TABLE-US-00035 (VIa) 5n.sub.p-N.sub.a-YYY-N.sub.a-n.sub.q3 3n.sub.p.sup.-N.sub.a.sup.-YYY-N.sub.a.sup.n.sub.q.sup.5 (VIb) 5n.sub.p-N.sub.a-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3 3n.sub.p.sup.-N.sub.a.sup.-YYY-N.sub.b.sup.-ZZZ-N.sub.a.sup.n.sub.q5 (VIc) 5n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.a-n.sub.q3 3n.sub.p.sup.-N.sub.a.sup.-XXX-N.sub.b.sup.-YYY-N.sub.a.sup.-n.sub.q.sup.5 (VId) 5n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q3 3n.sub.p.sup.-N.sub.a.sup.-XXX-N.sub.b.sup.-YYY-N.sub.b.sup.-ZZZ-N.sub.a.sup.-n.sub.q.sup.5

(332) When the RNAi agent is represented by formula (VIa), each N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(333) When the RNAi agent is represented by formula (VIb), each N.sub.b independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5, or 1-4 modified nucleosides. Each N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(334) When the RNAi agent is represented as formula (VIc), each N.sub.b, N.sub.b independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Each N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides.

(335) When the RNAi agent is represented as formula (VId), each N.sub.b, N.sub.b independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleosides. Each N.sub.a, N.sub.a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleosides. Each of N.sub.a, N.sub.a, N.sub.b, and N.sub.b independently comprises an alternating pattern of modified nucleosides.

(336) Each of X, Y, and Z in formulas (VI), (VIa), (VIb), (VIc), and (VId) may be the same or different from each other.

(337) When the RNAi agent is represented by formula (VI), (VIa), (VIb), (VIc), and (VId), at least one of the Y nucleosides may form a base pair with one of the Y nucleosides. Alternatively, at least two of the Y nucleosides form base pairs with the corresponding Y nucleosides; or all three of the Y nucleosides all form base pairs with the corresponding Y nucleosides.

(338) When the RNAi agent is represented by formula (VIb) or (VId), at least one of the Z nucleosides may form a base pair with one of the Z nucleosides. Alternatively, at least two of the Z nucleosides form base pairs with the corresponding Z nucleosides; or all three of the Z nucleosides all form base pairs with the corresponding Z nucleosides.

(339) When the RNAi agent is represented as formula (VIc) or (VId), at least one of the X nucleosides may form a base pair with one of the X nucleosides. Alternatively, at least two of the X nucleosides form base pairs with the corresponding X nucleosides; or all three of the X nucleosides all form base pairs with the corresponding X nucleosides.

(340) In some embodiments, the modification on the Y nucleoside is different than the modification on the Y nucleoside, the modification on the Z nucleoside is different than the modification on the Z nucleoside, or the modification on the X nucleoside is different than the modification on the X nucleoside.

(341) In some embodiments, when the RNAi agent is represented by formula (VId), N.sub.a modified nucleosides are 2-O-Me modified nucleosides or 2-F modified nucleosides. In some embodiments, when the RNAi agent is represented by formula (VId), the N.sub.a modified nucleosides are 2-O-Me or 2-F modified nucleosides and n.sub.p>0 and at least one n.sub.p is linked to a neighboring nucleoside via a phosphorothioate linkage. In some embodiments, when the RNAi agent is represented by formula (VId), the N.sub.a modified nucleosides are 2-O-Me or 2-F modified nucleosides, n.sub.p>0 and at least one n.sub.p is linked to a neighboring nucleoside via phosphorothioate linkage. In some embodiments, when the RNAi agent is represented by formula (VId), the N.sub.a modified nucleosides are 2-O-Me or 2-F modifications, n.sub.p>0 and at least one n.sub.p is linked to a neighboring nucleoside via phosphorothioate linkage, and the sense strand comprises at least one phosphorothioate linkage.

(342) In some embodiments, when the RNAi agent is represented by formula (VIa), the N.sub.a modified nucleosides are 2-O-Me or 2-F modifications, n.sub.p>0 and at least one n.sub.p is linked to a neighboring nucleoside via phosphorothioate linkage, and the sense strand comprises at least one phosphorothioate linkage.

(343) In some embodiments, the RNAi agent is a multimer containing at least two duplexes represented by formula (VI), (VIa), (VIb), (VIc), and (VId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

(344) In some embodiments, the RNAi agent is a multimer containing three, four, five, six, or more duplexes represented by formula (VI), (VIa), (VIb), (VIc), and (VId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

(345) In one embodiment, two RNAi agents represented by at least one of formulas (VI), (VIa), (VIb), (VIc), and (VId) are linked to each other at the 5 end, and one or both of the 3 ends.

(346) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises ten or fewer nucleosides with a 2-F modification. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleosides with a 2-F modification. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises ten 2-F modified nucleosides, e.g., four 2-F modified nucleosides in the sense strand and six 2-F modified nucleosides in the antisense strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises six 2-F modified nucleosides, e.g., four 2-F modified nucleosides in the sense strand and two 2-F modified nucleosides in the antisense strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises eight 2-F modified nucleosides, e.g., four 2-F modified nucleosides in the sense strand and four 2-F modified nucleosides in the antisense strand. In some embodiments, the rest of the nucleosides in the sense strand and the antisense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides.

(347) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 7, 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 6, 8, 9, 14, and 16 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages and wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(348) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 7, 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 6, 14, and 16 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages and wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(349) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein can be optimized for RNA interference by incorporating thermally destabilizing modifications at one or more positions selected from positions 2, 3, 4, 5, 6, 7, 8, and 9 (counting 5.fwdarw.3) of the 5-end of the antisense strand to reduce or inhibit off-target gene silencing. In some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleoside positions (counting 5.fwdarw.3) of the antisense strand. Such thermally destabilizing modifications are described, for example, in U.S. Pat. No. 10,233,448 and 11,504,391. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises thermally destabilizing modifications as described in U.S. Pat. No. 10,233,448 and 11,504,391. In some embodiments, the thermally destabilizing modification is an abasic modification, mismatch with the opposing nucleoside in the duplex, or a sugar modification (e.g., a 2-deoxy nucleoside or acyclic nucleoside e.g., unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA)). In some embodiments, the thermally destabilizing modification is a GNA. In some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) GNA within the first 9 nucleoside positions (counting 5.fwdarw.3) of the antisense strand.

(350) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 7, 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 6, 8, 9, 14, and 16 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 4, 5, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the nucleoside at position 7 of the antisense strand is a glycol modified nucleoside (GNA), and wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages and wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(351) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the nucleoside at the 3-end of the sense strand is a deoxy nucleoside (e.g., deoxy-thymine (dT)) or the nucleoside at the 3-end of the antisense strand is a deoxy nucleoside (e.g., dT). For example, in some embodiments, there is a short sequence of dT nucleosides, for example, two dT nucleosides on the 3-end of the sense, antisense strand, or both strands. In some embodiments there is one dT nucleoside at the 3-end of the sense, antisense strand, or both strands. In some embodiments, the dT nucleoside is an inverted dT nucleoside.

(352) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 7 and 9 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, and wherein the nucleoside at position 11 (counting 5.fwdarw.3) is a deoxy nucleoside (e.g. dT), wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 7, 9, 11, 13, 15, 17, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(353) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 3, 5, 7, 9, 10, 11, 13, 16, and 18 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 25 nucleosides in length, wherein the nucleosides at positions 2, 3, 5, 8, 10, 14, 16, and 18 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 4, 6, 7, 9, 11, 12, 13, 15, 17, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the nucleosides at position 24 and 25 (counting 5.fwdarw.3) are deoxy nucleosides (e.g. dT), wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the internucleoside linkages between positions 21 and 22 and between positions 22 and 23 of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a four nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(354) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 1, 3, 5, 7, 9, 10, 11, 13, 17, 19, and 21 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 2, 4, 6, 8, 12, 14, 15, 16, 18, and 20 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 4, 6, 7, 8, 10, 14, 16, 18, 20, and 22 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 5, 9, 11, 12, 13, 15, 17, 19, 21, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(355) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 7, 9, 11, 13, and 15 (counting 5 .fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(356) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 10 and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 5, 7, 9, 11, 12, 13, 15, 17, 19, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(357) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 1, 3, 5, 7, 9, 10, 11, and 13 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 2, 4, 6, 8, 12, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 4, 8, 10, 14, 16, and 20 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 17, 18, 19, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(358) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 21 nucleosides in length, wherein the nucleosides at positions 7, 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 23 nucleosides in length, wherein the nucleosides at positions 2, 6, 9, 14, and 16 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 4, 5, 7, 8, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages and wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(359) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the sense strand is 19 nucleosides in length, wherein the nucleosides at positions 5, 7, 8, and 9 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and wherein the nucleosides at positions 1, 2, 3, 4, 6, and 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, wherein the antisense strand is 21 nucleosides in length, wherein the nucleosides at positions 2, 6, 8, 9, 14, and 16 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages and wherein the first two and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages, and wherein the RNAi agent has a two nucleoside overhang at the 3-end of the antisense strand, and a blunt end at the 5-end of the antisense strand.

(360) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises the chemical modifications and/or modification patterns as described in U.S. Pat. Nos. 10,676,742, 11,661,604, and 11,655,473. For example, in some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the sense strand comprises a mix of different 2-modified nucleosides (e.g., 2-F modified nucleosides and 2-O-Me modified nucleosides). In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at three consecutive positions of the sense strand are 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 8 and 12 (counting 5.fwdarw.3) are not 2-F modified nucleosides, e.g., are 2-O-Me modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 8 and 12 (counting 5.fwdarw.3) are 2-O-Me modified nucleosides. In some embodiments, the rest of the nucleosides in the sense strand are a mix of 2-F modified nucleosides and 2-O-Me modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, and 11 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the nucleosides at positions 8 and 12 (counting 5.fwdarw.3) are 2-O-Me modified nucleosides and the rest of the nucleosides in the sense strand comprise alternating motifs of 2-O-Me modified nucleosides and 2-F modified nucleosides.

(361) In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the antisense strand comprises a mix of different 2-modified nucleosides (e.g., any combination of 2-F modified nucleosides, 2-deoxy nucleosides, and 2-O-Me modified nucleosides). In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at one or more of positions 2, 12, 16, 18, 20, and 21 (counting 5.fwdarw.3) of the antisense strand are 2-deoxy nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 2, 12, 16, 18, 20, and 21 (counting 5.fwdarw.3) of the antisense strand are 2-deoxy nucleosides and the rest of the nucleosides in the antisense strand are not 2-deoxy nucleosides, e.g., are 2-O-Me modified nucleosides or 2-F modified nucleosides. In some embodiments, an RNAi agent (e.g., a CYP7A1 RNAi agent described herein) comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 17, and 19 of the antisense strand are a mix of 2-F modified nucleosides and 2-O-Me modified nucleosides. In some embodiments, the nucleosides at positions 3, 4, 5, 6, 7, 8, 9, 10, and 11 of the antisense strand comprise alternating motifs of 2-O-Me modified nucleosides and 2-F modified nucleosides.

(362) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) phosphorothioate internucleoside linkages in the antisense strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises six phosphorothioate internucleoside linkages in the antisense strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein does not comprise any phosphorothioate internucleoside linkages in the sense strand. In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein further comprises six phosphorothioate internucleoside linkages in the antisense strand (e.g., the first two internucleoside linkages and the last two internucleoside linkages counting 5.fwdarw.3 and the internucleoside linkages between nucleosides at positions 15-16 and 17-18 counting 5.fwdarw.3) and no phosphorothioate internucleoside linkages in the sense strand. It is to be understood that, in any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein, the rest of the internucleoside linkages, unless otherwise specified, are all phosphodiester internucleoside linkages.

(363) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the nucleosides at positions 1, 3, 5, 7, 9, 10, 11, 13, 15, 17, and 19 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 3, 5, 7, 9, 11, 14, 17, and 19 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 12, 16, 18, 20, and 21 (counting 5.fwdarw.3) of the antisense strand are 2-deoxy nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides or 2-deoxy nucleosides are 2-O-Me modified nucleosides, and wherein the first two and the last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) and the internucleoside linkages between positions 15-16 and 17-18 of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(364) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein nucleosides at positions 8, 9, 10, and 11 of the sense strand are 2-F modified nucleosides and nucleosides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are 2-F modified nucleosides, wherein the rest of the nucleosides in the sense strand and antisense strand are 2-O-Me modified nucleosides, wherein the first two or first three internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(365) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein nucleosides at one or more of positions 3, 5, 8, 9, 10, 11, 13, 15, or 17 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides. In some embodiments, nucleosides at one or more of positions 1, 2, 4, 6, 7, 12, 14, 16, 18, 19, or 20 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides. In some embodiments, nucleosides at one or more of positions 2, 3, 4, 5, 7, 9, 10, 14, 16, or 18 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides. In some embodiments, nucleosides at one or more of positions 1, 6, 8, 11, 12, 13, 15, 17, 19, 20, 21, or 22 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides.

(366) In some embodiments, nucleosides at all of positions 3, 5, 8, 9, 10, 11, 13, 15, or 17 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and the rest of the nucleosides in the sense strand are 2-O-Me modified nucleosides. In some embodiments, nucleosides at all of positions 2, 3, 4, 5, 7, 9, 10, 14, 16, and 18 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides and the rest of the nucleosides in the antisense strand are 2-O-Me modified nucleosides. In some embodiments, the first internucleoside linkage of the sense strand (counting 5.fwdarw.3) is a phosphorothioate internucleoside linkage and the first three and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages. In some embodiments, nucleosides at all of positions 3, 5, 8, 9, 10, 11, 13, 15, or 17 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides and all of positions 2, 3, 4, 5, 7, 9, 10, 14, 16, and 18 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, and the rest of the nucleosides in the sense strand and antisense strand are 2-O-Me modified nucleosides, wherein the first internucleoside linkage of the sense strand (counting 5.fwdarw.3) is a phosphorothioate internucleoside linkage and the first three and last two internucleoside linkages of the antisense strand (counting 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(367) In some embodiments, any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a phosphorylated nucleoside at the 5 terminus, wherein the phosphorylated nucleoside is selected from uridine and adenosine. In some embodiments, the phosphorylated nucleoside is uridine. In some embodiments, the 5-nucleoside of the antisense strand comprises a 4-phosphate analog. In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In some embodiments, the phosphate analog is a 4-phosphate analog comprising 5-methoxyphosphonate-4-oxy.

(368) Conjugated CYP7A1 RNAi Agents

(369) In some embodiments, any one of the RNAi agents described herein is conjugated (e.g., covalently linked) to another chemical moiety (e.g., a targeting moiety for, e.g., improved delivery, cellular uptake and distribution). In some embodiments, any one of the RNAi agents described herein is conjugated (e.g., covalently linked) to a targeting moiety via a linker. Any of the linkers described herein may be used. In some embodiments, the targeting moiety is conjugated (e.g., covalently linked) to the 5 end of the antisense strand of an RNAi agent. In some embodiments, the targeting moiety is conjugated (e.g., covalently linked) to the 3 end of the antisense strand of an RNAi agent. In some embodiments, the targeting moiety is conjugated (e.g., covalently linked) to the 5 end of the sense strand of an RNAi agent. In some embodiments, the targeting moiety is conjugated (e.g., covalently linked) to the 3 end of the sense strand of an RNAi agent.

(370) Any suitable targeting moiety in the field of RNA interference may be conjugated (e.g., covalently linked) to any one of the RNAi agents described herein. Non-limiting examples of targeting moieties include carbohydrates (e.g., monosaccharides (such as GalNAc)), disaccharides, trisaccharides, tetrasaccharides, polysaccharides), folate, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, an aptamer, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, asialoglycoprotein receptor ligands, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.

(371) In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof that specifically binds to a cell surface protein, e.g., a cell surface receptor that promotes receptor mediated endocytosis and internalization of the RNAi agents described herein. Cell surface proteins that may be targeted by the targeting moiety include, without limitation: asialoglycoprotein receptors, CD63, MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL-receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein-2 (APLP2), apelin receptor (APL R), PRLR (prolactin receptor), MAL (Myelin And Lymphocyte protein, a.k.a. VIP 17), IGF2R, vacuolar-type H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptors, glutathione receptor, leptin receptors, scavenger receptors, SCARAl-5, SCARBI-3, CD36, CDH16 (Cadheri-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (Solute carrier family 22 member 13), SLC5A2 (Sodium/glucose cotransporter 2), UMOD (Uromodulin), BMPRIA (Bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4/GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadheri-15), ITGA7 (Integrin alpha-7), CACNG1 (L-type calcium channel subunit gamma-1), CACNA1s (L-type calcium channel subunit alpha-15), CACNG6 (L-type calcium channel subunit gamma-6), SCN1B (Sodium channel subunit beta-1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (Leucine-rich repeat-containing protein 14B), and POPDC3 (Popeye domain-containing protein 3). In some embodiments, antibodies or antigen binding fragments that may be used as a targeting moiety according to the present disclosure is a Fab fragment, a Fab, a F(ab)2 fragment, a Fv fragment, a scFv, a VHH, or a diabody. In some embodiments, the antibody or antigen binding fragment thereof is covalently linked directly or indirectly (e.g., via a linker) to an RNAi agent described herein.

(372) In some embodiments, the targeting moiety is a hepatospecific targeting moiety (i.e., it directs the RNAi agent composition to liver cells). In some embodiments, the targeting moiety comprises a monosaccharide. In some embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). In some embodiments, the targeting moiety comprises one or more (e.g, 1, 2, 3, 4, or more) GalNAc. In some embodiments, the one or more GalNAc in the targeting moiety are linked via phosphorothioate linkages. In some embodiments, the GalNAc targeting moiety serves as a ligand that targets the RNAi agent to particular cells. In some embodiments, the GalNAc targeting moiety targets the RNAi agent to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes). GalNAc targeting moieties, which comprise one or more GalNAc, have been described, for example, in the following references:

(373) TABLE-US-00036 Reference No. Application No. Filing Date U.S. Pat. No. 8,106,022 U.S. Pat. No. 12/328,528 Dec. 4, 2008 U.S. Pat. No. 10,246,709 U.S. Pat. No. 15/452,423 Mar. 7, 2017 U.S. Pat. No. 5,994,517 U.S. Pat. No. 08/755,062 Nov. 22, 1996 U.S. Pat. No. 6,906,182 U.S. Pat. No. 09/998,497 Nov. 30, 2001 U.S. Pat. No. 10,294,474 U.S. Pat. No. 15/452,324 Mar. 7, 2017 U.S. Pat. No. 2017305956 U.S. Pat. No. 15/621,395 Jun. 13, 2017 U.S. Pat. No. 10,233,448 U.S. Pat. No. 15/504,855 Feb. 17, 2017 U.S. Pat. No. 2016122761 U.S. Pat. No. 14/898,873 Jun. 23, 2014 U.S. Pat. No. 10,808,246 U.S. Pat. No. 14/901,945 Dec. 29, 2015 WO 2022/159158A1 PCT/US2021/057016 Oct. 28, 2021
The disclosures in these references related to GalNAc are hereby incorporated herein by reference.

(374) In some embodiments, a targeting moiety comprises the structure below:

(375) ##STR00078##
or a pharmaceutically acceptable salt thereof.

(376) In some embodiments a targeting moiety comprises the structure below:

(377) ##STR00079##
or a pharmaceutically acceptable salt thereof.

(378) In some embodiments, a targeting moiety comprises the structure below:

(379) ##STR00080##
or a pharmaceutically acceptable salt thereof.

(380) In some embodiments, an RNAi agent is conjugated to the targeting moiety via a tether, also referred to as a linker. A non-limiting example of a tether includes a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide and polyethylene glycol (PEG) groups in any combination. In some embodiments, a targeting moiety covalently linked to a tether shown below:

(381) ##STR00081##

(382) Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in U.S. Pat. Nos. 9,127,276, 9,145,558, and 10,683,499. Disclosures in U.S. Pat. No. 9,127,276 (U.S. patent application Ser. No. 14/267,842 filed May 1, 2014), 9,145,558 (U.S. patent application Ser. No. 14/633,491 filed Feb. 27, 2015), and 10,683,499 (U.S. patent application Ser. No. 15/687,306 filed Aug. 25, 2017) regarding carbohydrate conjugates and linkers are incorporated herein by reference.

(383) Some aspects of the present disclosure further provide additional targeting moieties comprising one or more GalNAc and structures that may be conjugated to any one of the CYP7A1 RNAi agents described herein. In some embodiments, a targeting moiety comprising one or more GalNAc is conjugated (attached) to a CYP7A1 RNAi agent.

(384) In some embodiments, the targeting moiety comprises a structure of Formula (I):

(385) ##STR00082##
or a pharmaceutically acceptable salt thereof;
wherein: each X is independently N(R.sup.A).sub.2, SR.sup.A, or OR.sup.A; each R.sup.1 is independently hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, OR.sup.A, or a group of formula:

(386) ##STR00083## each R.sup.2 is independently hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, OR.sup.A, or a group of formula:

(387) ##STR00084## each R.sup.3 is independently hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, OR.sup.A, or a group of formula:

(388) ##STR00085## R.sup.4 is hydrogen or substituted or unsubstituted alkyl; each P.sup.1 is independently hydrogen or an oxygen protecting group;

(389) each L is independently a bond, substituted or unsubstituted aliphatic, substituted or unsubstituted substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted carbocyclylene, substituted or unsubstituted arylene, O, N(R.sup.A), S, C(O), C(O)O, C(O)NR.sup.A, NR.sup.AC(O), NR.sup.AC(O)R.sup.A, C(O)R.sup.A, NR.sup.AC(O)O, NR.sup.AC(O)N(R.sup.A), OC(O), OC(O)O, OC(O)N(R.sup.A), S(O).sub.2NR.sup.A, NR.sup.AS(O).sub.2, or a combination thereof;

(390) each R.sup.A is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two R.sup.A groups are joined to form a substituted or unsubstituted heterocyclyl ring, or a substituted or unsubstituted heteroaryl ring; and n is 1-20; wherein the phosphorous labeled y is conjugated to the 3 end of the sense strand; and wherein when n is greater than 1, with the exception of the phosphorous directly attached to an RNAi agent; each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit; provided that each repeat unit of Formula (I) in the targeting moiety comprises one group of formula:

(391) ##STR00086##

(392) In some embodiments, the targeting moiety comprises a structure of Formula:

(393) ##STR00087## ##STR00088##
or a pharmaceutically acceptable salt thereof; wherein R.sup.10 is substituted or unsubstituted C.sub.1-8 alkylene having 0-3 carbon atoms replaced with O; and R.sup.20 is substituted or unsubstituted C.sub.1-8 alkylene having 0-3 carbon atoms replaced with O; and P.sup.1, R.sup.2, R.sup.1, R.sup.4, R.sup.A, n, and X are as defined for Formula (I).

(394) In some embodiments, the targeting moiety comprises a structure of formula:

(395) ##STR00089##
or a pharmaceutically acceptable salt thereof, wherein X is as defined for Formula (I).

(396) In some embodiments, the targeting moiety does not comprise a structure of:

(397) ##STR00090##
or a salt thereof, wherein X is OR.sup.A or SR.sup.A (e.g., where R.sup.A is H).

(398) In some embodiments, the targeting moiety does not comprise a structure of:

(399) ##STR00091##
or a salt thereof.

(400) In some embodiments, in a targeting moiety comprising a structure of Formula (I), when R.sup.1 and R.sup.2 are hydrogen, and R.sup.3 is a group of formula

(401) ##STR00092##
L does not attach to the 1 position of the ribose by an oxygen atom.

(402) In some embodiments, in a targeting moiety comprising a structure of Formula (I), when R.sup.3 is a group of formula

(403) ##STR00093##
L does not attach to the 1 position of the ribose by a nitrogen atom.

(404) In some embodiments, the targeting moiety does not comprise a structure of formula:

(405) ##STR00094##
or a salt thereof, wherein X is OR.sup.A or SR.sup.A (e.g., where R.sup.A is H); and R.sup.2 is hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, or OR.sup.A. In some embodiments, the targeting moiety does not comprise a group of the above formula, wherein X is OR.sup.A or SR.sup.A (e.g., where R.sup.A is H); and R.sup.2 is hydrogen, fluoro, OCH.sub.3, OTBS, or OCH.sub.2CH.sub.2OCH.sub.3.

(406) In some embodiments, the targeting moiety does not comprise a structure of formula:

(407) ##STR00095##
or a salt thereof, wherein R.sup.2 is hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, or OR.sup.A. In some embodiments, the targeting moiety does not comprise a group of the above formula, wherein R.sup.2 is hydrogen, fluoro, OCH.sub.3, OTBS, or OCH.sub.2CH.sub.2OCH.sub.3.

(408) In some embodiments, the targeting moiety does not comprise a group of formula:

(409) ##STR00096##
or a salt thereof, wherein X is OR.sup.A or SR.sup.A (e.g., where R.sup.A is H); Y is O, S, CH.sub.2, NH(CO), (CO)NH, or NH(CO)O; and R.sup.2 is hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, or OR.sup.A. In some embodiments, the targeting moiety does not comprise a group of the above formula, wherein X is OR.sup.A or SR.sup.A (e.g., where R.sup.A is H); Y is O, S, CH.sub.2, NH(CO), (CO)NH, or NH(CO)O; and R.sup.2 is hydrogen, fluoro, OCH.sub.3, OTBS, or OCH.sub.2CH.sub.2OCH.sub.3.

(410) In some embodiments, the targeting moiety does not comprise a structure of formula:

(411) ##STR00097##
or a salt thereof, wherein Y is O, S, CH.sub.2, NH(CO), (CO)NH, or NH(CO)O; and R.sup.2 is hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, or OR.sup.A. In some embodiments, the targeting moiety does not comprise a group of the above formula, wherein Y is O, S, CH.sub.2, NH(CO), (CO)NH, or NH(CO)O; and R.sup.2 is hydrogen, fluoro, OCH.sub.3, OTBS, or OCH.sub.2CH.sub.2OCH.sub.3.

(412) In some embodiments, the targeting moiety comprises a structure of Formula (II):

(413) ##STR00098##
or a pharmaceutically acceptable salt thereof;
wherein: R.sup.4 is hydrogen or substituted or unsubstituted alkyl; each X is independently N(R.sup.A).sub.2, SR.sup.A, or OR.sup.A; each P.sup.1 is independently hydrogen or an oxygen protecting group;
each L is independently a bond, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, O; N(R.sup.A), S, C(O), C(O)O, C(O)NR.sup.A, NR.sup.AC(O), NR.sup.AC(O)R.sup.A, C(O)R.sup.A, NR.sup.AC(O)O, NR.sup.AC(O)N(R.sup.A), OC(O), OC(O)O, OC(O)N(R.sup.A), S(O).sub.2NR.sup.A, NR.sup.AS(O).sub.2, or a combination thereof;
each R.sup.A is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two R.sup.A groups are joined to form a substituted or unsubstituted heterocyclyl ring, or a substituted or unsubstituted heteroaryl ring; and n is 1-20; wherein the phosphorous labeled y is conjugated to the 3 end of the sense strand; and wherein when n is greater than 1, with the exception of the phosphorous directly attached to the RNAi agent, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit.

(414) In some embodiments, the targeting moiety comprises a structure of Formula:

(415) ##STR00099##
or a pharmaceutically acceptable salt thereof; wherein R.sup.10 is substituted or unsubstituted C.sub.1-8 alkylene having 0-3 carbon atoms replaced with O; and R.sup.20 is substituted or unsubstituted C.sub.1-8 alkylene having 0-3 carbon atoms replaced with O; and P.sup.1, R.sup.4, R.sup.A, n, and X are as defined for Formula (II).

(416) In some embodiments, the targeting moiety comprises a structure of formula:

(417) ##STR00100##
or a pharmaceutically acceptable salt thereof, wherein X is as defined for Formula (II).

(418) In some embodiments, the targeting moiety comprises a structure of Formula (III):

(419) ##STR00101##
or a pharmaceutically acceptable salt thereof,
wherein: R.sup.4 is hydrogen or substituted or unsubstituted alkyl; each X is independently N(R.sup.A).sub.2, SR.sup.A, or OR.sup.A; each P.sup.1 is independently hydrogen or an oxygen protecting group; each R.sup.A is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; and n is 1-20; wherein the phosphorous labeled y is conjugated to the 3 end of the sense strand; and
wherein when n is greater than 1, with the exception of the phosphorous directly attached to the RNAi agent, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit.

(420) In some embodiments, the targeting moiety comprises a structure of formula:

(421) ##STR00102##
or a pharmaceutically acceptable salt thereof, wherein X is as defined for Formula (III).

(422) In some embodiments, the targeting moiety comprises a structure of Formula (IV):

(423) ##STR00103##
wherein: each X is independently N(R.sup.A).sub.2, SR.sup.A, or OR.sup.A; each R.sup.1 is independently hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, or OR.sup.A; each R.sup.2 is independently hydrogen, halogen, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, or OR.sup.A; each R.sup.3 is independently of formula:

(424) ##STR00104## R.sup.4 is hydrogen or substituted or unsubstituted alkyl; each P.sup.1 is independently hydrogen or an oxygen protecting group; each L is independently a bond, substituted or unsubstituted aliphatic, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heteroalkynyl, substituted or unsubstituted carbocyclylene, substituted or unsubstituted arylene, O, N(R.sup.A), S, C(O), C(O)O, C(O)NR.sup.A, NR.sup.AC(O), NR.sup.AC(O)R.sup.A, C(O)R.sup.A, NR.sup.AC(O)O, NR.sup.AC(O)N(R.sup.A), OC(O), OC(O)O, OC(O)N(R.sup.A), S(O).sub.2NR.sup.A, NR.sup.AS(O).sub.2, or a combination thereof; each R.sup.A is independently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two R.sup.A groups are joined to form a substituted or unsubstituted heterocyclyl ring, or a substituted or unsubstituted heteroaryl ring; and n is 1-20; wherein the phosphorous labeled y is conjugated to the 3 end of the sense strand; and wherein when n is greater than 1, with the exception of the phosphorous directly attached to the RNAi agent, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit.

(425) In some embodiments, the targeting moiety comprises a structure of formula:

(426) ##STR00105##
or a pharmaceutically acceptable salt thereof; wherein R.sup.4, L, P.sup.1, n, and X are as defined for Formula (IV). For any one of Formulas (IV-a), (IV-b), (IV-c), and (IV-d), when n is greater than 1, with the exception of the phosphorous directly attached to the RNAi agent, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit, and with the exception of the oxygen directly attached to R.sup.4, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(427) In some embodiments, the targeting moiety comprises a structure of formula:

(428) ##STR00106##
or a pharmaceutically acceptable salt thereof, wherein X is as defined for Formula (IV). For Formula (IV-e), with the exception of the phosphorous directly attached to the RNAi agent, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit, and with the exception of the oxygen directly attached to hydrogen, the oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(429) In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), n is 1-6. In some embodiments, in a targeting moiety comprising a structure of Formula ((I), (II), (III), or (IV), n is 1-3. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), n is 2-3. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), n is 1-4. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), n is 2-4. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), n is 1. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (I), or (IV), n is 2. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), n is 3. In some embodiments, the targeting moiety is conjugated to the sense strand via the phosphorous labeled y. In some embodiments, the targeting moiety is conjugated to the 3 end of the sense strand via the phosphorous labeled y.

(430) In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), each X is independently SR.sup.A. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), each X is independently OR.sup.A. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), each X is independently SH or OH. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), each X is independently SH. In some embodiments, in a targeting moiety comprising a structure of Formula (I), (II), (III), or (IV), each X is independently OH.

(431) Table 4 lists various exemplary GalNAc targeting moieties. Included are pharmaceutically acceptable salts of the exemplary targeting moieties.

(432) TABLE-US-00037 TABLE 4 Exemplary targeting moieties comprising GalNAc Tar- geting Moiety ID: Structure (Z.sup.1) 07 (Z.sup.2) 08 (Z.sup.3) 09 (Z.sup.4) 0 (Z.sup.5) (Z.sup.6) (Z.sup.7) (Z.sup.8) (Z.sup.9) (Z.sup.10) (Z.sup.11) (Z.sup.12) (Z.sup.13) (Z.sup.14) 0 (Z.sup.15) (Z.sup.16) For any one of Formulas (Z.sup.1)-(Z.sup.16), with the exception of the phosphorous directly attached to the RNAi agent, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(433) In some embodiments, a targeting moiety is linked to the RNAi agent via a linker. Various linkers may be used to conjugate a targeting moiety to any one of the RNAi agents described herein. The linker facilitates covalent linkage of the agent to a targeting moiety. The linker is conjugated to the 5 or 3 end of an RNAi agent sense strand or antisense strand. In some embodiments, the linker is conjugated to the 5 or 3 end of an RNAi agent sense strand. In some embodiments, a linker is conjugated to the 5 end of an RNAi agent sense strand. In some embodiments, a linker is conjugated to the 3 end of an RNAi agent sense strand. In some embodiments, a linker is conjugated to the 5 end of an RNAi agent antisense strand. In some embodiments, a linker is conjugated to the 3 end of an RNAi agent antisense strand. Examples of linkers can include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleotides, ribitol (abasic ribose), non-nucleotidic linker, and/or PEG groups. In some embodiments, the linker is a monovalent, bivalent, trivalent, or tetravalent branched linker. In some embodiments, the linker is a phosphorus-containing linker (e.g., phosphate, phosphodiester, phosphorothioate, phosphorodithioate, phosphoroamidate, etc), or other linker. Non-limiting examples of other linkers can include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleotides, ribitol (abasic ribose), and/or PEG groups. In some embodiments, the linker is a phosphorothioate linkage.

(434) In some embodiments, any one of the CYP7A1 RNAi agents described herein is conjugated (e.g., covalently linked) linked to a targeting moiety comprising a structure of any one of Formulae (Z.sup.1)-(Z.sup.16). For any one of Formulas (Z.sup.1)-(Z.sup.16) referred to herein, with the exception of the phosphorous directly attached to the RNAi agent, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit. In some embodiments, any one of the CYP7A1 RNAi agents described herein is (e.g., covalently linked) to a targeting moiety comprising a structure of Formula (Z.sup.6). In some embodiments, any one of the CYP7A1 RNAi agents described herein is (e.g., covalently linked) at the 3 end (e.g., to the 3 terminal nucleoside) of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6). For any one of Formula (Z.sup.6) referred to herein, with the exception of the phosphorous directly attached to the sense strand, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent repeat unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(435) Unless stated otherwise, use of the symbol

(436) ##STR00123##
means that any group or groups may be linked thereto that is in accordance with the scope of the inventions described herein. For example, in any one of the structures of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), and (Z.sup.1)-(Z.sup.16), the symbol

(437) ##STR00124##
means the attachment point at which the RNAi agent (e.g., at the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand) is attached to the target moiety.

(438) It is to be understood that in an RNAi agent (e.g., CYP7A1 RNAi agent) that further comprises a targeting moiety conjugated to the 3 terminal nucleoside of the sense strand, the 3-terminal nucleoside is linked to the targeting moiety via a phosphorothioate linkage. As such, when the 3-terminal nucleoside of the sense strand is represented as [mAs] (e.g., the sense strands of the siRNAs provided in Table 9 and elsewhere in the present disclosure), the s corresponds to the phosphorothioate linkage (e.g.,

(439) ##STR00125##
wherein X is SH, i.e.,

(440) ##STR00126##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formulae: (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16).
Examples of RNAi Agents Conjugated to Targeting Moieties

(441) Aspects of the present disclosure provide conjugates comprising any one of the RNAi agents described herein conjugated to any one of the targeting moieties described herein (e.g., comprising one or more GalNAc, such as targeting moieties comprising a structure of Formula (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-J), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16)). In some embodiments, an RNAi agent (e.g., CYP7A1 RNAi agent) is conjugated to a targeting moiety (e.g., comprising one or more GalNAc, such as targeting moieties of Formula (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16)) via a linker described herein. In some embodiments, an RNAi agent (e.g., CYP7A1 RNAi agent) is conjugated to a targeting moiety via a monovalent, bivalent, trivalent, or tetravalent branched linker. In some embodiments, an RNAi agent (e.g., CYP7A1 RNAi agent) is conjugated to a targeting moiety via a phosphorus-containing linker (e.g., a phosphorothioate linkage).

(442) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to any one of the targeting moieties described herein (e.g., comprising one or more GalNAc, such as targeting moieties of Formula (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16)) via a linker (e.g., a phosphorothioate linkage), wherein the CYP7A1 RNAi agent comprises a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises a region of complementarity comprising a region of complementarity of at least 15 nucleosides to a target sequence in CYP7A1 mRNA, (e.g., a target sequence listed in Table 2), wherein the region of complementarity comprises a nucleoside sequence that contains no more than 3 mismatches to the CYP7A1 target sequence, and wherein the sense strand is at least substantially complementary to the antisense strand.

(443) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to any of the targeting moieties described herein (e.g., comprising one or more GalNAc, such as targeting moieties of Formula (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16)) via a linker (e.g., a phosphorothioate linkage), wherein the CYP7A1 RNAi agent comprises a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises a region of complementarity of at least 15 nucleotides to a CYP7A1 target sequence set forth in nucleosides 113-133, 221-241, 249-269, 290-310, 301-321, 475-495, 476-496, 504-524, 593-613, 600-620, 671-691, 779-799, 839-859, 842-862, 1003-1023, 1009-1029, 1037-1057, 1082-1102,1189-1209, 1207-1227, 1215-1235, 1225-1245, 1226-1246, 1235-1255, 1289-1309, 1296-1316, 1384-1404, 1415-1435, 1431-1451, or 1559-1579 of SEQ ID NO: 1, wherein the region of complementarity comprises a nucleoside sequence that contains no more than 3 mismatches to the CYP7A1 target sequence, and wherein the sense strand is at least substantially complementary to the antisense strand.

(444) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to a targeting moiety comprising a structure of any one of Formula (Z.sup.1)-(Z.sup.16) (e.g., Formula (Z.sup.6)), wherein the CYP7A1 RNAi agent comprises an antisense strand comprising the nucleobase sequence of any one of SEQ ID Nos: 777-1190 and a sense strand substantially complementary to the antisense strand and comprising the nucleobase sequence of any one of SEQ ID Nos: 393-776, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand. In some embodiments, the targeting moiety comprises a structure of Formula (Z.sup.6).

(445) ##STR00127##
or a pharmaceutically acceptable salt thereof, wherein the

(446) ##STR00128##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand. For Formula (Z.sup.6), with the exception of the phosphorous directly attached to the sense strand, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(447) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to a targeting moiety comprising a structure of any one of Formula (Z.sup.1)-(Z.sup.16) (e.g., Formula (Z.sup.6)), wherein the CYP7A1 RNAi agent comprises nucleobase sequences (e.g., nucleobase sequences of the sense strand and antisense strand) of an siRNA selected from siRNA1-siRNA384, siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand. In some embodiments, the targeting moiety comprises a structure of Formula (Z.sup.6).

(448) ##STR00129##
or a pharmaceutically acceptable salt thereof, wherein the

(449) ##STR00130##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand. For Formula (Z.sup.6), with the exception of the phosphorous directly attached to the sense strand, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(450) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to a targeting moiety, wherein the CYP7A1 RNAi agent comprises nucleobase sequences (e.g., nucleobase sequences of the sense strand and antisense strand) of an siRNA selected from siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand, and wherein the targeting moiety comprises a structure of Formula (Z.sup.6):

(451) ##STR00131##
or a pharmaceutically acceptable salt thereof, wherein the

(452) ##STR00132##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand. For Formula (Z.sup.6), with the exception of the phosphorous directly attached to the sense strand, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(453) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to a targeting moiety, wherein the CYP7A1 RNAi agent comprises nucleobase sequences (e.g., nucleobase sequences of the sense strand and antisense strand) of an siRNA selected from siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand, and wherein the targeting moiety comprises a structure of Formula (Z.sup.6):

(454) ##STR00133##
or a pharmaceutically acceptable salt thereof, wherein the

(455) ##STR00134##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand. For Formula (Z.sup.6), with the exception of the phosphorous directly attached to the sense strand, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(456) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to a targeting moiety, wherein the CYP7A1 RNAi agent comprises nucleobase sequences (e.g., nucleobase sequences of the sense strand and antisense strand) of an siRNA selected from siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand, and wherein the targeting moiety comprises a structure of Formula (Z.sup.6):

(457) ##STR00135##
or a pharmaceutically acceptable salt thereof, wherein the

(458) ##STR00136##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand. For Formula (Z.sup.6), with the exception of the phosphorous directly attached to the sense strand, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(459) In some embodiments, a CYP7A1 RNAi agent described herein is conjugated (e.g., covalently linked) to a targeting moiety, wherein the CYP7A1 RNAi agent comprises nucleobase sequences (e.g., nucleobase sequences of the sense strand and antisense strand) of an siRNA selected from siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand, and wherein the targeting moiety comprises a structure of Formula (Z.sup.6):

(460) ##STR00137##
or a pharmaceutically acceptable salt thereof, wherein the

(461) ##STR00138##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand. For Formula (Z.sup.6), with the exception of the phosphorous directly attached to the sense strand, each instance of phosphorous labeled y is bound to the oxygen labeled z of the adjacent unit, and with the exception of the oxygen directly attached to hydrogen, each instance of oxygen labeled z is bound to the phosphorous labeled y of the adjacent unit.

(462) In some embodiments, any one of the CYP7A1 RNAi agents described herein is conjugated (e.g., covalently linked) to a targeting moiety, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand, wherein the targeting moiety comprising a structure of formula:

(463) ##STR00139##
or a pharmaceutically acceptable salt thereof, wherein the

(464) ##STR00140##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand. In some embodiments, a conjugate described herein comprises a CYP7A1 RNAi agent conjugated (e.g., covalently linked) to a targeting moiety, wherein the targeting moiety is conjugated (e.g., covalently linked) to the 3 terminal nucleoside of the sense strand, wherein the conjugate comprises a structure of:

(465) ##STR00141##
or a pharmaceutically acceptable salt thereof,
wherein a CYP7A1 RNAi agent comprises nucleobase sequences (e.g., nucleobase sequences of the sense strand and antisense strand) of an siRNA selected from siRNA1-siRNA384, siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272, and wherein: (i) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages; (ii) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages; (iii) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages; or (iv) the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.

(466) In some embodiments, a CYP7A1 RNAi agent described herein comprises: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1173 and a structure (5.fwdarw.3) of [mUs][fCs][fU][mA][fA][mG][fU][fG][mC][fA][mU][mU][mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG] (SEQ ID NO: 2016); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 604 and a structure (5.fwdarw.3) of

(467) TABLE-US-00038 (SEQIDNO:1425) [mCs][mCs][mC][mA][mC][mA][mG][mU][fU][fA][fA] [fU][mG][mC][mA][mC][mU][mU][mA][mG][mAs]; wherein mA, mC, mG and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage; and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a targeting moiety comprising a structure of Formula (Z.sup.6), wherein the conjugated CYP7A1 RNAi agent comprises a structure as shown below:

(468) ##STR00142## or a pharmaceutically acceptable salt thereof. It is to be understood that the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(469) ##STR00143## wherein X is SH, i.e.,

(470) ##STR00144## of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).

(471) In some embodiments, a CYP7A1 RNAi agent described herein comprises: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1174 and a structure (5.fwdarw.3) of [mUs][fUs][fU][mA][fA][mC][fU][fG][mU][fG][mG][mG][mU][fA][mA][mA][mG][mA][mG][mC][mUs][mAs][mG] (SEQ ID NO: 2020); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 598 and a structure (5.fwdarw.3) of

(472) TABLE-US-00039 (SEQIDNO:1418) [mAs][mGs][mC][mU][mC][mU][mU][mU][fA][fC][fC] [fC][mA][mC][mA][mG][mU][mU][mA][mA][mAs], wherein mA, mC, mG and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage; and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a targeting moiety comprising a structure of Formula (Z.sup.6), wherein the conjugated CYP7A1 RNAi agent comprises a structure as shown below:

(473) ##STR00145##
or a pharmaceutically acceptable salt thereof. It is to be understood that the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(474) ##STR00146##
wherein X is SH, i.e.,

(475) ##STR00147##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).

(476) In some embodiments, a CYP7A1 RNAi agent described herein comprises: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1181 and a structure (5.fwdarw.3) of [mUs][fUs][fU][mC][fC][fG][fU][mG][mA][fG][mG][mG][mA][fA][mU][mU][mC][mA][mA][mG][mGs][mAs][mG] (SEQ ID NO: 2033); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 464 and a structure (5.fwdarw.3) of

(477) TABLE-US-00040 (SEQIDNO:1267) [mCs][mCs][mU][mU][mG][mA][mA][mU][fU][fC][fC] [fC][mU][mC][mA][mC][mG][mG][mA][mA][mAs], wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage; and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a targeting moiety comprising a structure of Formula (Z.sup.6), wherein the conjugated CYP7A1 RNAi agent comprises a structure as shown below:

(478) ##STR00148##
or a pharmaceutically acceptable salt thereof. It is to be understood that the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage (e.g.,

(479) ##STR00149##
wherein X is SH, i.e.,

(480) ##STR00150##
of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).

(481) In some embodiments, an RNAi agent comprising a targeting moiety described herein (e.g., a targeting moiety of Formulae (I), (I-a), (I-a-1), (I-b), (I-b-1), (I-c), (I-c-1), (I-d), (I-d-1), (I-e), (I-e-1), (I-f), (I-f-1), (I-g), (I-h), (I-i), (I-j), (II), (II-a), (II-b), (II-c), (III), (III-a), (IV), (IV-a), (IV-b), (IV-c), (IV-d), (IV-e), (IV-f), or (Z.sup.1)-(Z.sup.16)) may be synthesized via including GalNAc-containing phosphoramidite during solids phase synthesis of the sense strand such that the GalNAc-containing repeat units of the targeting moiety is directly added to the 5 or 3 end of the sense strand during synthesis.

(482) Pharmaceutical Compositions

(483) Pharmaceutical compositions comprising one or more RNAi agents (e.g., CYP7A1 RNAi agents), either alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided. The pharmaceutical compositions comprising RNAi agents (e.g., CYP7A1 RNAi agents) provided herein are for use in, but not limited to, diagnosing, detecting, or monitoring a disease, in preventing, treating, managing, or ameliorating a disease or one or more symptoms thereof, and/or in research. In some embodiments, a pharmaceutical composition may further comprise any other suitable therapeutic agent for treatment of a subject, e.g., a human subject having a CYP7A1 disease or CYP7A1-associated disease. In some embodiments, the other therapeutic agents may enhance or supplement the effectiveness of the complexes described herein. Non-limiting examples of other therapeutic agents include another RNAi agent, a small molecule drug, an antibody, an antibody fragment, peptide and/or aptamer. In some embodiments, the other therapeutic agents may function to treat a different symptom or disease than the CYP7A1 RNAi agents described herein. The formulation of pharmaceutical compositions, either alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers, are known to one skilled in the art.

(484) An aspect of the disclosure includes pharmaceutical compositions for inhibiting expression of a gene encoding CYP7A1 comprising any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein. In some embodiments, an RNAi agent (e.g., CYP7A1 RNAi agent) is in an unbuffered solution. In some embodiments, an RNAi agent (e.g., CYP7A1 RNAi agent) is formulated in water or in an aqueous solution (e.g., water with pH adjustments). In some embodiments, the unbuffered solution is saline. In some embodiments, an RNAi agent (e.g., CYP7A1 RNAi agent) is formulated in a buffer solution such as a phosphate-buffered saline solution, liposome, micellar structure, and capsid. In some embodiments, an RNAi agent (e.g., CYP7A1 RNAi agent) is formulated in a basic buffered aqueous solution (e.g., PBS). In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and/or (e.g., and) therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). Any one of the described RNAi agents (e.g., CYP7A1 RNAi agents), when added to pharmaceutically acceptable excipients, can be packaged into kits, containers, packs, or dispensers. The pharmaceutical compositions described herein can be packaged in pre-filled syringes or vials.

(485) In some embodiments, the delivery vehicle can be used to deliver a CYP7A1 RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves delivery of a CYP7A1 RNAi agent to a cell or tissue. A delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine. In some embodiments, any one of the CYP7A1 RNAi agents or pharmaceutical compositions described herein can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art. The CYP7A1 RNAi agents can also be chemically conjugated to targeting moiety, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example US2004171044, U.S. Pat. Nos. 8,137,695, 8,313,772, 8,501,930, 8,932,572, 8,933,047, US2016015824, U.S. Pat. Nos. 9,750,819, and 9,561,286, each of which is incorporated herein by reference), or other delivery systems available in the art.

(486) In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Non-limiting examples of routes of administration include intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the route of administration is subcutaneous.

(487) Kits

(488) An aspect of the disclosure includes kits comprising any one of the RNAi agents (e.g., CYP7A1 RNAi agents) described herein or a pharmaceutical composition described herein. In some embodiments, the kit further comprises instructions for administration. In some embodiments, the kit is for treating a CYP7A1 disease or a CYP7A1-associated disease described herein. In some embodiments, the kit further comprises an additional agent described herein.

(489) Methods

(490) Some aspects of the present disclosure provide methods for inhibiting or reducing expression level of CYP7A1 (e.g., mRNA level and/or protein level) in a cell. In some embodiments, a method for inhibiting or reducing CYP7A1 expression comprises contacting the cell with any one of the CYP7A1 RNAi agents described herein or any one of the pharmaceutical compositions described herein, thereby inhibiting or reducing expression level (e.g., mRNA level and/or protein level) of the CYP7A1 in the cell. In some embodiments, contacting the cell with any one of the CYP7A1 RNAi agents described herein inhibits the expression level (e.g., mRNA level and/or protein level) of CYP7A1 by at least 40%, 50%, 60%, 70%, 80%, 90%, or 95%, relative to the CYP7A1 level in cells not contacted with a CYP7A1 RNAi agent. In some embodiments, the cell is in vitro (e.g., in a cell culture). In some embodiments, the cell is in vivo (e.g., in a subject).

(491) The disclosure also provides methods of decreasing or reducing the expression level of CYP7A1 (e.g., mRNA level and/or protein level) in a subject compared to baseline pre-treatment levels. In some embodiments, methods comprise administering to the subject an effective amount any one of the CYP7A1 RNAi agents described herein or any one of the pharmaceutical compositions described herein. The expression level (e.g., mRNA level and/or protein level) in the subject is reduced in a cell, group of cells, tissue, blood, and/or other fluid of the subject. In some embodiments, the above described methods further comprise determining the level of CYP7A1 (e.g., mRNA level and/or protein level) in a sample(s) from the subject (e.g., CYP7A1 level in a blood or serum sample(s)). The level of CYP7A1 in a sample may be measured by general methods known in the art.

(492) The Examples disclosed herein set forth generally known methods for assessing inhibition of CYP7A1 expression and reduction in CYP7A1 expression levels. In some embodiments, a CYP7A1 RNAi agent described herein is administered to a subject at an effective concentration sufficient to inhibit activity or expression of CYP7A1 by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to a control, e.g., baseline level of gene expression prior to treatment. Some aspects of the disclosure provide methods of maintaining bile acid homeostasis by preventing overproduction of bile acid in the liver, comprising administering to the subject an effective amount any one of the CYP7A1 RNAi agents described herein or any one of the pharmaceutical compositions described herein.

(493) The disclosure also provides methods for reducing inflammation of the bile ducts in a subject, comprising administering to the subject an effective amount any one of the CYP7A1 RNAi agents described herein or any one of the pharmaceutical compositions described herein. In some embodiments, administering to the subject an effective amount any one of the CYP7A1 RNAi agents described herein or any one of the pharmaceutical compositions described herein, reduces inflammation of the bile ducts in a subject by at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% relative to the baseline level of inflammation.

(494) In some embodiments, in any one of the methods described herein, a subject is non-human primate, or rodent. In some embodiments, a subject is a human. In some embodiments, a subject is a patient, e.g., a human patient that has or is suspected of having a disease. In some embodiments, the subject is a human patient who has or is suspected of having a CYP7A1 disease or CYP7A1-associated disease. In some embodiments, the CYP7A1 disease or CYP7A1-associated disease is a liver disease. In some embodiments, the CYP7A1 disease or CYP7A1-associated disease is a cholestatic liver disease such as primary sclerosing cholangitis (PSC), familial intrahepatic cholestasis (PFIC, including Type 1, PFIC1, Type 2, PFIC2, and Type 3, PFIC3), primary biliary cholangitis (PBC), Alagille syndrome, and biliary atresia, and other liver diseases such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD).

(495) An aspect of the disclosure includes a method of treating a subject having a CYP7A1 disease or CYP7A1-associated disease. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of any one of the CYP7A1 RNAi agents described herein or a pharmaceutical composition described herein. In some embodiments the method results in treating the subject having the CYP7A1 disease or CYP7A1-associated disease. In some embodiments, the CYP7A1 disease or CYP7A1-associated disease is a liver disease. In some embodiments, the CYP7A1 disease or CYP7A1-associated disease is a cholestatic liver disease such as primary sclerosing cholangitis (PSC), familial intrahepatic cholestasis (PFIC, including Type 1, PFIC1, Type 2, PFIC2, and Type 3, PFIC3), primary biliary cholangitis (PBC), Alagille syndrome, and biliary atresia, and other liver diseases such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD).

(496) An aspect of the disclosure includes treating at least one symptom in a subject having a CYP7A1 disease or CYP7A1-associated disease. In some embodiments, the method comprises administering to the subject an effective amount of any one of the CYP7A1 RNAi agents described herein, or the pharmaceutical composition described herein, thereby treating at least one symptom in the subject having the CYP7A1 disease or CYP7A1-associated disease.

(497) In some embodiments, the CYP7A1 disease or the CYP7A1-associated disease symptoms to be treated by the administration of an effective amount of any one of the CYP7A1 RNAi agents described herein, or the pharmaceutical composition described herein, include, but are not limited to, biochemical symptoms, cellular symptoms, histological symptoms, physiological symptoms and/or medical symptoms. In some embodiments, the CYP7A1 disease or the CYP7A1-associated disease symptoms to be treated by the administration of an effective amount of any one of the CYP7A1 RNAi agents described herein, or the pharmaceutical composition described herein are selected from the group consisting of increased liver enzymes (ALP, ALT, AST, GGT), liver inflammation and scarring (fibrosis), increased liver stiffness, bile duct strictures, cirrhosis, liver failure, need for liver transplantation, portal hypertension, variceal bleeding, jaundice, and pruritus.

(498) In some embodiments, any one of the CYP7A1 RNAi agents or any one of the pharmaceutical compositions described herein are effective in treating CYP7A1 disease and CYP7A1-associated disease. In some embodiments, the CYP7A1 disease or CYP7A1-associated disease is a liver disease. In some embodiments, the CYP7A1 disease or CYP7A1-associated disease is a cholestatic liver disease such as primary sclerosing cholangitis (PSC), familial intrahepatic cholestasis (PFIC, including Type 1, PFIC1, Type 2, PFIC2, and Type 3, PFIC3), primary biliary cholangitis (PBC), Alagille syndrome, and biliary atresia, and other liver diseases such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and alcoholic liver disease (ALD).

(499) In some embodiments, the above described methods further comprise administering an additional agent for the treatment of a CYP7A1 disease or CYP7A1-associated disease.

(500) Having now described some embodiments in detail, practice of the invention will be more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.

EXAMPLES

Example 1: Identification of Active RNAi Sequences from Preliminary In-Vitro Screening

(501) A bioinformatics method assuming a canonical 19mer dsRNA was first employed to generate a potential set of 19 mer-containing antisense oligonucleotides targeting various regions of the human CYP7A1 mRNA transcript that were, in certain instances, also complementary to either cynomolgus monkey, rat, and/or mouse CYP7A1 mRNA.

(502) The sequence of the human CYP7A1 mRNA transcript (NM_000780.4), upon which the 19mer-containing antisense oligonucleotides were designed, is set forth in SEQ ID NO. 1. Of the potential 19mer antisense oligonucleotide sequences generated, 384 were selected for incorporation into chemically modified RNAi agents that could be prepared and evaluated in an in-vitro assay to assess target gene knockdown in primary human hepatocytes expressing CYP7A1. The CYP7A1 19-mer mRNA target sequences are listed in Table 2.

Example 2: siRNA Synthesis

(503) Generic 21/23-mer double stranded siRNAs (Table 5A) were designed based on the target sequences in Table 2. siRNAs with modifications as indicated in Table 5B were prepared by Integrated DNA Technologies Inc. (Iowa, USA) using standard solid phase oligonucleotide synthesis.

(504) Table 5A provides the nucleobase sequences of the siRNAs. It is to be understood that any chemical modification pattern may be applied to the nucleobase sequences of the siRNAs in Table 5A. For example, Table 5B lists the siRNA synthesized for in vitro screening, which all have a modification pattern M0 (the nucleosides at positions 9-13 and 19 in the sense strand are ribonucleosides and the rest of the nucleosides in the sense strand are 2-O-Me modified nucleosides; the nucleosides at positions 2, 3, 5-10, 13-15, 17, 19, and 20 of the antisense strand are ribonucleosides and the rest of the nucleosides in the antisense strand are 2O-Me modified nucleosides).

(505) TABLE-US-00041 TABLE5A NucleobasesequencesofsiRNAsusedininvitroscreening SEQ SEQ ID ID Position siRNA# Sense(5-3) NO: Antisense(5-3) NO 34 siRNA1 UUCUUCCUCAGAGAUUUUGGA 393 UCCAAAAUCUCUGAGGAAGAAUU 777 40 siRNA2 CUCAGAGAUUUUGGCCUAGAA 394 UUCUAGGCCAAAAUCUCUGAGUU 778 41 siRNA3 UCAGAGAUUUUGGCCUAGAUA 395 UAUCUAGGCCAAAAUCUCUGAUU 779 42 siRNA4 CAGAGAUUUUGGCCUAGAUUA 396 UAAUCUAGGCCAAAAUCUCUGUU 780 43 siRNA5 AGAGAUUUUGGCCUAGAUUUA 397 UAAAUCUAGGCCAAAAUCUCUUU 781 45 siRNA6 AGAUUUUGGCCUAGAUUUGCA 398 UGCAAAUCUAGGCCAAAAUCUUU 782 105 siRNA7 CAGCAUGCUGUUGUCUAUGGA 399 UCCAUAGACAACAGCAUGCUGUU 783 110 siRNA8 UGCUGUUGUCUAUGGCUUAUA 400 UAUAAGCCAUAGACAACAGCAUU 784 111 siRNA9 GCUGUUGUCUAUGGCUUAUUA 401 UAAUAAGCCAUAGACAACAGCUU 785 112 siRNA10 CUGUUGUCUAUGGCUUAUUCA 402 UGAAUAAGCCAUAGACAACAGUU 786 113 siRNA11 UGUUGUCUAUGGCUUAUUCUA 403 UAGAAUAAGCCAUAGACAACAUU 787 115 siRNA12 UUGUCUAUGGCUUAUUCUUGA 404 UCAAGAAUAAGCCAUAGACAAUU 788 116 siRNA13 UGUCUAUGGCUUAUUCUUGGA 405 UCCAAGAAUAAGCCAUAGACAUU 789 117 siRNA14 GUCUAUGGCUUAUUCUUGGAA 406 UUCCAAGAAUAAGCCAUAGACUU 790 118 siRNA15 UCUAUGGCUUAUUCUUGGAAA 407 UUUCCAAGAAUAAGCCAUAGAUU 791 139 siRNA16 UAGGAGAAGGCAAACGGGUGA 408 UCACCCGUUUGCCUUCUCCUAUU 792 141 siRNA17 GGAGAAGGCAAACGGGUGAAA 409 UUUCACCCGUUUGCCUUCUCCUU 793 142 siRNA18 GAGAAGGCAAACGGGUGAACA 410 UGUUCACCCGUUUGCCUUCUCUU 794 143 siRNA19 AGAAGGCAAACGGGUGAACCA 411 UGGUUCACCCGUUUGCCUUCUUU 795 181 siRNA20 AAUUCCAUACCUGGGCUGUGA 412 UCACAGCCCAGGUAUGGAAUUUU 796 182 siRNA21 AUUCCAUACCUGGGCUGUGCA 413 UGCACAGCCCAGGUAUGGAAUUU 797 183 siRNA22 UUCCAUACCUGGGCUGUGCUA 414 UAGCACAGCCCAGGUAUGGAAUU 798 201 siRNA23 CUCUGCAAUUUGGUGCCAAUA 415 UAUUGGCACCAAAUUGCAGAGUU 799 203 siRNA24 CUGCAAUUUGGUGCCAAUCCA 416 UGGAUUGGCACCAAAUUGCAGUU 800 210 siRNA25 UUGGUGCCAAUCCUCUUGAGA 417 UCUCAAGAGGAUUGGCACCAAUU 801 214 siRNA26 UGCCAAUCCUCUUGAGUUCCA 418 UGGAACUCAAGAGGAUUGGCAUU 802 223 siRNA27 UCUUGAGUUCCUCAGAGCAAA 419 UUUGCUCUGAGGAACUCAAGAUU 803 225 siRNA28 UUGAGUUCCUCAGAGCAAAUA 420 UAUUUGCUCUGAGGAACUCAAUU 804 229 siRNA29 GUUCCUCAGAGCAAAUCAAAA 421 UUUUGAUUUGCUCUGAGGAACUU 805 230 siRNA30 UUCCUCAGAGCAAAUCAAAGA 422 UCUUUGAUUUGCUCUGAGGAAUU 806 236 siRNA31 AGAGCAAAUCAAAGGAAACAA 423 UUGUUUCCUUUGAUUUGCUCUUU 807 238 siRNA32 AGCAAAUCAAAGGAAACAUGA 424 UCAUGUUUCCUUUGAUUUGCUUU 808 242 siRNA33 AAUCAAAGGAAACAUGGUCAA 425 UUGACCAUGUUUCCUUUGAUUUU 809 243 siRNA34 AUCAAAGGAAACAUGGUCAUA 426 UAUGACCAUGUUUCCUUUGAUUU 810 244 siRNA35 UCAAAGGAAACAUGGUCAUGA 427 UCAUGACCAUGUUUCCUUUGAUU 811 245 siRNA36 CAAAGGAAACAUGGUCAUGUA 428 UACAUGACCAUGUUUCCUUUGUU 812 246 siRNA37 AAAGGAAACAUGGUCAUGUUA 429 UAACAUGACCAUGUUUCCUUUUU 813 251 siRNA38 AAACAUGGUCAUGUUUUUACA 430 UGUAAAAACAUGACCAUGUUUUU 814 253 siRNA39 ACAUGGUCAUGUUUUUACCUA 431 UAGGUAAAAACAUGACCAUGUUU 815 254 siRNA40 CAUGGUCAUGUUUUUACCUGA 432 UCAGGUAAAAACAUGACCAUGUU 816 262 siRNA41 UGUUUUUACCUGCAAACUAAA 433 UUUAGUUUGCAGGUAAAAACAUU 817 264 siRNA42 UUUUUACCUGCAAACUAAUGA 434 UCAUUAGUUUGCAGGUAAAAAUU 818 266 siRNA43 UUUACCUGCAAACUAAUGGGA 435 UCCCAUUAGUUUGCAGGUAAAUU 819 267 siRNA44 UUACCUGCAAACUAAUGGGAA 436 UUCCCAUUAGUUUGCAGGUAAUU 820 268 siRNA45 UACCUGCAAACUAAUGGGAAA 437 UUUCCCAUUAGUUUGCAGGUAUU 821 291 siRNA46 AUGUCCAUUUCAUCACAAAUA 438 UAUUUGUGAUGAAAUGGACAUUU 822 292 siRNA47 UGUCCAUUUCAUCACAAAUCA 439 UGAUUUGUGAUGAAAUGGACAUU 823 294 siRNA48 UCCAUUUCAUCACAAAUCCCA 440 UGGGAUUUGUGAUGAAAUGGAUU 824 300 siRNA49 UCAUCACAAAUCCCUUGUCAA 441 UUGACAAGGGAUUUGUGAUGAUU 825 302 siRNA50 AUCACAAAUCCCUUGUCAUAA 442 UUAUGACAAGGGAUUUGUGAUUU 826 303 siRNA51 UCACAAAUCCCUUGUCAUACA 443 UGUAUGACAAGGGAUUUGUGAUU 827 304 siRNA52 CACAAAUCCCUUGUCAUACCA 444 UGGUAUGACAAGGGAUUUGUGUU 828 305 siRNA53 ACAAAUCCCUUGUCAUACCAA 445 UUGGUAUGACAAGGGAUUUGUUU 829 309 siRNA54 AUCCCUUGUCAUACCAUAAGA 446 UCUUAUGGUAUGACAAGGGAUUU 830 310 siRNA55 UCCCUUGUCAUACCAUAAGGA 447 UCCUUAUGGUAUGACAAGGGAUU 831 311 siRNA56 CCCUUGUCAUACCAUAAGGUA 448 UACCUUAUGGUAUGACAAGGGUU 832 312 siRNA57 CCUUGUCAUACCAUAAGGUGA 449 UCACCUUAUGGUAUGACAAGGUU 833 313 siRNA58 CUUGUCAUACCAUAAGGUGUA 450 UACACCUUAUGGUAUGACAAGUU 834 315 siRNA59 UGUCAUACCAUAAGGUGUUGA 451 UCAACACCUUAUGGUAUGACAUU 835 319 siRNA60 AUACCAUAAGGUGUUGUGCCA 452 UGGCACAACACCUUAUGGUAUUU 836 360 siRNA61 AAAAAUUUCACUUUGCUACUA 453 UAGUAGCAAAGUGAAAUUUUUUU 837 363 siRNA62 AAUUUCACUUUGCUACUUCUA 454 UAGAAGUAGCAAAGUGAAAUUUU 838 372 siRNA63 UUGCUACUUCUGCGAAGGCAA 455 UUGCCUUCGCAGAAGUAGCAAUU 839 375 siRNA64 CUACUUCUGCGAAGGCAUUUA 456 UAAAUGCCUUCGCAGAAGUAGUU 840 377 siRNA65 ACUUCUGCGAAGGCAUUUGGA 457 UCCAAAUGCCUUCGCAGAAGUUU 841 390 siRNA66 CAUUUGGGCACAGAAGCAUUA 458 UAAUGCUUCUGUGCCCAAAUGUU 842 392 siRNA67 UUUGGGCACAGAAGCAUUGAA 459 UUCAAUGCUUCUGUGCCCAAAUU 843 394 siRNA68 UGGGCACAGAAGCAUUGACCA 460 UGGUCAAUGCUUCUGUGCCCAUU 844 474 siRNA69 AUGCCUUGAAUUCCCUCACGA 461 UCGUGAGGGAAUUCAAGGCAUUU 845 475 siRNA70 UGCCUUGAAUUCCCUCACGGA 462 UCCGUGAGGGAAUUCAAGGCAUU 846 476 siRNA71 GCCUUGAAUUCCCUCACGGAA 463 UUCCGUGAGGGAAUUCAAGGCUU 847 477 siRNA72 CCUUGAAUUCCCUCACGGAAA 464 UUUCCGUGAGGGAAUUCAAGGUU 848 478 siRNA73 CUUGAAUUCCCUCACGGAAAA 465 UUUUCCGUGAGGGAAUUCAAGUU 849 482 siRNA74 AAUUCCCUCACGGAAAGCAUA 466 UAUGCUUUCCGUGAGGGAAUUUU 850 484 siRNA75 UUCCCUCACGGAAAGCAUGAA 467 UUCAUGCUUUCCGUGAGGGAAUU 851 486 siRNA76 CCCUCACGGAAAGCAUGAUGA 468 UCAUCAUGCUUUCCGUGAGGGUU 852 488 siRNA77 CUCACGGAAAGCAUGAUGGAA 469 UUCCAUCAUGCUUUCCGUGAGUU 853 501 siRNA78 UGAUGGAAAACCUCCAACGUA 470 UACGUUGGAGGUUUUCCAUCAUU 854 503 siRNA79 AUGGAAAACCUCCAACGUAUA 471 UAUACGUUGGAGGUUUUCCAUUU 855 505 siRNA80 GGAAAACCUCCAACGUAUCAA 472 UUGAUACGUUGGAGGUUUUCCUU 856 506 siRNA81 GAAAACCUCCAACGUAUCAUA 473 UAUGAUACGUUGGAGGUUUUCUU 857 507 siRNA82 AAAACCUCCAACGUAUCAUGA 474 UCAUGAUACGUUGGAGGUUUUUU 858 509 siRNA83 AACCUCCAACGUAUCAUGAGA 475 UCUCAUGAUACGUUGGAGGUUUU 859 510 siRNA84 ACCUCCAACGUAUCAUGAGAA 476 UUCUCAUGAUACGUUGGAGGUUU 860 511 siRNA85 CCUCCAACGUAUCAUGAGACA 477 UGUCUCAUGAUACGUUGGAGGUU 861 512 siRNA86 CUCCAACGUAUCAUGAGACCA 478 UGGUCUCAUGAUACGUUGGAGUU 862 513 siRNA87 UCCAACGUAUCAUGAGACCUA 479 UAGGUCUCAUGAUACGUUGGAUU 863 514 siRNA88 CCAACGUAUCAUGAGACCUCA 480 UGAGGUCUCAUGAUACGUUGGUU 864 539 siRNA89 UCCUCUAACUCAAAGACCGCA 481 UGCGGUCUUUGAGUUAGAGGAUU 865 552 siRNA90 AGACCGCUGCCUGGGUGACAA 482 UUGUCACCCAGGCAGCGGUCUUU 866 562 siRNA91 CUGGGUGACAGAAGGGAUGUA 483 UACAUCCCUUCUGUCACCCAGUU 867 563 siRNA92 UGGGUGACAGAAGGGAUGUAA 484 UUACAUCCCUUCUGUCACCCAUU 868 564 siRNA93 GGGUGACAGAAGGGAUGUAUA 485 UAUACAUCCCUUCUGUCACCCUU 869 585 siRNA94 CUUUCUGCUACCGAGUGAUGA 486 UCAUCACUCGGUAGCAGAAAGUU 870 587 siRNA95 UUCUGCUACCGAGUGAUGUUA 487 UAACAUCACUCGGUAGCAGAAUU 871 588 siRNA96 UCUGCUACCGAGUGAUGUUUA 488 UAAACAUCACUCGGUAGCAGAUU 872 589 siRNA97 CUGCUACCGAGUGAUGUUUGA 489 UCAAACAUCACUCGGUAGCAGUU 873 593 siRNA98 UACCGAGUGAUGUUUGAAGCA 490 UGCUUCAAACAUCACUCGGUAUU 874 594 siRNA99 ACCGAGUGAUGUUUGAAGCUA 491 UAGCUUCAAACAUCACUCGGUUU 875 595 siRNA100 CCGAGUGAUGUUUGAAGCUGA 492 UCAGCUUCAAACAUCACUCGGUU 876 602 siRNA101 AUGUUUGAAGCUGGGUAUUUA 493 UAAAUACCCAGCUUCAAACAUUU 877 605 siRNA102 UUUGAAGCUGGGUAUUUAACA 494 UGUUAAAUACCCAGCUUCAAAUU 878 606 siRNA103 UUGAAGCUGGGUAUUUAACUA 495 UAGUUAAAUACCCAGCUUCAAUU 879 607 siRNA104 UGAAGCUGGGUAUUUAACUAA 496 UUAGUUAAAUACCCAGCUUCAUU 880 608 siRNA105 GAAGCUGGGUAUUUAACUAUA 497 UAUAGUUAAAUACCCAGCUUCUU 881 609 siRNA106 AAGCUGGGUAUUUAACUAUCA 498 UGAUAGUUAAAUACCCAGCUUUU 882 610 siRNA107 AGCUGGGUAUUUAACUAUCUA 499 UAGAUAGUUAAAUACCCAGCUUU 883 612 siRNA108 CUGGGUAUUUAACUAUCUUUA 500 UAAAGAUAGUUAAAUACCCAGUU 884 613 siRNA109 UGGGUAUUUAACUAUCUUUGA 501 UCAAAGAUAGUUAAAUACCCAUU 885 653 siRNA110 GACACACAGAAAGCACAUAUA 502 UAUAUGUGCUUUCUGUGUGUCUU 886 663 siRNA111 AAGCACAUAUUCUAAACAAUA 503 UAUUGUUUAGAAUAUGUGCUUUU 887 665 siRNA112 GCACAUAUUCUAAACAAUCUA 504 UAGAUUGUUUAGAAUAUGUGCUU 888 666 siRNA113 CACAUAUUCUAAACAAUCUUA 505 UAAGAUUGUUUAGAAUAUGUGUU 889 667 siRNA114 ACAUAUUCUAAACAAUCUUGA 506 UCAAGAUUGUUUAGAAUAUGUUU 890 669 siRNA115 AUAUUCUAAACAAUCUUGACA 507 UGUCAAGAUUGUUUAGAAUAUUU 891 670 siRNA116 UAUUCUAAACAAUCUUGACAA 508 UUGUCAAGAUUGUUUAGAAUAUU 892 671 siRNA117 AUUCUAAACAAUCUUGACAAA 509 UUUGUCAAGAUUGUUUAGAAUUU 893 673 siRNA118 UCUAAACAAUCUUGACAACUA 510 UAGUUGUCAAGAUUGUUUAGAUU 894 679 siRNA119 CAAUCUUGACAACUUCAAGCA 511 UGCUUGAAGUUGUCAAGAUUGUU 895 712 siRNA120 CUUUCCAGCCCUGGUAGCAGA 512 UCUGCUACCAGGGCUGGAAAGUU 896 771 siRNA121 GGGAGAAACUGGCAGAGAGCA 513 UGCUCUCUGCCAGUUUCUCCCUU 897 781 siRNA122 GGCAGAGAGCUUGAGGCACGA 514 UCGUGCCUCAAGCUCUCUGCCUU 898 788 siRNA123 AGCUUGAGGCACGAGAACCUA 515 UAGGUUCUCGUGCCUCAAGCUUU 899 841 siRNA124 CCUGCGCAUGUUUCUCAAUGA 516 UCAUUGAGAAACAUGCGCAGGUU 900 844 siRNA125 GCGCAUGUUUCUCAAUGACAA 517 UUGUCAUUGAGAAACAUGCGCUU 901 846 siRNA126 GCAUGUUUCUCAAUGACACUA 518 UAGUGUCAUUGAGAAACAUGCUU 902 850 siRNA127 GUUUCUCAAUGACACUUUGUA 519 UACAAAGUGUCAUUGAGAAACUU 903 851 siRNA128 UUUCUCAAUGACACUUUGUCA 520 UGACAAAGUGUCAUUGAGAAAUU 904 852 siRNA129 UUCUCAAUGACACUUUGUCCA 521 UGGACAAAGUGUCAUUGAGAAUU 905 861 siRNA130 ACACUUUGUCCACCUUUGAUA 522 UAUCAAAGGUGGACAAAGUGUUU 906 862 siRNA131 CACUUUGUCCACCUUUGAUGA 523 UCAUCAAAGGUGGACAAAGUGUU 907 901 siRNA132 ACACCUCGUGGUCCUCUGGGA 524 UCCCAGAGGACCACGAGGUGUUU 908 903 siRNA133 ACCUCGUGGUCCUCUGGGCAA 525 UUGCCCAGAGGACCACGAGGUUU 909 904 siRNA134 CCUCGUGGUCCUCUGGGCAUA 526 UAUGCCCAGAGGACCACGAGGUU 910 905 siRNA135 CUCGUGGUCCUCUGGGCAUCA 527 UGAUGCCCAGAGGACCACGAGUU 911 906 siRNA136 UCGUGGUCCUCUGGGCAUCGA 528 UCGAUGCCCAGAGGACCACGAUU 912 912 siRNA137 UCCUCUGGGCAUCGCAAGCAA 529 UUGCUUGCGAUGCCCAGAGGAUU 913 915 siRNA138 UCUGGGCAUCGCAAGCAAACA 530 UGUUUGCUUGCGAUGCCCAGAUU 914 916 siRNA139 CUGGGCAUCGCAAGCAAACAA 531 UUGUUUGCUUGCGAUGCCCAGUU 915 917 siRNA140 UGGGCAUCGCAAGCAAACACA 532 UGUGUUUGCUUGCGAUGCCCAUU 916 920 siRNA141 GCAUCGCAAGCAAACACCAUA 533 UAUGGUGUUUGCUUGCGAUGCUU 917 921 siRNA142 CAUCGCAAGCAAACACCAUUA 534 UAAUGGUGUUUGCUUGCGAUGUU 918 950 siRNA143 UUCUGGAGUUUAUUUCAAAUA 535 UAUUUGAAAUAAACUCCAGAAUU 919 954 siRNA144 GGAGUUUAUUUCAAAUGAUUA 536 UAAUCAUUUGAAAUAAACUCCUU 920 955 siRNA145 GAGUUUAUUUCAAAUGAUUAA 537 UUAAUCAUUUGAAAUAAACUCUU 921 956 siRNA146 AGUUUAUUUCAAAUGAUUAGA 538 UCUAAUCAUUUGAAAUAAACUUU 922 957 siRNA147 GUUUAUUUCAAAUGAUUAGGA 539 UCCUAAUCAUUUGAAAUAAACUU 923 980 siRNA148 CCAGAAGCAAUGAAAGCAGCA 540 UGCUGCUUUCAUUGCUUCUGGUU 924 982 siRNA149 AGAAGCAAUGAAAGCAGCUAA 541 UUAGCUGCUUUCAUUGCUUCUUU 925 983 siRNA150 GAAGCAAUGAAAGCAGCUACA 542 UGUAGCUGCUUUCAUUGCUUCUU 926 988 siRNA151 AAUGAAAGCAGCUACUGAAGA 543 UCUUCAGUAGCUGCUUUCAUUUU 927 989 siRNA152 AUGAAAGCAGCUACUGAAGAA 544 UUCUUCAGUAGCUGCUUUCAUUU 928 992 siRNA153 AAAGCAGCUACUGAAGAAGUA 545 UACUUCUUCAGUAGCUGCUUUUU 929 993 siRNA154 AAGCAGCUACUGAAGAAGUGA 546 UCACUUCUUCAGUAGCUGCUUUU 930 994 siRNA155 AGCAGCUACUGAAGAAGUGAA 547 UUCACUUCUUCAGUAGCUGCUUU 931 995 siRNA156 GCAGCUACUGAAGAAGUGAAA 548 UUUCACUUCUUCAGUAGCUGCUU 932 996 siRNA157 CAGCUACUGAAGAAGUGAAAA 549 UUUUCACUUCUUCAGUAGCUGUU 933 1005 siRNA158 AAGAAGUGAAAAGAACAUUAA 550 UUAAUGUUCUUUUCACUUCUUUU 934 1006 siRNA159 AGAAGUGAAAAGAACAUUAGA 551 UCUAAUGUUCUUUUCACUUCUUU 935 1007 siRNA160 GAAGUGAAAAGAACAUUAGAA 552 UUCUAAUGUUCUUUUCACUUCUU 936 1011 siRNA161 UGAAAAGAACAUUAGAGAAUA 553 UAUUCUCUAAUGUUCUUUUCAUU 937 1012 siRNA162 GAAAAGAACAUUAGAGAAUGA 554 UCAUUCUCUAAUGUUCUUUUCUU 938 1013 siRNA163 AAAAGAACAUUAGAGAAUGCA 555 UGCAUUCUCUAAUGUUCUUUUUU 939 1018 siRNA164 AACAUUAGAGAAUGCUGGUCA 556 UGACCAGCAUUCUCUAAUGUUUU 940 1019 siRNA165 ACAUUAGAGAAUGCUGGUCAA 557 UUGACCAGCAUUCUCUAAUGUUU 941 1020 siRNA166 CAUUAGAGAAUGCUGGUCAAA 558 UUUGACCAGCAUUCUCUAAUGUU 942 1021 siRNA167 AUUAGAGAAUGCUGGUCAAAA 559 UUUUGACCAGCAUUCUCUAAUUU 943 1026 siRNA168 AGAAUGCUGGUCAAAAAGUCA 560 UGACUUUUUGACCAGCAUUCUUU 944 1027 siRNA169 GAAUGCUGGUCAAAAAGUCAA 561 UUGACUUUUUGACCAGCAUUCUU 945 1029 siRNA170 AUGCUGGUCAAAAAGUCAGCA 562 UGCUGACUUUUUGACCAGCAUUU 946 1036 siRNA171 UCAAAAAGUCAGCUUGGAAGA 563 UCUUCCAAGCUGACUUUUUGAUU 947 1039 siRNA172 AAAAGUCAGCUUGGAAGGCAA 564 UUGCCUUCCAAGCUGACUUUUUU 948 1042 siRNA173 AGUCAGCUUGGAAGGCAAUCA 565 UGAUUGCCUUCCAAGCUGACUUU 949 1084 siRNA174 ACUGAAUGACCUGCCAGUAUA 566 UAUACUGGCAGGUCAUUCAGUUU 950 1087 siRNA175 GAAUGACCUGCCAGUAUUAGA 567 UCUAAUACUGGCAGGUCAUUCUU 951 1134 siRNA176 UUUCCAGUGCCUCCCUCAACA 568 UGUUGAGGGAGGCACUGGAAAUU 952 1151 siRNA177 AACAUCCGGACAGCUAAGGAA 569 UUCCUUAGCUGUCCGGAUGUUUU 953 1152 siRNA178 ACAUCCGGACAGCUAAGGAGA 570 UCUCCUUAGCUGUCCGGAUGUUU 954 1155 siRNA179 UCCGGACAGCUAAGGAGGAUA 571 UAUCCUCCUUAGCUGUCCGGAUU 955 1158 siRNA180 GGACAGCUAAGGAGGAUUUCA 572 UGAAAUCCUCCUUAGCUGUCCUU 956 1160 siRNA181 ACAGCUAAGGAGGAUUUCACA 573 UGUGAAAUCCUCCUUAGCUGUUU 957 1162 siRNA182 AGCUAAGGAGGAUUUCACUUA 574 UAAGUGAAAUCCUCCUUAGCUUU 958 1165 siRNA183 UAAGGAGGAUUUCACUUUGCA 575 UGCAAAGUGAAAUCCUCCUUAUU 959 1168 siRNA184 GGAGGAUUUCACUUUGCACCA 576 UGGUGCAAAGUGAAAUCCUCCUU 960 1169 siRNA185 GAGGAUUUCACUUUGCACCUA 577 UAGGUGCAAAGUGAAAUCCUCUU 961 1174 siRNA186 UUUCACUUUGCACCUUGAGGA 578 UCCUCAAGGUGCAAAGUGAAAUU 962 1177 siRNA187 CACUUUGCACCUUGAGGACGA 579 UCGUCCUCAAGGUGCAAAGUGUU 963 1178 siRNA188 ACUUUGCACCUUGAGGACGGA 580 UCCGUCCUCAAGGUGCAAAGUUU 964 1189 siRNA189 UGAGGACGGUUCCUACAACAA 581 UUGUUGUAGGAACCGUCCUCAUU 965 1191 siRNA190 AGGACGGUUCCUACAACAUCA 582 UGAUGUUGUAGGAACCGUCCUUU 966 1194 siRNA191 ACGGUUCCUACAACAUCCGAA 583 UUCGGAUGUUGUAGGAACCGUUU 967 1196 siRNA192 GGUUCCUACAACAUCCGAAAA 584 UUUUCGGAUGUUGUAGGAACCUU 968 1200 siRNA193 CCUACAACAUCCGAAAAGAUA 585 UAUCUUUUCGGAUGUUGUAGGUU 969 1201 siRNA194 CUACAACAUCCGAAAAGAUGA 586 UCAUCUUUUCGGAUGUUGUAGUU 970 1203 siRNA195 ACAACAUCCGAAAAGAUGACA 587 UGUCAUCUUUUCGGAUGUUGUUU 971 1204 siRNA196 CAACAUCCGAAAAGAUGACAA 588 UUGUCAUCUUUUCGGAUGUUGUU 972 1205 siRNA197 AACAUCCGAAAAGAUGACAUA 589 UAUGUCAUCUUUUCGGAUGUUUU 973 1207 siRNA198 CAUCCGAAAAGAUGACAUCAA 590 UUGAUGUCAUCUUUUCGGAUGUU 974 1209 siRNA199 UCCGAAAAGAUGACAUCAUAA 591 UUAUGAUGUCAUCUUUUCGGAUU 975 1212 siRNA200 GAAAAGAUGACAUCAUAGCUA 592 UAGCUAUGAUGUCAUCUUUUCUU 976 1214 siRNA201 AAAGAUGACAUCAUAGCUCUA 593 UAGAGCUAUGAUGUCAUCUUUUU 977 1215 siRNA202 AAGAUGACAUCAUAGCUCUUA 594 UAAGAGCUAUGAUGUCAUCUUUU 978 1217 siRNA203 GAUGACAUCAUAGCUCUUUAA 595 UUAAAGAGCUAUGAUGUCAUCUU 979 1226 siRNA204 AUAGCUCUUUACCCACAGUUA 596 UAACUGUGGGUAAAGAGCUAUUU 980 1227 siRNA205 UAGCUCUUUACCCACAGUUAA 597 UUAACUGUGGGUAAAGAGCUAUU 981 1228 siRNA206 AGCUCUUUACCCACAGUUAAA 598 UUUAACUGUGGGUAAAGAGCUUU 982 1229 siRNA207 GCUCUUUACCCACAGUUAAUA 599 UAUUAACUGUGGGUAAAGAGCUU 983 1230 siRNA208 CUCUUUACCCACAGUUAAUGA 600 UCAUUAACUGUGGGUAAAGAGUU 984 1231 siRNA209 UCUUUACCCACAGUUAAUGCA 601 UGCAUUAACUGUGGGUAAAGAUU 985 1233 siRNA210 UUUACCCACAGUUAAUGCACA 602 UGUGCAUUAACUGUGGGUAAAUU 986 1236 siRNA211 ACCCACAGUUAAUGCACUUAA 603 UUAAGUGCAUUAACUGUGGGUUU 987 1237 siRNA212 CCCACAGUUAAUGCACUUAGA 604 UCUAAGUGCAUUAACUGUGGGUU 988 1238 siRNA213 CCACAGUUAAUGCACUUAGAA 605 UUCUAAGUGCAUUAACUGUGGUU 989 1239 siRNA214 CACAGUUAAUGCACUUAGAUA 606 UAUCUAAGUGCAUUAACUGUGUU 990 1240 siRNA215 ACAGUUAAUGCACUUAGAUCA 607 UGAUCUAAGUGCAUUAACUGUUU 991 1241 siRNA216 CAGUUAAUGCACUUAGAUCCA 608 UGGAUCUAAGUGCAUUAACUGUU 992 1242 siRNA217 AGUUAAUGCACUUAGAUCCAA 609 UUGGAUCUAAGUGCAUUAACUUU 993 1248 siRNA218 UGCACUUAGAUCCAGAAAUCA 610 UGAUUUCUGGAUCUAAGUGCAUU 994 1249 siRNA219 GCACUUAGAUCCAGAAAUCUA 611 UAGAUUUCUGGAUCUAAGUGCUU 995 1261 siRNA220 AGAAAUCUACCCAGACCCUUA 612 UAAGGGUCUGGGUAGAUUUCUUU 996 1283 siRNA221 ACUUUUAAAUAUGAUAGGUAA 613 UUACCUAUCAUAUUUAAAAGUUU 997 1285 siRNA222 UUUUAAAUAUGAUAGGUAUCA 614 UGAUACCUAUCAUAUUUAAAAUU 998 1286 siRNA223 UUUAAAUAUGAUAGGUAUCUA 615 UAGAUACCUAUCAUAUUUAAAUU 999 1288 siRNA224 UAAAUAUGAUAGGUAUCUUGA 616 UCAAGAUACCUAUCAUAUUUAUU 1000 1290 siRNA225 AAUAUGAUAGGUAUCUUGAUA 617 UAUCAAGAUACCUAUCAUAUUUU 1001 1291 siRNA226 AUAUGAUAGGUAUCUUGAUGA 618 UCAUCAAGAUACCUAUCAUAUUU 1002 1292 siRNA227 UAUGAUAGGUAUCUUGAUGAA 619 UUCAUCAAGAUACCUAUCAUAUU 1003 1293 siRNA228 AUGAUAGGUAUCUUGAUGAAA 620 UUUCAUCAAGAUACCUAUCAUUU 1004 1296 siRNA229 AUAGGUAUCUUGAUGAAAACA 621 UGUUUUCAUCAAGAUACCUAUUU 1005 1297 siRNA230 UAGGUAUCUUGAUGAAAACGA 622 UCGUUUUCAUCAAGAUACCUAUU 1006 1298 siRNA231 AGGUAUCUUGAUGAAAACGGA 623 UCCGUUUUCAUCAAGAUACCUUU 1007 1299 siRNA232 GGUAUCUUGAUGAAAACGGGA 624 UCCCGUUUUCAUCAAGAUACCUU 1008 1300 siRNA233 GUAUCUUGAUGAAAACGGGAA 625 UUCCCGUUUUCAUCAAGAUACUU 1009 1301 siRNA234 UAUCUUGAUGAAAACGGGAAA 626 UUUCCCGUUUUCAUCAAGAUAUU 1010 1302 siRNA235 AUCUUGAUGAAAACGGGAAGA 627 UCUUCCCGUUUUCAUCAAGAUUU 1011 1303 siRNA236 UCUUGAUGAAAACGGGAAGAA 628 UUCUUCCCGUUUUCAUCAAGAUU 1012 1306 siRNA237 UGAUGAAAACGGGAAGACAAA 629 UUUGUCUUCCCGUUUUCAUCAUU 1013 1307 siRNA238 GAUGAAAACGGGAAGACAAAA 630 UUUUGUCUUCCCGUUUUCAUCUU 1014 1311 siRNA239 AAAACGGGAAGACAAAGACUA 631 UAGUCUUUGUCUUCCCGUUUUUU 1015 1313 siRNA240 AACGGGAAGACAAAGACUACA 632 UGUAGUCUUUGUCUUCCCGUUUU 1016 1314 siRNA241 ACGGGAAGACAAAGACUACCA 633 UGGUAGUCUUUGUCUUCCCGUUU 1017 1353 siRNA242 AGUUAAAGUAUUACUACAUGA 634 UCAUGUAGUAAUACUUUAACUUU 1018 1354 siRNA243 GUUAAAGUAUUACUACAUGCA 635 UGCAUGUAGUAAUACUUUAACUU 1019 1358 siRNA244 AAGUAUUACUACAUGCCCUUA 636 UAAGGGCAUGUAGUAAUACUUUU 1020 1360 siRNA245 GUAUUACUACAUGCCCUUUGA 637 UCAAAGGGCAUGUAGUAAUACUU 1021 1361 siRNA246 UAUUACUACAUGCCCUUUGGA 638 UCCAAAGGGCAUGUAGUAAUAUU 1022 1363 siRNA247 UUACUACAUGCCCUUUGGAUA 639 UAUCCAAAGGGCAUGUAGUAAUU 1023 1364 siRNA248 UACUACAUGCCCUUUGGAUCA 640 UGAUCCAAAGGGCAUGUAGUAUU 1024 1382 siRNA249 UCGGGAGCUACAAUAUGUCCA 641 UGGACAUAUUGUAGCUCCCGAUU 1025 1386 siRNA250 GAGCUACAAUAUGUCCUGGAA 642 UUCCAGGACAUAUUGUAGCUCUU 1026 1388 siRNA251 GCUACAAUAUGUCCUGGAAGA 643 UCUUCCAGGACAUAUUGUAGCUU 1027 1389 siRNA252 CUACAAUAUGUCCUGGAAGAA 644 UUCUUCCAGGACAUAUUGUAGUU 1028 1413 siRNA253 UCGCUAUCCACGAAAUCAAGA 645 UCUUGAUUUCGUGGAUAGCGAUU 1029 1415 siRNA254 GCUAUCCACGAAAUCAAGCAA 646 UUGCUUGAUUUCGUGGAUAGCUU 1030 1416 siRNA255 CUAUCCACGAAAUCAAGCAAA 647 UUUGCUUGAUUUCGUGGAUAGUU 1031 1417 siRNA256 UAUCCACGAAAUCAAGCAAUA 648 UAUUGCUUGAUUUCGUGGAUAUU 1032 1424 siRNA257 GAAAUCAAGCAAUUUUUGAUA 649 UAUCAAAAAUUGCUUGAUUUCUU 1033 1425 siRNA258 AAAUCAAGCAAUUUUUGAUUA 650 UAAUCAAAAAUUGCUUGAUUUUU 1034 1431 siRNA259 AGCAAUUUUUGAUUCUGAUGA 651 UCAUCAGAAUCAAAAAUUGCUUU 1035 1433 siRNA260 CAAUUUUUGAUUCUGAUGCUA 652 UAGCAUCAGAAUCAAAAAUUGUU 1036 1506 siRNA261 ACCAGUCCCGGGCAGGCUUGA 653 UCAAGCCUGCCCGGGACUGGUUU 1037 1507 siRNA262 CCAGUCCCGGGCAGGCUUGGA 654 UCCAAGCCUGCCCGGGACUGGUU 1038 1510 siRNA263 GUCCCGGGCAGGCUUGGGCAA 655 UUGCCCAAGCCUGCCCGGGACUU 1039 1511 siRNA264 UCCCGGGCAGGCUUGGGCAUA 656 UAUGCCCAAGCCUGCCCGGGAUU 1040 1513 siRNA265 CCGGGCAGGCUUGGGCAUUUA 657 UAAAUGCCCAAGCCUGCCCGGUU 1041 1518 siRNA266 CAGGCUUGGGCAUUUUGCCGA 658 UCGGCAAAAUGCCCAAGCCUGUU 1042 1555 siRNA267 AUUUAAAUAUAAAUUCAAGCA 659 UGCUUGAAUUUAUAUUUAAAUUU 1043 1556 siRNA268 UUUAAAUAUAAAUUCAAGCAA 660 UUGCUUGAAUUUAUAUUUAAAUU 1044 1557 siRNA269 UUAAAUAUAAAUUCAAGCAUA 661 UAUGCUUGAAUUUAUAUUUAAUU 1045 1558 siRNA270 UAAAUAUAAAUUCAAGCAUUA 662 UAAUGCUUGAAUUUAUAUUUAUU 1046 1559 siRNA271 AAAUAUAAAUUCAAGCAUUUA 663 UAAAUGCUUGAAUUUAUAUUUUU 1047 1561 siRNA272 AUAUAAAUUCAAGCAUUUGUA 664 UACAAAUGCUUGAAUUUAUAUUU 1048 1593 siRNA273 GGAAUAAGAGGACACUAGAUA 665 UAUCUAGUGUCCUCUUAUUCCUU 1049 1594 siRNA274 GAAUAAGAGGACACUAGAUGA 666 UCAUCUAGUGUCCUCUUAUUCUU 1050 1597 siRNA275 UAAGAGGACACUAGAUGAUAA 667 UUAUCAUCUAGUGUCCUCUUAUU 1051 1599 siRNA276 AGAGGACACUAGAUGAUAUUA 668 UAAUAUCAUCUAGUGUCCUCUUU 1052 1601 siRNA277 AGGACACUAGAUGAUAUUACA 669 UGUAAUAUCAUCUAGUGUCCUUU 1053 1605 siRNA278 CACUAGAUGAUAUUACAGGAA 670 UUCCUGUAAUAUCAUCUAGUGUU 1054 1606 siRNA279 ACUAGAUGAUAUUACAGGACA 671 UGUCCUGUAAUAUCAUCUAGUUU 1055 1609 siRNA280 AGAUGAUAUUACAGGACUGCA 672 UGCAGUCCUGUAAUAUCAUCUUU 1056 1610 siRNA281 GAUGAUAUUACAGGACUGCAA 673 UUGCAGUCCUGUAAUAUCAUCUU 1057 1614 siRNA282 AUAUUACAGGACUGCAGAACA 674 UGUUCUGCAGUCCUGUAAUAUUU 1058 1638 siRNA283 UCACCACACAGUCCCUUUGGA 675 UCCAAAGGGACUGUGUGGUGAUU 1059 1660 siRNA284 AAAUGCAUUUAGUGGUGGUAA 676 UUACCACCACUAAAUGCAUUUUU 1060 1661 siRNA285 AAUGCAUUUAGUGGUGGUAGA 677 UCUACCACCACUAAAUGCAUUUU 1061 1662 siRNA286 AUGCAUUUAGUGGUGGUAGAA 678 UUCUACCACCACUAAAUGCAUUU 1062 1663 siRNA287 UGCAUUUAGUGGUGGUAGAAA 679 UUUCUACCACCACUAAAUGCAUU 1063 1669 siRNA288 UAGUGGUGGUAGAAAUGAUUA 680 UAAUCAUUUCUACCACCACUAUU 1064 1671 siRNA289 GUGGUGGUAGAAAUGAUUCAA 681 UUGAAUCAUUUCUACCACCACUU 1065 1673 siRNA290 GGUGGUAGAAAUGAUUCACCA 682 UGGUGAAUCAUUUCUACCACCUU 1066 1676 siRNA291 GGUAGAAAUGAUUCACCAGGA 683 UCCUGGUGAAUCAUUUCUACCUU 1067 1683 siRNA292 AUGAUUCACCAGGUCCAAUGA 684 UCAUUGGACCUGGUGAAUCAUUU 1068 1686 siRNA293 AUUCACCAGGUCCAAUGUUGA 685 UCAACAUUGGACCUGGUGAAUUU 1069 1689 siRNA294 CACCAGGUCCAAUGUUGUUCA 686 UGAACAACAUUGGACCUGGUGUU 1070 1690 siRNA295 ACCAGGUCCAAUGUUGUUCAA 687 UUGAACAACAUUGGACCUGGUUU 1071 1692 siRNA296 CAGGUCCAAUGUUGUUCACCA 688 UGGUGAACAACAUUGGACCUGUU 1072 1712 siRNA297 AGUGCUUGCUUGUGAAUCUUA 689 UAAGAUUCACAAGCAAGCACUUU 1073 1713 siRNA298 GUGCUUGCUUGUGAAUCUUAA 690 UUAAGAUUCACAAGCAAGCACUU 1074 1769 siRNA299 UCUGCUAGUGAAAAGAACUAA 691 UUAGUUCUUUUCACUAGCAGAUU 1075 1770 siRNA300 CUGCUAGUGAAAAGAACUAGA 692 UCUAGUUCUUUUCACUAGCAGUU 1076 1824 siRNA301 UAAGUCCAUGAAUGUUCAUAA 693 UUAUGAACAUUCAUGGACUUAUU 1077 1825 siRNA302 AAGUCCAUGAAUGUUCAUAUA 694 UAUAUGAACAUUCAUGGACUUUU 1078 1826 siRNA303 AGUCCAUGAAUGUUCAUAUAA 695 UUAUAUGAACAUUCAUGGACUUU 1079 1827 siRNA304 GUCCAUGAAUGUUCAUAUAGA 696 UCUAUAUGAACAUUCAUGGACUU 1080 1828 siRNA305 UCCAUGAAUGUUCAUAUAGCA 697 UGCUAUAUGAACAUUCAUGGAUU 1081 1829 siRNA306 CCAUGAAUGUUCAUAUAGCCA 698 UGGCUAUAUGAACAUUCAUGGUU 1082 1830 siRNA307 CAUGAAUGUUCAUAUAGCCAA 699 UUGGCUAUAUGAACAUUCAUGUU 1083 1895 siRNA308 UUUUUUUCAAAAUGAAGAUAA 700 UUAUCUUCAUUUUGAAAAAAAUU 1084 2052 siRNA309 UGUAUUCUAAUUGGCAGAUUA 701 UAAUCUGCCAAUUAGAAUACAUU 1085 2053 siRNA310 GUAUUCUAAUUGGCAGAUUGA 702 UCAAUCUGCCAAUUAGAAUACUU 1086 2075 siRNA311 UUUUCCUAAGGAAACUGCUUA 703 UAAGCAGUUUCCUUAGGAAAAUU 1087 2127 siRNA312 AAAUGUUCAAAUUCACGUUCA 704 UGAACGUGAAUUUGAACAUUUUU 1088 2128 siRNA313 AAUGUUCAAAUUCACGUUCUA 705 UAGAACGUGAAUUUGAACAUUUU 1089 2132 siRNA314 UUCAAAUUCACGUUCUAGUGA 706 UCACUAGAACGUGAAUUUGAAUU 1090 2133 siRNA315 UCAAAUUCACGUUCUAGUGAA 707 UUCACUAGAACGUGAAUUUGAUU 1091 2134 siRNA316 CAAAUUCACGUUCUAGUGAAA 708 UUUCACUAGAACGUGAAUUUGUU 1092 2135 siRNA317 AAAUUCACGUUCUAGUGAAAA 709 UUUUCACUAGAACGUGAAUUUUU 1093 2136 siRNA318 AAUUCACGUUCUAGUGAAACA 710 UGUUUCACUAGAACGUGAAUUUU 1094 2137 siRNA319 AUUCACGUUCUAGUGAAACUA 711 UAGUUUCACUAGAACGUGAAUUU 1095 2139 siRNA320 UCACGUUCUAGUGAAACUGCA 712 UGCAGUUUCACUAGAACGUGAUU 1096 2140 siRNA321 CACGUUCUAGUGAAACUGCAA 713 UUGCAGUUUCACUAGAACGUGUU 1097 2144 siRNA322 UUCUAGUGAAACUGCAUUAUA 714 UAUAAUGCAGUUUCACUAGAAUU 1098 2189 siRNA323 CGGGUGUGAUCAUAUAUCAUA 715 UAUGAUAUAUGAUCACACCCGUU 1099 2190 siRNA324 GGGUGUGAUCAUAUAUCAUAA 716 UUAUGAUAUAUGAUCACACCCUU 1100 2191 siRNA325 GGUGUGAUCAUAUAUCAUAAA 717 UUUAUGAUAUAUGAUCACACCUU 1101 2192 siRNA326 GUGUGAUCAUAUAUCAUAAAA 718 UUUUAUGAUAUAUGAUCACACUU 1102 2193 siRNA327 UGUGAUCAUAUAUCAUAAAGA 719 UCUUUAUGAUAUAUGAUCACAUU 1103 2194 siRNA328 GUGAUCAUAUAUCAUAAAGGA 720 UCCUUUAUGAUAUAUGAUCACUU 1104 2195 siRNA329 UGAUCAUAUAUCAUAAAGGAA 721 UUCCUUUAUGAUAUAUGAUCAUU 1105 2199 siRNA330 CAUAUAUCAUAAAGGAUAUUA 722 UAAUAUCCUUUAUGAUAUAUGUU 1106 2209 siRNA331 AAAGGAUAUUUCAAAUGAUUA 723 UAAUCAUUUGAAAUAUCCUUUUU 1107 2210 siRNA332 AAGGAUAUUUCAAAUGAUUAA 724 UUAAUCAUUUGAAAUAUCCUUUU 1108 2211 siRNA333 AGGAUAUUUCAAAUGAUUAUA 725 UAUAAUCAUUUGAAAUAUCCUUU 1109 2215 siRNA334 UAUUUCAAAUGAUUAUGAUUA 726 UAAUCAUAAUCAUUUGAAAUAUU 1110 2216 siRNA335 AUUUCAAAUGAUUAUGAUUAA 727 UUAAUCAUAAUCAUUUGAAAUUU 1111 2217 siRNA336 UUUCAAAUGAUUAUGAUUAGA 728 UCUAAUCAUAAUCAUUUGAAAUU 1112 2218 siRNA337 UUCAAAUGAUUAUGAUUAGUA 729 UACUAAUCAUAAUCAUUUGAAUU 1113 2220 siRNA338 CAAAUGAUUAUGAUUAGUUAA 730 UUAACUAAUCAUAAUCAUUUGUU 1114 2221 siRNA339 AAAUGAUUAUGAUUAGUUAUA 731 UAUAACUAAUCAUAAUCAUUUUU 1115 2223 siRNA340 AUGAUUAUGAUUAGUUAUGUA 732 UACAUAACUAAUCAUAAUCAUUU 1116 2224 siRNA341 UGAUUAUGAUUAGUUAUGUCA 733 UGACAUAACUAAUCAUAAUCAUU 1117 2225 siRNA342 GAUUAUGAUUAGUUAUGUCUA 734 UAGACAUAACUAAUCAUAAUCUU 1118 2313 siRNA343 UUGAUUUCCCAAAAACACUAA 735 UUAGUGUUUUUGGGAAAUCAAUU 1119 2316 siRNA344 AUUUCCCAAAAACACUAAAGA 736 UCUUUAGUGUUUUUGGGAAAUUU 1120 2317 siRNA345 UUUCCCAAAAACACUAAAGGA 737 UCCUUUAGUGUUUUUGGGAAAUU 1121 2319 siRNA346 UCCCAAAAACACUAAAGGUGA 738 UCACCUUUAGUGUUUUUGGGAUU 1122 2320 siRNA347 CCCAAAAACACUAAAGGUGGA 739 UCCACCUUUAGUGUUUUUGGGUU 1123 2321 siRNA348 CCAAAAACACUAAAGGUGGUA 740 UACCACCUUUAGUGUUUUUGGUU 1124 2353 siRNA349 UCAUGUUUUAACUUAUUGUUA 741 UAACAAUAAGUUAAAACAUGAUU 1125 2355 siRNA350 AUGUUUUAACUUAUUGUUGCA 742 UGCAACAAUAAGUUAAAACAUUU 1126 2356 siRNA351 UGUUUUAACUUAUUGUUGCUA 743 UAGCAACAAUAAGUUAAAACAUU 1127 2362 siRNA352 AACUUAUUGUUGCUGAAAACA 744 UGUUUUCAGCAACAAUAAGUUUU 1128 2364 siRNA353 CUUAUUGUUGCUGAAAACUCA 745 UGAGUUUUCAGCAACAAUAAGUU 1129 2365 siRNA354 UUAUUGUUGCUGAAAACUCUA 746 UAGAGUUUUCAGCAACAAUAAUU 1130 2366 siRNA355 UAUUGUUGCUGAAAACUCUAA 747 UUAGAGUUUUCAGCAACAAUAUU 1131 2370 siRNA356 GUUGCUGAAAACUCUAUGUCA 748 UGACAUAGAGUUUUCAGCAACUU 1132 2503 siRNA357 AAAAAUGUAGCUUUUAUGUGA 749 UCACAUAAAAGCUACAUUUUUUU 1133 2562 siRNA358 GGAAGCUUUGGUUAUGAAACA 750 UGUUUCAUAACCAAAGCUUCCUU 1134 2563 siRNA359 GAAGCUUUGGUUAUGAAACAA 751 UUGUUUCAUAACCAAAGCUUCUU 1135 2620 siRNA360 AUUUAAAUGCUUUUUAUCGCA 752 UGCGAUAAAAAGCAUUUAAAUUU 1136 2621 siRNA361 UUUAAAUGCUUUUUAUCGCUA 753 UAGCGAUAAAAAGCAUUUAAAUU 1137 2622 siRNA362 UUAAAUGCUUUUUAUCGCUAA 754 UUAGCGAUAAAAAGCAUUUAAUU 1138 2623 siRNA363 UAAAUGCUUUUUAUCGCUAAA 755 UUUAGCGAUAAAAAGCAUUUAUU 1139 2624 siRNA364 AAAUGCUUUUUAUCGCUAAAA 756 UUUUAGCGAUAAAAAGCAUUUUU 1140 2625 siRNA365 AAUGCUUUUUAUCGCUAAAUA 757 UAUUUAGCGAUAAAAAGCAUUUU 1141 2629 siRNA366 CUUUUUAUCGCUAAAUGACUA 758 UAGUCAUUUAGCGAUAAAAAGUU 1142 2630 siRNA367 UUUUUAUCGCUAAAUGACUUA 759 UAAGUCAUUUAGCGAUAAAAAUU 1143 2632 siRNA368 UUUAUCGCUAAAUGACUUGCA 760 UGCAAGUCAUUUAGCGAUAAAUU 1144 2633 siRNA369 UUAUCGCUAAAUGACUUGCAA 761 UUGCAAGUCAUUUAGCGAUAAUU 1145 2635 siRNA370 AUCGCUAAAUGACUUGCAGAA 762 UUCUGCAAGUCAUUUAGCGAUUU 1146 2639 siRNA371 CUAAAUGACUUGCAGAUGAAA 763 UUUCAUCUGCAAGUCAUUUAGUU 1147 2679 siRNA372 UGUUUAAAUGCUGUGUACAAA 764 UUUGUACACAGCAUUUAAACAUU 1148 2682 siRNA373 UUAAAUGCUGUGUACAACAAA 765 UUUGUUGUACACAGCAUUUAAUU 1149 2683 siRNA374 UAAAUGCUGUGUACAACAAUA 766 UAUUGUUGUACACAGCAUUUAUU 1150 2687 siRNA375 UGCUGUGUACAACAAUGCUUA 767 UAAGCAUUGUUGUACACAGCAUU 1151 2690 siRNA376 UGUGUACAACAAUGCUUUGAA 768 UUCAAAGCAUUGUUGUACACAUU 1152 2691 siRNA377 GUGUACAACAAUGCUUUGAUA 769 UAUCAAAGCAUUGUUGUACACUU 1153 2692 siRNA378 UGUACAACAAUGCUUUGAUAA 770 UUAUCAAAGCAUUGUUGUACAUU 1154 2833 siRNA379 GAUAAUUUUGAAAUGAUUCAA 771 UUGAAUCAUUUCAAAAUUAUCUU 1155 2834 siRNA380 AUAAUUUUGAAAUGAUUCAUA 772 UAUGAAUCAUUUCAAAAUUAUUU 1156 2835 siRNA381 UAAUUUUGAAAUGAUUCAUCA 773 UGAUGAAUCAUUUCAAAAUUAUU 1157 2838 siRNA382 UUUUGAAAUGAUUCAUCUUUA 774 UAAAGAUGAAUCAUUUCAAAAUU 1158 2839 siRNA383 UUUGAAAUGAUUCAUCUUUCA 775 UGAAAGAUGAAUCAUUUCAAAUU 1159 2858 siRNA384 CAGAAAUAAAAGUAUGAAUCA 776 UGAUUCAUACUUUUAUUUCUGUU 1160 Each uracil base (U) in any one of the sequences provided in Table 5A may independently and optionally be replaced with a thymine base (T).

(506) TABLE-US-00042 TABLE5B siRNAsforinvitroScreening SEQID SEQID NOof NOof modified modified Nucleobase sense Nucleobase antisense Position siRNA#* Sense(5-3) SEQIDNO: strand: Antisense(5-3) SEQIDNO: strand: 34 siRNA1- [mU][mU][mC][mU][mU][m 393 1191 [mU][C][C][mA][A][A][A 777 1605 M0 C][mC][mU][C][A][G][A][G] ]U[C][U][mC][mU][G][ [mA][mU][mU][mU][mU][G] A][G][mG][A][mA][G][A] [mG][mA] [mA][mU][mU] 40 siRNA2- [mC][mU][mC][mA][mG][m 394 1192 [mU][U][C][mU][A][G][G 778 1606 M0 A][mG][mA][U][U][U][U][G ][C][C][A][mA][mA][A][ ][mG][mC][mC][mU][mA][G U][C][mU][C][mU][G][A] ][mA][mA] [mG][mU][mU] 41 siRNA3- [mU][mC][mA][mG][mA][m 395 1193 [mU][A][U][mC][U][A][G 779 1607 M0 G][mA][mU][U][U][U][G][G ][G][C][C][mA][mA][A][ ][mC][mC][mU][mA][mG][A A][U][mC][U][mC][U][G] ][mU][mA] [mA][mU][mU] 42 siRNA4- [mC][mA][mG][mA][mG][m 396 1194 [mU][A][A][mU][C][U][A 780 1608 M0 G][mU][mU][U][U][G][G][C] ][G][G][C][mC][mA][A][ [mC][mU][mA][mG][mA][U] A][A][mU][C][mU][C][U] [mU][mA] [mG][mU][mU] 43 siRNA5- [mA][mG][mA][mG][mA][m 397 1195 [mU][A][A][mA][U][U][U 781 1609 M0 U][mU][mU][U][G][G][C][C] ][A][G][G][mC][mC][A][ [mU][mA][mG][mA][mU][U] A][A][mA][U][mC][U][C] [mU][mA] [mU][mU][mU] 45 siRNA6- [mA][mG][mA][mU][mU][m 398 1196 [mU][G][C][mA][A][A][U 782 1610 M0 U][mU][mG][G][C][C][U][A] ][C][U][A][mG][mG][C][ [mG][mA][mU][mU][mU][G] C][A][mA][A][mA][U][C] [mC][mA] [mU][mU][mU] 105 siRNA7- [mC][mA][mG][mC][mA][m 399 1197 [mU][C][C][mA][U][A][G 783 1611 M0 U][mG][mC][U][G][U][U][G] ][A][C][A][mA][mC][A][ [mU][mC][mU][mA][mU][G] G][C][mA][U][mG][C][U] [mG][mA] [mG][mU][mU] 110 siRNA8- [mU][mG][mC][mU][mG][m 400 1198 [mU][A][U][mA][A][G][C 784 1612 M0 U][mU][mG][U][C][U][A][U] ][C][A][U][mA][mG][A][ [mG][mG][mC][mU][mU][A] C][A][mA][C][mA][G][C] [mU][mA] [mA][mU][mU] 111 siRNA9- [mG][mC][mU][mG][mU][m 401 1199 [mU][A][A][mU][A][A][G 785 1613 M0 U][mG][mU][C][U][A][U][G] ][C][C][A][mU][mA][G][ [mG][mC][mU][mU][mA][U] A][C][mA][A][mC][A][G] [mU][mA] [mC][mU][mU] 112 siRNA10- [mC][mU][mG][mU][mU][m 402 1200 [mU][G][A][mA][U][A][A] 786 1614 M0 G][mU][mC][U][A][U][G][G] ][G][C][C][mA][mU][A][ [mC][mU][mU][mA][mU][U] G][A][mC][A][mA][C][A] [mC][mA] [mG][mU][mU] 113 siRNA11- [mU][mG][mU][mU][mG][m 403 1201 [mU][A][G][mA][A][U][A 787 1615 M0 U][mC][mU][A][U][G][G][C] ][A][G][C][mC][mA][U][ [mU][mU][mA][mU][mU][C] A][G][mA][C][mA][A][C] [mU][mA] [mA][mU][mU] 115 siRNA12- [mU][mU][mG][mU][mC][m 404 1203 [mU][C][A][mA][G][A][A 788 1616 M0 U][mA][mU][G][G][C][U][U] ][U][A][A][mG][mC][C][ [mA][mU][mU][mC][mU][U] A][G][mA][C][mA][A][C] [mG][mA] [mA][mU][mU] 116 siRNA13- [mU][mG][mU][mC][mU][m 405 1204 [mU][C][C][mA][A][G][A 789 1617 M0 A][mU][mG][G][C][U][U][A] ][A][U][A][mA][mG][C][ [mU][mU][mC][mU][mU][G] C][A][mU][A][mG][A][C] [mG][mA] [mA][mU][mU] 117 siRNA14- [mG][mU][mC][mU][mA][m 406 1205 [mU][U][C][mC][A][A][G 790 1618 M0 U][mG][mG][C][U][U][A][U] ][A][A][U][mA][mA][G][ [mU][mC][mU][mU][mG][G] C][C][mA][U][mA][G][A] [mA][mA] [mC][mU][mU] 118 siRNA15- [mU][mC][mU][mA][mU][m 407 1206 [mU][U][U][mC][C][A][A 791 1619 M0 G][mG][mC][U][U][A][U][U] ][G][A][A][mU][mA][A][ [mC][mU][mU][mG][mG][A] G][C][mC][A][mU][A][G] [mA][mA] [mA][mU][mU] 139 siRNA16- [mU][mA][mG][mG][mA][m 408 1207 [mU][C][A][mC][C][C][G 792 1620 M0 G][mA][mA][G][G][C][A][A] ][U][U][U][mG][mC][C][ [mA][mC][mG][mG][mG][U] ][U][U][U][mG][mC][C][ [mG][mA] [mA][mU][mU] 141 siRNA17- [mG][mG][mA][mG][mA][m 409 1208 [mU][U][U][mC][A][C][C 793 1621 M0 A][mG][mG][C][A][A][A][C] ][C][G][U][mU][mU][G][ [mG][mG][mG][mU][mG][A] C][C][mU][U][mC][U][C] [mA][mA] [mC][mU][mU] 142 siRNA18- [mG][mA][mG][mA][mA][m 410 1209 [mU][G][U][mU][C][A][C 794 1622 M0 G][mG][mC][A][A][A][C][G] ][C][C][G][mU][mU][U][ [mG][mG][mU][mG][mA][A] G][C][mC][U][mU][C][U] [mC][mA] [mC][mU][mU] 143 siRNA19- [mA][mG][mA][mA][mG][m 411 1210 [mU][G][G][mU][U][C][A 795 1623 M0 G][mC][mA][A][A][C][G][G] ][C][C][C][mG][mU][U][ [mG][mU][mG][mA][mA][C] U][G][mC][C][mU][U][C] [mC][mA] [mU][mU][mU 181 siRNA20- [mA][mA][mU][mU][mC][m 412 1211 [mU][C][A][mC][A][G][C 796 1624 M0 C][mA][mU][A][C][C][U][G] ][C][C][A][mG][mG][U][ [mG][mG][mC][mU][mG][U] A][U][mG][G][mA][A][U] [mG][mA] [mU][mU][mU] 182 siRNA21- [mA][mU][mU][mC][mC][m 413 1212 [mU][G][C][mA][C][A][G 797 1625 M0 A][mU][mA][C][C][U][G][G] ][C][C][C][mA][mG][G][ [mG][mC][mU][mG][mU][G] U][A][mU][G][mG][A][A] [mC][mA] [mU][mU][mU] 183 siRNA22- [mU][mU][mC][mC][mA][m 414 1213 [mU][A][G][mC][A][C][A 798 1626 M0 U][mA][mC][C][U][G][G][G] ][G][C][C][mC][mA][G][ [mC][mU][mG][mU][mG][C] G][U][mA][U][mG][G][A] [mU][mA] [mA][mU][mU] 201 siRNA23- [mC][mU][mC][mU][mG][m 415 1214 [mU][A][U][mU][G][G][C 799 1627 M0 C][mA][mA][U][U][U][G][G] ][A][C][C][mA][mA][A][ [mU][mG][mC][mC][mA][A] U][U][mG][C][mA][G][A] [mU][mA] [mG][mU][mU] 203 siRNA24- [mC][mU][mG][mC][mA][m 416 1215 [mU][G][G][mA][U][U][G 800 1628 M0 A][mU][mU][U][G][G][U][G] ][G][C][A][mC][mC][A][ ][mC][mC][mA][mA][mU][C A][A][mU][U][mG][C][A] ][mC][mA] [mG][mU][mU] 210 siRNA25- [mU][mU][mG][mG][mU][m 417 1216 [mU][C][U][mC][A][A][G 801 1629 M0 G][mC][mC][A][A][U][C][C] ][A][G][G][mA][mU][U][ [mU][mC][mU][mU][mG][A] G][G][mC][A][mC][C][A] [mG][mA] [mA][mU][mU] 214 siRNA26- [mU][mG][mC][mC][mA][m 418 1217 [mU][G][G][mA][A][C][U 802 1630 M0 A][mU][mC][C][U][C][U][U] ][C][A][A][mG][mA][G][ [mG][mA][mG][mU][mU][C] G][A][mU][U][mG][G][C] [mC][mA] [mA][mU][mU] 223 siRNA27- [mU][mC][mU][mU][mG][m 419 1218 [mU][U][U][mG][C][U][C 803 1631 M0 A][mG][mU][U][C][C][U][C] ][U][G][A][mG][mG][A][ [mA][mG][mA][mG][mC][A] A][C][mU][C][mA][A][G] [mA][mA] [mA][mU][mU 225 siRNA28- [mU][mU][mG][mA][mG][m 420 1220 [mU][A][U][mU][U][G][C 804 1632 M0 U][mU][mC][C][U][C][A][G] ][U][C][U][mG][mA][G][ [mA][mG][mC][mA][mA][A] G][A][mA][C][mU][C][A] [mU][mA] [mA][mU][mU] 229 siRNA29- [mG][mU][mU][mC][mC][m 421 1221 [mU][U][U][mU][G][A][U 805 1633 M0 U][mC][mA][G][A][G][C][A] ][U][U][G][mC][mU][C][ [mA][mA][mU][mC][mA][A] U][G][mA][G][mG][A][A] [mA][mA] [mC][mU][mU] 230 siRNA30- [mU][mU][mC][mC][mU][m 422 1222 [mU][C][U][mU][U][G][A 806 1634 M0 C][mA][mG][A][G][C][A][A] ][U][U][U][mG][mC][U][ [mA][mU][mC][mA][mA][A] C][U][mG][A][mG][G][A] [mG][mA] [mA][mU][mU] 236 siRNA31- [mA][mG][mA][mG][mC][m 423 1223 [mU][U][G][mU][U][U][C 807 1635 M0 A][mA][mA][U][C][A][A][A] ][C][U][U][mG][mG][A][ [mG][mG][mA][mA][mA][C] U][U][mU][G][mC][U][C] [mA][mA] [mU][mU][mU] 238 siRNA32- [mA][mG][mC][mA][mA][m 424 1224 [mU][C][A][mU][G][U][U 808 1636 M0 A][mU][mC][A][A][A][G][G] ][U][C][C][mU][mU][U][ [mA][mA][mA][mC][mA][U] G][A][mU][U][mU][G][C] [mG][mA] [mU][mU][mU] 242 siRNA33- [mA][mA][mU][mC][mA][m 425 1225 [mU][U][G][mA][C][C][A 809 1637 M0 A][mA][mG][G][A][A][A][C] ][U][G][U][mU][mU][C][ [mA][mU][mG][mG][mU][C] C][U][mU][U][mG][A][U] [mA][mA] [mU][mU][mU] 243 siRNA34- [mA][mU][mC][mA][mA][m 426 1226 [mU][A][U][mG][A][C][C 810 1638 M0 A][mG][mG][A][A][A][C][A] ][A][U][G][mU][mU][U][ [mU][mG][mG][mU][mC][A] C][C][mU][U][mU][G][A] [mU][mA] [mU][mU][mU 244 siRNA35- [mU][mC][mA][mA][mA][m 427 1227 [mU][C][A][mU][G][A][C 811 1639 M0 G][mG][mA][A][A][C][A][U] ][C][A][U][mG][mU][U][ [mG][mG][mU][mC][mA][U] U][C][mC][U][mU][U][G] [mG][mA] [mA][mU][mU] 245 siRNA36- [mC][mA][mA][mA][mG][m 428 1228 [mU][A][C][mA][U][G][A 812 1640 M0 G][mA][mA][A][C][A][U][G] ][C][C][A][mU][mG][U][ [mG][mU][mC][mA][mU][G] U][U][mC][C][mU][U][U] [mU][mA] [mG][mU][mU] 246 siRNA37- [mA][mA][mA][mG][mG][m 429 1229 [mU][A][A][mC][A][U][G 813 1641 M0 A][mA][mA][C][A][U][G][G] ][A][C][C][mA][mU][G][ [mU][mC][mA][mU][mG][U] U][U][mU][C][mC][U][U] [mU][mA] [mU][mU][mU] 251 siRNA38- [mA][mA][mA][mC][mA][m 430 1230 [mU][G][U][mA][A][A][A] 814 1642 M0 U][mG][mG][U][C][A][U][G] ][A][C][A][mU][mG][A][ [mU][mU][mU][mU][mU][A] C][C][mA][U][mG][U][U] [mC][mA] [mU][mU][mU 253 siRNA39- [mA][mC][mA][mU][mG][m 431 1232 [mU][A][G][mG][U][A][A 815 1643 M0 G][mU][mC][A][U][G][U][U] ][A][A][A][mC][mA][U][ [mU][mU][mU][mA][mC][C] G][A][mC][C][mA][U][G] [mU][mA] [mU][mU][mU] 254 siRNA40- [mC][mA][mU][mG][mG][m 432 1233 [mU][C][A][mG][G][U][A 816 1644 M0 U][mC][mA][U][G][U][U][U] ][A][A][A][mA][mC][A][ [mU][mU][mA][mC][mC][U] U][G][mA][C][mC][A][U] [mG][mA] [mG][mU][mU] 262 siRNA41- [mU][mG][mU][mU][mU][m 433 1234 [mU][U][U][mA][G][U][U 817 1645 M0 U][mU][mA][C][C][U][G][C] ][U][G][C][mA][mG][G][ [mA][mA][mA][mC][mU][A] U][A][mA][A][mA][A][C] [mA][mA] [mA][mU][mU] 264 siRNA42- [mU][mU][mU][mU][mU][m 434 1235 [mU][C][A][mU][U][A][G 818 1646 M0 A][mC][mC][U][G][C][A][A] ][U][U][U][mG][mC][A][ [mA][mC][mU][mA][mA][U] G][G][mU][A][mA][A][A] [mG][mA] [mA][mU][mU] 266 siRNA43- [mU][mU][mU][mA][mC][m 435 1236 [mU][C][C][mC][A][U][U 819 1647 M0 C][mU][mG][C][A][A][A][C] ][A][G][U][mU][mU][G][ [mU][mA][mA][mU][mG][G] C][A][mG][G][mU][A][A] [mG][mA] [mA][mU][mU] 267 siRNA44- [mU][mU][mA][mC][mC][m 436 1237 [mU][U][C][mC][C][A][U 820 1648 M0 U][mG][mC][A][A][A][C][U] ][U][A][G][mU][mU][U][ [mA][mA][mU][mG][mG][G] G][C][mA][G][mG][U][A] [mA][mA] [mA][mU][mU] 268 siRNA45- [mU][mA][mC][mC][mU][m 437 1238 [mU][U][U][mC][C][C][A 821 1649 M0 G][mC][mA][A][A][C][U][A] ][U][U][A][mG][mU][U][ [mA][mU][mG][mG][mG][A] U][G][mC][A][mG][G][U] [mA][mA] [mA][mU][mU] 291 siRNA46- [mA][mU][mG][mU][mC][m 438 1239 [mU][A][U][mU][U][G][U 822 1650 M0 C][mA][mU][U][U][C][A][U] ][G][A][U][mG][mA][A][ [mC][mA][mC][mA][mA][A] A][U][mG][G][mA][C][A] [mU][mA] [mU][mU][mU] 292 siRNA47- [mU][mG][mU][mC][mC][m 439 1240 [mU][G][A][mU][U][U][G 823 1651 M0 A][mU][mU][U][C][A][U][C] ][U][G][A][mU][mG][A][ [mA][mC][mA][mA][mA][U] A][A][mU][G][mG][A][C] [mC][mA] [mA][mU][mU] 294 siRNA48- [mU][mC][mC][mA][mU][m 440 1242 [mU][G][G][mG][A][U][U 824 1652 M0 U][mU][mC][A][U][C][A][C] ][U][G][U][mG][mA][U][ [mA][mA][mA][mU][mC][C] G][A][mA][A][mU][G][G] [mC][mA] [mA][mU][mU] 300 siRNA49- [mU][mC][mA][mU][mC][m 441 1243 [mU][U][G][mA][C][A][A 825 1653 M0 A][mC][mA][A][A][U][C][C] ][G][G][G][mA][mU][U][ [mC][mU][mU][mG][mU][C] U][G][mU][G][mA][U][G] [mA][mA] [mA][mU][mU] 302 siRNA50- [mA][mU][mC][mA][mC][m 442 1244 [mU][U][A][mU][G][A][C 826 1654 M0 A][mA][mA][U][C][C][C][U] ][A][A][G][mG][mG][A][ [mU][mG][mU][mC][mA][U] U][U][mU][G][mU][G][A] [mA][mA] [mU][mU][mU] 303 siRNA51- [mU][mC][mA][mC][mA][m 443 1245 [mU][G][U][mA][U][G][A 827 1655 M0 A][mA][mU][C][C][C][U][U] ][C][A][A][mG][mG][G][ [mG][mU][mC][mA][mU][A] A][U][mU][U][mG][U][G] [mC][mA] [mA][mU][mU] 304 siRNA52- [mC][mA][mC][mA][mA][m 444 1247 [mU][G][G][mU][A][U][G 828 1656 M0 A][mU][mC][C][C][U][U][G] ][A][C][A][mA][mG][G][ [mU][mC][mA][mU][mA][C] G][A][mU][U][mU][G][U] [mC][mA] [mG][mU][mU] 305 siRNA53- [mA][mC][mA][mA][mA][m 445 1248 [mU][U][G][mG][U][A][U 829 1657 M0 U][mC][mC][C][U][U][G][U] ][G][A][C][mA][mA][G][ [mC][mA][mU][mA][mC][C] G][G][mA][U][mU][U][G] [mA][mA] [mU][mU][mU] 309 siRNA54- [mA][mU][mC][mC][mC][m 446 1249 [mU][C][U][mU][A][U][G 830 1658 M0 U][mU][mG][U][C][A][U][A] ][G][U][A][mU][mG][A][ [mC][mC][mA][mU][mA][A] C][A][mA][G][mG][G][A] [mG][mA] [mU][mU][mU] 310 siRNA55- [mU][mC][mC][mC][mU][m 447 1250 [mU][C][C][mU][U][A][U 831 1659 M0 U][mG][mU][C][A][U][A][C] ][G][G][U][mA][mU][G][ [mC][mA][mU][mA][mA][G] A][C][mA][A][mG][G][G] [mG][mA] [mA][mU][mU] 311 siRNA56- [mC][mC][mC][mU][mU][m 448 1251 [mU][A][C][mC][U][U][A 832 1660 M0 G][mU][mC][A][U][A][C][C] ][U][G][G][mU][mA][U][ [mA][mU][mA][mA][mG][G] G][A][mC][A][mA][G][G] [mU][mA] [mG][mU][mU] 312 siRNA57- [mC][mC][mU][mU][mG][m 449 1252 [mU][C][A][mC][C][U][U 833 1661 M0 U][mC][mA][U][A][C][C][A] ][A][U][G][mG][mU][A][ [mU][mA][mA][mG][mG][U] U][G][mA][C][mA][A][G] [mG][mA] [mG][mU][mU] 313 siRNA58- [mC][mU][mU][mG][mU][m 450 1253 [mU][A][C][mA][C][C][U 834 1662 M0 C][mA][mU][A][C][C][A][U] ][U][A][U][mG][mG][U][ [mA][mA][mG][mG][mU][G] A][U][mG][A][mC][A][A] [mU][mA] [mG][mU][mU] 315 siRNA59- [mU][mG][mU][mC][mA][m 451 1254 [mU][C][A][mA][C][A][C 835 1663 M0 U][mA][mC][C][A][U][A][A] ][C][U][U][mA][mU][G][ [mG][mG][mU][mG][mU][U] G][U][mA][U][mG][A][C] [mG][mA] [mA][mU][mU] 319 siRNA60- [mA][mU][mA][mC][mC][m 452 1255 [mU][G][G][mC][A][C][A 836 1664 M0 A][mU][mA][A][G][G][U][G ][A][C][A][mC][mC][U][ ][mU][mU][mG][mU][mG][C U][A][mU][G][mG][U][A] ][mC][mA] [mU][mU][mU] 360 siRNA61- [mA][mA][mA][mA][mA][m 453 1256 [mU][A][G][mU][A][G][C 837 1665 M0 U][mU][mU][C][A][C][U][U] ][A][A][A][mG][mU][G][ [mU][mG][mC][mU][mA][C] A][A][mA][U][mU][U][U] [mU][mA] [mU][mU][mU] 363 siRNA62- [mA][mA][mU][mU][mU][m 454 1257 [mU][A][G][mA][A][G][U 838 1666 M0 C][mA][mC][U][U][U][G][C] ][A][G][C][mA][mA][A][ [mU][mA][mC][mU][mU][C] G][U][mG][A][mA][A][U] [mU][mA] [mU][mU][mU] 372 siRNA63- [mU][m][mG][mC][mU][m 455 1258 [mU][U][G][mC][C][U][U 839 1667 M0 A][mC][mU][U][C][U][G][C] ][C][G][C][mA][mG][A][ [mG][mA][mA][mG][mG][C] A][G][mU][A][mG][C][A] [mA][mA] [mA][mU][mU] 375 siRNA64- [mC][mU][mA][mC][mU][m 456 1259 [mU][A][A][mA][U][G][C 840 1668 M0 U][mC][mU][G][C][G][A][A] ][C][U][U][mC][mG][C][ [mG][mG][mC][mA][mU][U] A][G][mA][A][mG][U][A] [mU][mA] [mG][mU][mU] 377 siRNA65- [mA][mC][mU][mU][mC][m 457 1260 [mU][C][C][mA][A][A][U 841 1669 M0 U][mG][mC][G][A][A][G][G] ][G][C][C][mU][mU][C][ [mC][mA][mU][mU][mU][G] G][C][mA][G][mA][A][G] [mG][mA] [mU][mU][mU] 390 siRNA66- [mC][mA][mU][mU][mU][m 458 1261 [mU][A][A][mU][G][C][U 842 1670 M0 G][mG][mG][C][A][C][A][G] ][U][C][U][mG][mU][G][ [mA][mA][mG][mC][mA][U] C][C][mC][A][mA][A][U] [mU][mA] [mG][mU][mU] 392 siRNA67- [mU][mU][mU][mG][mG][m 459 1262 [mU][U][C][mA][A][U][G 843 1671 M0 G][mC][mA][C][A][G][A][A] ][C][U][U][mC][mU][G][ [mG][mC][mA][mU][mU][G] U][G][mC][C][mC][A][A] [mA][mA] [mA][mU][mU] 394 siRNA68- [mU][mG][mG][mG][mC][m 460 1263 [mU][G][G][mU][C][A][A 844 1672 M0 A][mC][mA][G][A][A][G][C] ][U][G][C][mU][mU][C][ [mA][mU][mU][mG][mA][C] U][G][mU][G][mC][C][C] [mC][mA] [mA][mU][mU] 474 siRNA69- [mA][mU][mG][mC][mC][m 461 1264 [mU][C][G][mU][G][A][G 845 1673 M0 U][mU][mG][A][A][U][U][C] ][G][G][A][mA][mU][U][ [mC][mC][mU][mC][mA][C] C][A][mA][G][mG][C][A] [mG][mA] [mU][mU][mU] 475 siRNA70- [mU][mG][mC][mC][mU][m 462 1265 [mU][C][C][mG][U][G][A 846 1674 M0 U][mG][mA][A][U][U][C][C] ][G][G][G][mA][mA][U][ [mC][mU][mC][mA][mC][G] U][C][mA][A][mG][G][C] [mG][mA] [mA][mU][mU] 476 siRNA71- [mG][mC][mC][mU][mU][m 463 1266 [mU][U][C][mC][G][U][G 847 1675 M0 G][mA][mA][U][U][C][C][C] ][A][G][G][mG][mA][A][ [mU][mC][mA][mC][mG][G] U][U][mC][A][mA][G][G] [mA][mA] [mC][mU][mU] 477 siRNA72- [mC][mC][mU][mU][mG][m 464 1268 [mU][U][U][mC][C][G][U 848 1676 M0 A][mA][mU][U][C][C][C][U] ][G][A][G][mG][mG][A][ [mC][mA][mC][mG][mG][A] A][U][mU][C][mA][A][G] [mA][mA] [mG][mU][mU] 478 siRNA73- [mC][mU][mU][mG][mA][m 465 1269 [mU][U][U][mU][C][C][G 849 1677 M0 A][mU][mU][C][C][C][U][C] ][U][G][A][mG][mG][G][ [mA][mC][mG][mG][mA][A] A][A][mU][U][mC][A][A] [mA][mA] [mG][mU][mU] 482 siRNA74- [mA][mA][mU][mU][mC][m 466 1271 [mU][A][U][mG][C][U][U 850 1678 M0 C][mC][mU][C][A][C][G][G] ][U][C][C][mG][mU][G][ [mA][mA][mA][mG][mC][A] A][G][mG][G][mA][A][U] [mU][mA] [mU][mU][mU] 484 siRNA75- [mU][mU][mC][mC][mC][m 467 1272 [mU][U][C][mA][U][G][C 851 1679 M0 U][mC][mA][C][G][G][A][A] ][U][U][U][mC][mC][G][ [mA][mG][mC][mA][mU][G] U][G][mA][G][mG][G][A] [mA][mA] [mA][mU][mU] 486 siRNA76- [mC][mC][mC][mU][mC][m 468 1273 [mU][C][A][mU][C][A][U 852 1680 M0 A][mC][mG][G][A][A][A][G] ][G][C][U][mU][mU][C][ [mC][mA][mU][mG][mA][U] C][G][mU][G][mA][G][G] [mG][mA] [mG][mU][mU] 488 siRNA77- [mC][mU][mC][mA][mC][m 469 1274 [mU][U][C][mC][A][U][C 853 1681 M0 G][mG][mA][A][A][G][C][A] ][A][U][G][mC][mU][U][ [mU][mG][mA][mU][mG][G] U][C][mC][G][mU][G][A] [mA][mA] [mG][mU][mU] 501 siRNA78- [mU][mG][mA][mU][mG][m 470 1275 [mU][A][C][mG][U][U][G 854 1682 M0 G][mA][mA][A][A][C][C][U] ][G][A][G][mG][mU][U][ [mC][mC][mA][mA][mC][G] U][U][mC][C][mA][U][C] [mU][mA] [mA][mU][mU] 503 siRNA79- [mA][mU][mG][mG][mA][m 471 1276 [mU][A][U][mA][C][G][U 855 1683 M0 A][mA][mA][C][C][U][C][C] ][U][G][G][mA][mG][G][ [mA][mA][mC][mG][mU][A] U][U][mU][U][mC][C][A] [mU][mA] [mU][mU][mU] 505 siRNA80- [mG][mG][mA][mA][mA][m 472 1277 [mU][U][G][mA][U][A][C 856 1684 M0 A][mC][mC][U][C][C][A][A] ][G][U][U][mG][mG][A][ [mC][mG][mU][mA][mU][C] G][G][mU][U][mU][U][C] [mA][mA] [mC][mU][mU] 506 siRNA81- [mG][mA][mA][mA][mA][m 473 1278 [mU][A][U][mG][A][U][A 857 1685 M0 C][mC][mU][C][C][A][A][C] ][C][G][U][mU][mG][G][ [mG][mU][mA][mU][mC][A] A][G][mG][U][mU][U][U] [mU][mA] [mC][mU][mU] 507 siRNA82- [mA][mA][mA][mA][mC][m 474 1280 [mU][C][A][mU][G][A][U 858 1686 M0 C][mU][mC][C][A][A][C][G] ][A][C][G][mU][mU][G][ [mU][mA][mU][mC][mA][U] G][A][mG][G][mU][U][U] [mG][mA] [mU][mU][mU] 509 siRNA83- [mA][mA][mC][mC][mU][m 475 1281 [mU][C][U][mC][A][U][G 859 1687 M0 C][mC][mA][A][C][G][U][A] ][A][U][A][mC][mG][U][ [m][mC][mA][mU][mG][A] U][G][mG][A][mG][G][U] [mG][mA] [mU][mU][mU] 510 siRNA84- [mA][mC][mC][mU][mC][m 476 1282 [mU][U][C][mU][C][A][U 860 1688 M0 C][mA][mA][C][G][U][A][U] ][G][A][U][mA][mC][G][ [mC][mA][mU][mG][mA][G] U][U][mG][G][mA][G][G] [mA][mA] [mU][mU][mU] 511 siRNA85- [mC][mC][mU][mC][mC][m 477 1283 [mU][G][U][mC][U][C][A 861 1689 M0 A][mA][mC][G][U][A][U][C] ][U][G][A][mU][mA][C][ [mA][mU][mG][mA][mG][A] G][U][mU][G][mG][A][G] [mC][mA] [mG][mU][mU] 512 siRNA86- [mC][mU][mC][mC][mA][m 478 1284 [mU][G][G][mU][C][U][C 862 1690 M0 A][mC][mG][U][A][U][C][A] ][A][U][G][mA][mU][A][ [mU][mG][mA][mG][mA][C] C][G][mU][U][mG][G][A] [mC][mA] [mG][mU][mU] 513 siRNA87- [mU][mC][mC][mA][mA][m 479 1285 [mU][A][G][mG][U][C][U 863 1691 M0 C][mG][mU][A][U][C][A][U] ][C][A][U][mG][mA][U][ [mG][mA][mG][mA][mC][C] A][C][mG][U][mU][G][G] [mU][mA] [mA][mU][mU] 514 siRNA88- [mC][mC][mA][mA][mC][m 480 1286 [mU][G][A][mG][G][U][C 864 1692 M0 G][mU][mA][U][C][A][U][G] ][U][C][A][mU][mG][A][ [mA][mG][mA][mC][mC][U] U][A][mC][G][mU][U][G] [mC][mA] [mG][mU][mU] 539 siRNA89- [m][mC][mC][mU][mC][m 481 1287 [mU][G][C][G][G][U][C 865 1693 M0 U][mA][mA][C][U][C][A][A] ][U][U][U][mH][mS][H][ [mA][mG][mA][mC][mC][G] U][U][mA][G][mA][G][G] [mC][mA] [mA][mU][mU] 552 siRNA90- [mA][mG][mA][mC][mC][m 482 1288 [mU][U][G][mU][C][A][C 866 1694 M0 G][mC][mU][G][C][C][U][G] ][C][C][A][mG][mG][C][ [mG][mG][mU][mG][mA][C] A][G][mC][G][mG][U][C] [mA][mA] [mU][mU][mU] 562 siRNA91- [mC][mU][mG][mG][mG][m 483 1289 [mU][A][C][mA][U][C][C 867 1695 M0 U][mG][mA][C][A][G][A][A] ][C][U][U][mC][mU][G][ [mG][mG][mG][mA][mU][G] U][C][mA][C][mC][C][A] [mU][mA] [mG][mU][mU] 563 siRNA92- [mU][mG][mG][mG][mU][m 484 1290 [mU][U][A][mC][A][U][C 868 1696 M0 G][mA][mC][A][G][A][A][G] ][C][C][U][mU][mC][U][ [mG][mG][mA][mU][mG][U] G][U][mC][A][mC][C][C] [mA][mA] [mA][mU][mU] 564 siRNA93- [mG][mG][mG][mU][mG][m 485 1291 [mU][A][U][mA][C][A][U 869 1697 M0 A][mC][mA][G][A][A][G][G] ][C][C][C][mU][mU][C][ [mG][mA][mU][mG][mU][A] U][G][mU][C][mA][C][C] [mU][mA] [mC][mU][mU] 585 siRNA94- [mC][mU][mU][mU][mC][m 486 1292 [mU][C][A][mU][C][A][C 870 1698 M0 U][mG][mC][U][A][C][C][G] ][U][C][G][mG][mU][A][ [mA][mG][mU][mG][mA][U] G][C][mA][G][mA][A][A] [mG][mA] [mG][mU][mU] 587 siRNA95- [mU][mU][mC][mU][mG][m 487 1293 [mU][A][A][mC][A][U][C 871 1699 M0 C][mU][mA][C][C][G][A][G] ][A][C][U][mC][mG][G][ [mU][mG][mA][mU][mG][U] U][A][mG][C][mA][G][A] [mU][mA] [mA][mU][mU] 588 siRNA96- [mU][mC][mU][mG][mC][m 488 1294 [mU][A][A][mA][C][A][U 872 1700 M0 U][mA][mC][C][G][A][G][U] ][C][A][C][mU][mC][G][ [mG][mA][mU][mG][mU][U] G][U][mA][G][mC][A][G] [mU][mA] [mA][mU][mU] 589 siRNA97- [mC][mU][mG][mC][mU][m 489 1295 [mU][C][A][mA][A][C][A 873 1701 M0 A][mC][mC][G][A][G][U][G] ][U][C][A][mC][mU][C][ [mA][mU][mG][mU][mU][U] G][G][mU][A][mG][C][A] [mG][mA] [mG][mU][mU] 593 siRNA98- [mU][mA][mC][mC][mG][m 490 1296 [mU][G][C][mU][U][C][A 874 1702 M0 A][mG][mU][G][A][U][G][U] ][A][A][C][mA][mU][C][ ][mU][mU][mG][mA][mA][G A][C][mU][C][mG][G][U] [mC][mA] [mA][mU][mU] 594 siRNA99- [mA][mC][mC][mG][mA][m 491 1297 [mU][A][G][mC][U][U][C 875 1703 M0 G][mU][mG][A][U][G][U][U] ][A][A][A][mC][mA][U][ ][mU][mG][mA][mA][mG][C C][A][mC][U][mC][G][G] ][mU][mA] [mU][mU][mU] 595 siRNA100- [mC][mC][mG][mA][mG][m 492 1298 [mU][C][A][mG][C][U][U 876 1704 M0 U][mG][mA][U][G][U][U][U] ][C][A][A][mA][mC][A][ ][mG][mA][mA][mG][mC][U U][C][mA][C][mU][C][G] ][mG][mA] [mG][mU][mU] 602 siRNA101- [mA][mU][mG][mU][mU][m 493 1300 [mU][A][A][mA][U][A][C 877 1705 M0 U][mG][mA][A][G][C][U][G] ][C][C][A][mG][mC][U][ [mG][mG][mU][mA][mU][U] U][C][mA][A][mA][C][A] [mU][mA] [mU][mU][mU] 605 siRNA102- [mU][mU][mU][mG][mA][m 494 1302 [mU][G][U][mU][A][A][A 878 1706 M0 A][mG][mC][U][G][G][G][U] ][U][A][C][mC][mC][A][ [mA][mU][mU][mU][mA][A] G][C][mU][U][mC][A][A] [mC][mA] [mA][mU][mU] 606 siRNA103- [mU][mU][mG][mA][mA][m 495 1303 [mU][A][G][mU][U][A][A 879 1707 M0 G][mC][mU][G][G][G][U][A] ][A][U][A][mC][mC][C][ [mU][mU][mU][mA][mA][C] A][G][mC][U][mU][C][A] [mU][mA] [mA][mU][mU] 607 siRNA104- [mU][mG][mA][mA][mG][m 496 1304 [mU][U][A][mG][U][U][A 880 1708 M0 C][mU][mG][G][G][U][A][U] ][A][A][U][mA][mC][C][ [mU][mU][mA][mA][mC][U] C][A][mG][C][mU][U][C] [mA][mA] [mA][mU][mU] 608 siRNA105- [mG][mA][mA][mG][mC][m 497 1305 [mU][A][U][mA][G][U][U 881 1709 M0 U][mG][mG][G][U][A][U][U ][A][A][A][mU][mA][C][ ][mU][mA][mA][mC][mU][A C][C][mA][G][mC][U][U] ][mU][mA] [mC][mU][mU] 609 siRNA106- [mA][mA][mG][mC][mU][m 498 1306 [mU][G][A][mU][A][G][U 882 1710 M0 ][mA][mA][mC][mU][mA][U ][U][A][A][mA][mU][A][ ][mA][mA][mC][mU][mA][U C][C][mC][A][mG][C][U] ][mC][mA] [mU][mU][mU] 610 siRNA107- [mA][mG][mC][mU][mG][m 499 1307 [mU][A][G][mA][U][A][G] 883 1711 M0 G][mG][mU][A][U][U][U][A ][U][U][A][mA][mA][U][ ][mA][mC][mU][mA][mU][C A][C][mC][C][mA][G][C] ][mU][mA] [mU][mU][mU] 612 siRNA108- [mC][mU][mG][mG][mG][m 500 1308 [mU][A][A][mA][G][A][U 884 1712 M0 U][mA][mU][U][U][A][A][C] ][A][G][U][mU][mA][A][ [mU][mA][mU][mC][mU][U] A][U][mA][C][mC][C][A] [mU][mA] [mG][mU][mU 613 siRNA109- [mU][mG][mG][mG][mU][m 501 1309 [mU][C][A][mA][A][G][A 885 1713 M0 A][mU][mU][U][A][A][C][U] ][U][A][G][mU][mU][A][ [mA][mU][mC][mU][mU][U] A][U][mA][C][mC][C][A] [mG][mA] [mA][mU][mU] 653 siRNA110- [mG][mA][mC][mA][mC][m 502 1310 [mU][A][U][mA][U][G][U 886 1714 M0 A][mC][mA][G][A][A][A][G] ][G][C][U][mU][mU][C][ [mC][mA][mC][mA][mU][A] U][G][mU][G][mU][G][U] [mU][mA] [mC][mU][mU] 663 siRNA111- [mA][mA][mG][mC][mA][m 503 1311 [mU][A][U][mU][G][U][U 887 1715 M0 C][mA][mU][A][U][U][C][U] ][U][A][G][mA][mA][U][ [mA][mA][mA][mC][mA][A] A][U][mG][U][mG][C][U] [mU][mA] [mU][mU][mU] 665 siRNA112- [mG][mC][mA][mC][mA][m 504 1312 [mU][A][G][m][U][U][G] 888 1716 M0 U][mA][mU][U][C][U][A][A] ][U][U][U][mA][mG][A][ [mA][mC][mA][mA][mU][C] A][U][mA][U][mG][U][G] [mU][mA] [mC][mU][mU] 666 siRNA113- [mC][mA][mC][mA][mU][m 505 1313 [mU][A][A][mG][A][U][U] 889 1717 M0 A][mU][mU][C][U][A][A][A] ][G][U][U][mU][mA][G][ [mC][mA][mA][mU][mC][U] A][A][mU][A][mU][G][U] [mU][mA] [mG][mU][mU] 667 siRNA114- [mA][mC][mA][mU][mA][m 506 1314 [mU][C][A][mA][G][A][U 890 1718 M0 U][mU][mC][U][A][A][A][C] ][U][G][U][mU][mU][A][ [mA][mA][mU][mC][mU][U] G][A][mA][U][mA][U][G] [mG][mA] [mU][mU][mU] 669 siRNA115- [mA][mU][mA][mU][mU][m 507 1315 [mU][G][U][mC][A][A][G 891 1719 M0 C][mU][mA][A][A][C][A][A] ][A][U][U][mG][mU][U][ [mU][mC][mU][mU][mG][A] U][A][mG][A][mA][U][A] [mC][mA] [mU][mU][mU] 670 siRNA116- [mU][mA][mU][mU][mC][m 508 1316 [mU][U][G][mU][C][A][A 892 1720 M0 U][mA][mA][A][C][A][A][U] ][G][A][U][mU][mG][U][ [mC][mU][mU][mG][mA][C] U][U][mA][G][mA][A][U] [mA][mA] [mA][mU][mU] 671 siRNA117- [mA][mU][mU][mC][mU][m 509 1317 [mU][U][U][mG][U][C][A 893 1721 M0 A][mA][mA][C][A][A][U][C] ][A][G][A][mU][mU][U][ [mU][mU][mG][mA][mC][A] U][U][mU][A][mG][A][A] [mA][mA] [mU][mU][mU] 673 siRNA118- [mU][mC][mU][mA][mA][m 510 1318 [mU][A][G][mU][U][G][U 894 1722 M0 A][mC][mA][A][U][C][U][U] ][C][A][A][mG][mA][U][ [mG][mA][mC][mA][mA][C] U][G][mU][U][mU][A][G] [mU][mA] [mA][mU][mU] 679 siRNA119- [mC][mA][mA][mU][mC][m 511 1320 [mU][G][C][mU][U][G][A 895 1723 M0 U][mU][mG][A][C][A][A][C] ][A][G][U][mU][mG][U][ [mU][mU][mC][mA][mA][G] C][A][mA][G][mA][U][U] [mC][mA] [mG][mU][mU] 712 siRNA120- [mC][m][mU][mU][mC][m 512 1321 [mU][C][U][mG][C][U][A 896 1724 M0 C][mA][mG][C][C][C][U][G] ][C][C][A][mG][mG][G][ [mG][mU][mA][mG][mC][A] C][U][mG][G][mA][A][A] [mG][mA] [mG][mU][mU] 771 siRNA121- [mG][mG][mG][mA][mG][m 513 1322 [mU][G][C][mU][C][U][C 897 1725 M0 A][mA][mA][C][U][G][G][C] ][U][G][C][mC][mA][G][ [mA][mG][mA][mG][mA][G] U][U][mU][C][mU][C][C] [mC][mA] [mC][mU][mU] 781 siRNA122- [mG][mG][mC][mA][mG][m 514 1323 [mU][C][G][mU][G][C][C 898 1726 M0 A][mG][mA][G][C][U][U][G] ][U][C][A][mA][mG][C][ [mA][mG][mG][mC][mA][C] U][C][mU][C][mU][G][C] [mG][mA] [mC][mU][mU] 788 siRNA123- [mA][mG][mC][mU][mU][m 515 1325 [mU][A][G][mG][U][U][C 899 1727 M0 G][mA][mG][G][C][A][C][G] ][U][C][G][mU][mG][C][ [mA][mG][mA][mA][mC][C] C][U][mC][A][mA][G][C] [mU][mA] [mU][mU][mU] 841 siRNA124- [mC][mC][mU][mG][mC][m 516 1326 [mU][C][A][mU][U][G][A 900 1728 M0 G][mC][mA][U][G][U][U][U] ][G][A][A][mA][mC][A][ [mC][mU][mC][mA][mA][U] U][G][mC][G][mC][A][G] [mG][mA] [mG][mU][mU] 844 siRNA125- [mG][mC][mG][mC][mA][m 517 1328 [mU][U][G][mU][C][A][U 901 1729 M0 U][mG][mU][U][U][C][U][C] ][U][G][A][mG][mA][A][ [mA][mA][mU][mG][mA][C] A][C][mA][U][mG][C][G] [mA][mA] [mC][mU][mU] 846 siRNA126- [mG][mC][mA][mU][mG][m 518 1330 [mU][A][G][mU][G][U][C 902 1730 M0 U][mU][mU][C][U][C][A][A] ][A][U][U][mG][mA][G][ [mU][mG][mA][mC][mA][C] A][A][mA][C][mA][U][G] [mU][mA] [mC][mU][mU] 850 siRNA127- [mG][mU][mU][mU][mC][m 519 1331 [mU][A][C][mA][A][A][G 903 1731 M0 U][mC][mA][A][U][G][A][C] ][U][G][U][mC][mA][U][ [mA][mC][mU][mU][mU][G] U][G][mA][G][mA][A][A] [mU][mA] [mC][mU][mU] 851 siRNA128- [mU][mU][mU][mC][mU][m 520 1332 [mU][G][A][mC][A][A][A 904 1732 M0 C][mA][mA][U][G][A][C][A] ][G][U][G][mU][mC][A][ [mC][mU][mU][mU][mG][U] U][U][mG][A][mG][A][A] [mC][mA] [mA][mU][mU] 852 siRNA129- [mU][mU][mC][mU][mC][m 521 1333 [mU][G][G][mA][C][A][A 905 1733 M0 A][mA][mU][G][A][C][A][C] ][A][G][U][mG][mU][C][ [mU][mU][mU][mG][mU][C] A][U][mU][G][mA][G][A] [mC][mA] [mA][mU][mU] 861 siRNA130- [mA][mC][mA][mC][mU][m 522 1334 [mU][A][U][mC][A][A][A 906 1734 M0 U][mU][mG][U][C][C][A][C] ][G][G][U][mG][mG][A][ [mC][mU][mU][mU][mG][A] C][A][mA][A][mG][U][G] [mU][mA] [mU][mU][mU] 862 siRNA131- [mC][mA][mC][mU][mU][m 523 1335 [mU][C][A][mU][C][A][A 907 1735 M0 U][mG][mU][C][C][A][C][C] ][A][G][G][mU][mG][G][ [mU][mU][mU][mG][mA][U] A][C][mA][A][mA][G][U] [mG][mA] [mG][mU][mU] 901 siRNA132- [mA][mC][mA][mC][mC][m 524 1336 [mU][C][C][mC][A][G][A 908 1736 M0 U][mC][mG][U][G][G][U][C] ][G][G][A][mC][mC][A][ [mC][mU][mC][mU][mG][G] C][G][mA][G][mG][U][G] [mG][mA] [mU][mU][mU] 903 siRNA133- [mA][mC][mC][mU][mC][m 525 1337 [mU][U][G][mC][C][C][A 909 1737 M0 G][mU][mG][G][U][C][C][U] ][G][A][G][mG][mA][C][ [mC][mU][mG][mG][mG][C] C][A][mC][G][mA][G][G] [mA][mA] [mU][mU][mU] 904 siRNA134- [mC][mC][m][mC][mG][m 526 1338 [mU][A][U][mG][C][C][C 910 1738 M0 U][mG][mG][U][C][C][U][C] ][A][G][A][mG][mG][A][ [mU][mG][mG][mG][mC][A] C][C][mA][C][mG][A][G] [mU][mA] [mG][mU][mU] 905 siRNA135- [mC][mU][mC][mG][mU][m 527 1339 [mU][G][A][mU][G][C][C 911 1739 M0 G][mU][mU][C][C][U][C][U] ][C][A][G][mA][mG][G][ [mG][mG][mG][mC][mA][U] A][C][mC][A][mC][G][A] [mC][mA] [mG][mU][mU] 906 siRNA136- [[mU][mC][mG][mU][mG][m 528 1340 [mU][C][G][mA][U][G][C 912 1740 M0 G][mU][mC][C][U][C][U][G] ][C][C][A][mG][mA][G][ [mG][mG][mC][mA][mU][C] G][A][mC][C][mA][C][G] [mG][mA] [mA][mU][mU] 912 siRNA137- [mU][mC][mC][mU][mC][m 529 1341 [mU][U][G][mC][U][U][G 913 1741 M0 U][mG][mG][G][C][A][U][C] ][C][G][A][mU][mG][C][ [mG][mC][mA][mA][mG][C] C][C][mA][G][mA][G][G] [mA][mA] [mA][mU][mU] 915 siRNA138- [mU][mC][mU][mG][mG][m 530 1342 [mU][G][U][mU][U][G][C 914 1742 M0 G][mC][mA][U][C][G][C][A] ][U][U][G][mC][mG][A][ [mA][mG][mC][mA][mA][A] U][G][mC][C][mC][A][G] [mC][mA] [mA][mU][mU] 916 siRNA139- [mC][mU][mG][mG][mG][m 531 1343 [mU][U][G][mU][U][U][G 915 1743 M0 C][mA][mU][C][G][C][A][A] ][C][U][U][mG][mC][G][ [mG][mC][mA][mA][mA][C] A][U][mG][C][mC][C][A] [mA][mA] [mG][mU][mU] 917 siRNA140- [mU][mG][mG][mG][mC][m 532 1344 [mU][G][U][mG][U][U][U 916 1744 M0 A][mU][mV][G][C][A][A][G] ][G][C][U][mU][mG][C][ [mC][mA][mA][mA][mC][A] ][G][C][U][mU][mG][C][ [mC][mA] [mA][mU][mU] 920 siRNA141- [mG][mC][mA][mU][mC][m 533 1345 [mU][A][U][mG][G][U][G 917 1745 M0 G][mC][mA][A][G][C][A][A] ][U][U][U][mG][mC][U][ [mA][mC][mA][mC][mC][A] U][G][mC][G][mA][U][G] [mU][mA] [mC][mU][mU] 921 siRNA142- [mC][mA][mU][mC][mG][m 534 1346 [mU][A][A][mU][G][G][U 918 1746 M0 C][mA][mA][G][C][A][A][A] ][G][U][U][mU][mG][C][ [mC][mA][mC][mC][mA][U] U][U][mG][C][mG][A][U] [mU][mA] [mG][mU][mU] 950 siRNA143- [mU][mU][mC][mU][mG][m 535 1347 [mU][A][U][mU][U][G][A 919 1747 M0 G][mA][mG][U][U][U][A][U ][A][A][U][mA][mA][A][ ][mU][mU][mC][mA][mA][A C][U][mC][C][mA][G][A] ][mU][mA] [mA][mU][mU] 954 siRNA144- [mG][mG][mA][mG][mU][m 536 1348 [mU][A][A][mU][C][A][U 920 1748 M0 U][mU][mA][U][U][U][C][A] ][U][U][G][mA][mA][A][ [mA][mA][mU][mG][mA][U] U][A][mA][A][mC][U][C] [mU][mA] [mC][mU][mU] 955 siRNA145- [mG][mA][mG][mU][mU][m 537 1349 [mU][U][A][mA][U][C][A 921 1749 M0 U][mA][mU][U][U][C][A][A] ][U][U][U][mG][mA][A][ [mA][mU][mG][mA][mU][U] A][U][mA][A][mA][C][U] [mA][mA] [mC][mU][mU] 956 siRNA146- [mA][mG][mU][mU][mU][m 538 1350 [mU][C][U][mA][A][U][C 922 1750 M0 A][mU][mU][U][C][A][A][A] ][A][U][U][mU][mG][A][ [mU][mG][mA][mU][mU][A] A][A][mU][A][mA][A][C] [mG][mA] [mU][mU][mU] 957 siRNA147- [mG][mU][mU][mU][mA][m 539 1351 [mU][C][C][mU][A][A][U 923 1751 M0 U][mU][mU][C][A][A][A][U] ][C][A][U][mU][mU][G][ [mG][mA][mU][mU][mA][G] A][A][mA][U][mA][A][A] [mG][mA] [mC][mU][mU] 980 siRNA148- [mC][mC][mA][mG][mA][m 540 1352 [mU][G][C][mU][G][C][U 924 1752 M0 A][mG][mC][A][A][U][G][A] ][U][U][C][mA][mU][U][ [mA][mA][mG][mC][mA][G] C][C][mU][U][mC][U][G] [mC][mA] [mG][mU][mU] 982 siRNA149- [mA][mG][mA][mA][mG][m 541 1353 [mU][U][A][mG][C][U][G 925 1753 M0 C][mA][mA][U][G][A][A][A] ][C][U][U][mU][mC][A][ [mG][mC][mA][mG][mC][U] U][U][G][C][mU][U][C] [mA][mA] [mU][mU][mU] 983 siRNA150- [mG][mA][mA][mG][mC][m 542 1354 [mU][G][U][mA][G][C][U 926 1754 M0 A][mA][mU][G][A][A][A][G] ][G][C][U][mU][mU][C][ ][mC][mA][mG][mC][mU][A A][U][mU][G][mC][U][U] ][mC][mA] [mC][mU][mU] 988 siRNA151- [mA][mA][mU][mG][mA][m 543 1355 [mU][C][U][mU][C][A][G 927 1755 M0 A][mA][mG][C][A][G][C][U] ][U][A][G][mC][mU][G][ [mA][mC][mU][mG][mA][A] C][U][mU][U][mC][A][U] [mG][mA] [mU][mU][mU] 989 siRNA152- [mA][mU][mG][mA][mA][m 544 1356 [mU][U][C][mU][U][C][A 928 1756 M0 A][mG][mC][A][G][C][U][A] ][G][U][A][mG][mC][U][ [mC][mU][mG][mA][mA][G] G][C][mU][U][mU][C][A] [mA][mA] [mU][mU][mU] 992 siRNA153- [mA][mA][mA][mG][mC][m 545 1357 [mU][A][C][mU][U][C][U 929 1757 M0 A][mG][mC][U][A][C][U][G] ][U][C][A][mG][mU][A][ [mA][mA][mG][mA][mA][G] G][C][mU][G][mC][U][U] [mU][mA] [mU][mU][mU] 993 siRNA154- [mA][mA][mG][mC][mA][m 546 1358 [mU][C][A][mC][U][U][C 930 1758 M0 G][mC][mU][A][C][U][G][A] ][U][U][C][mA][mG][U][ [mA][mG][mA][mA][mG][U] A][G][mC][U][mG][C][U] [mG][mA] [mU][mU][mU] 994 siRNA155- [mA][mG][mC][mA][mG][m 547 1359 [mU][U][C][mA][C][U][U 931 1759 M0 C][mU][mA][C][U][G][A][A] ][C][U][U][mC][mA][G][ [mG][mA][mA][mG][mU][G] U][A][mG][C][mU][G][C] [mA][mA] [mU][mU][mU] 995 siRNA156- [mG][mC][mA][mG][mC][m 548 1360 [mU][U][U][mC][A][C][U 932 1760 M0 U][mA][mC][U][G][A][A][G] ][U][C][U][mU][mC][A][ [mA][mA][mG][mU][mG][A] G][U][mA][G][mC][U][G] [mA][mA] [mC][mU][mU] 996 siRNA157- [mC][mA][mG][mC][mU][m 549 1361 [mU][U][U][mU][C][A][C 933 1761 M0 A][mC][mU][G][A][A][G][A] ][U][U][C][mU][mU][C][ [mA][mG][mU][mG][mA][A] A][G][mU][A][mG][C][U] [mA][mA] [mG][mU][mU] 1005 siRNA158- [mA][mA][mG][mA][mA][m 550 1362 [mU][U][A][mA][U][G][U 934 1762 M0 G][mU][mG][A][A][A][A][G] ][U][C][U][mU][mU][U][ ][mA][mA][mC][mA][mU][U C][A][mC][U][mU][C][U] ][mA][mA] [mU][mU][mU] 1006 siRNA159- [mA][mG][mA][mA][mG][m 551 1364 [mU][C][U][mA][A][U][G 935 1763 M0 U][mG][mA][A][A][A][G][A ][U][U][C][mU][mU][U][ ][mA][mC][mA][mU][mU][A U][C][mA][C][mU][U][C] ][mG][mA] [mU][mU][mU] 1007 siRNA160- [mG][mA][mA][mG][mU][m 552 1365 [mU][U][C][mU][A][A][U 936 1764 M0 G][mA][mA][A][A][G][A][A ][G][U][U][mC][mU][U][ ][mC][mA][mU][mU][mA][G U][U][mC][A][mC][U][U] ][mA][mA] [mC][mU][mU] 1011 siRNA161- [mU][mG][mA][mA][mA][m 553 1366 [mU][A][U][mU][C][U][C 937 1765 M0 A][mG][mA][A][C][A][U][U] ][U][A][A][mU][mG][U][ [mA][mG][mA][mG][mA][A] U][C][mU][U][mU][U][C] [mU][mA] [mA][mU][mU] 1012 siRNA162- [mG][mA][mA][mA][mA][m 554 1368 [mU][C][A][mU][U][C][U 938 1766 M0 G][mA][mA][C][A][U][U][A] ][C][U][A][mA][mU][G][ [mG][mA][mG][mA][mA][U] U][U][mC][U][mU][U][U] [mG][mA] [mC][mU][mU] 1013 siRNA163- [mA][mA][mA][mA][mG][m 555 1369 [mU][G][C][mA][U][U][C 939 1767 M0 A][mA][mC][A][U][U][A][G] ][U][C][U][mA][mA][U][ [mA][mG][mA][mA][mU][G] G][U][mU][C][mU][U][U] [mC][mA] [mU][mU][mU] 1018 siRNA164- [mA][mA][mC][mA][mU][m 556 1370 [mU][G][A][mC][C][A][G 940 1768 M0 U][mA][mG][A][G][A][A][U ][C][A][U][mU][mC][U][ ][mG][mC][mU][mG][mG][U C][U][mA][A][mU][G][U] ][mC][mA] [mU][mU][mU] 1019 siRNA165- [mA][mC][mA][mU][mU][m 557 1371 [mU][U][G][mA][C][C][A 941 1769 M0 A][mG][mA][G][G][A][U][G ][G][C][A][mU][mU][C][ ][mC][mU][mG][mG][mU][C U][C][mU][A][mA][U][G] ][mA][mA] [mU][mU][mU] 1020 siRNA166- [mC][mA][mU][mU][mA][m 558 1372 [mU][U][U][mG][A][C][C 942 1770 M0 G][mA][mG][A][A][U][G][C] ][A][G][C][mA][mU][U][ [mU][mG][mG][mU][mC][A] C][U][mC][U][mA][A][U] [mA][mA] [mG][mU][mU] 1021 siRNA167- [mA][mU][mU][mA][mG][m 559 1373 [mU][U][U][mU][G][A][C 943 1771 M0 A][mG][mA][A][U][G][C][U] ][C][A][G][mC][mA][U][ [mG][mG][mU][mC][mA][A] U][C][mU][C][mU][A][A] [mA][mA] [mU][mU][mU] 1026 siRNA168- [mA][mG][mA][mA][mU][m 560 1374 [mU][G][A][m][U][U][U 944 1772 M0 G][mC][mU][G][G][U][C][A] ][U][U][G][mA][mC][C][ [mA][mA][mA][mA][mG][U] A][G][mC][A][mU][U][C] [mC][mA] [mU][mU][mU] 1027 siRNA169- [mG][mA][mA][mU][mG][m 561 1375 [mU][U][G][mA][C][U][U 945 1773 M0 C][mU][mG][G][U][C][A][A] ][U][U][U][mG][mA][C][ [mA][mA][mA][mG][mU][C] C][A][mG][C][mA][U][U] [mA][mA] [mC][mU][mU] 1029 siRNA170- [mA][mU][mG][mC][mU][m 562 1376 [mU][G][C][mU][G][A][C 946 1774 M0 G][mG][mU][C][A][A][A][A] ][U][U][U][mU][mU][G][ [mA][mG][mU][mC][mA][G] A][C][mC][A][mG][C][C] [mC][mA] [mU][mU][mU] 1036 siRNA171- [mU][mC][mA][mA][mA][m 563 1377 [mU][C][U][mU][C][C][A 947 1775 M0 A][mA][mG][U][C][A][G][C] ][A][G][C][mU][mG][A][ [mU][mU][mG][mG][mA][A] C][U][mU][U][mU][U][G] [mG][mA] [mA][mU][mU] 1039 siRNA172- [mA][mA][mA][mA][mG][m 564 1378 [mU][U][G][mC][C][U][U 948 1776 M0 U][mC][mA][G][C][U][U][G] ][C][C][A][mA][mG][C][ [mG][mA][mA][mG][mG][C] U][G][mA][C][mU][U][U] [mA][mA] [mU][mU][mU] 1042 siRNA173- [mA][mG][mU][mC][mA][m 565 1380 [mU][G][A][mU][U][G][C 949 1777 M0 G][mC][mU][U][G][G][A][A] ][C][U][U][mC][mC][A][ [mG][mG][mC][mA][mA][U] A][G][mC][U][mG][A][C] [mC][mA] [mU][mU][mU] 1084 siRNA174- [mA][mC][mU][mG][mA][m 566 1381 [mU][A][U][mA][C][U][G 950 1778 M0 A][mU][mG][A][C][C][U][G] ][G][C][A][mG][mG][U][ [mC][mC][mA][mG][mU][A] C][A][mU][U][mC][A][G] [mU][mA] [mU][mU][mU] 1087 siRNA175- [mG][mA][mA][mU][mG][m 567 1383 [mU][C][U][mA][A][U][A 951 1779 M0 A][mC][mC][U][G][C][C][A] ][C][U][G][mG][mC][A][ [mG][mU][mA][mU][mU][A] G][G][mU][C][mA][U][U] [mG][mA] [mC][mU][mU] 1134 siRNA176- [mU][mU][mU][mC][mC][m 568 1384 [mU][G][U][mU][G][A][G 952 1780 M0 A][mG][mU][G][C][C][U][C] ][G][G][A][mG][mG][C][ [mC][mC][mU][mC][mA][A] A][C][mU][G][mG][A][A] [mC][mA] [mA][mU][mU] 1151 siRNA177- [mA][mA][mC][mA][mU][m 569 1385 [mU][U][C][mC][U][U][A 953 1781 M0 C][mC][mG][G][A][C][A][G] ][G][G][A][mG][mG][C][ [mC][mU][mA][mA][mG][G] C][G][mG][A][mU][G][U] [mA][mA] [mU][mU][mU] 1152 siRNA178- [mA][mC][mA][mU][mC][m 570 1386 [mU][C][U][mC][C][U][U 954 1782 M0 C][mG][mG][A][C][A][G][C] ][A][G][C][mU][mG][U][ [mU][mA][mA][mG][mG][A] C][C][mG][G][mA][U][G] [mG][mA] [mU][mU][mU] 1155 siRNA179- [mU][mC][mC][mG][mG][m 571 1387 [mU][A][U][mC][C][U][C 955 1783 M0 A][mC][mA][G][C][U][A][A] ][C][U][U][mA][mG][C][ [mG][mG][mA][mG][mG][A] U][G][mU][C][mC][G][G] [mU][mA] [mA][mU][mU] 1158 siRNA180- [mG][mG][mA][mC][mA][m 572 1388 [mU][G][A][mA][A][U][C 956 1784 M0 G][mC][mU][A][A][G][G][A] ][C][U][C][mC][mU][U][ [mG][mG][mA][mU][mU][U] A][G][mC][U][mG][U][C] [mC][mA] [mC][mU][mU] 1160 siRNA181- [mA][mC][mA][mG][mC][m 573 1389 [mU][G][U][mG][A][A][A 957 1785 M0 U][mA][mA][G][G][A][G][G ][U][C][C][mU][mC][C][ ][mA][mU][mU][mU][mC][A U][U][mA][G][mC][U][G] ][mC][mA] [mU][mU][mU] 1162 siRNA182- [mA][mG][mC][mU][mA][m 574 1390 [mU][A][A][mG][U][G][A 958 1786 M0 A][mG][mG][A][G][G][A][U ][A][A][U][mC][mC][U][ ][mU][mU][mC][mA][mC][U C][C][mU][U][mA][G][C] ][mU][mA] [mU][mU][mU] 1165 siRNA183- [mU][mA][mA][mG][mG][m 575 1391 [mU][G][C][mA][A][A][G 959 1787 M0 A][mG][mG][A][U][U][U][C] ][U][G][A][mA][mA][U][ [mA][mC][mU][mU][mU][G] C][C][mU][C][mC][U][U] [mC][mA] [mA][mU][mU] 1168 siRNA184- [mG][mG][mA][mG][mG][m 576 1392 [mU][G][G][mU][G][C][A 960 1788 M0 A][mU][mU][U][C][A][C][U] ][A][A][G][mU][mG][A][ [mU][mU][mG][mC][mA][C] A][A][mU][C][mC][U][C] [mC][mA] [mC][mU][mU] 1169 siRNA185- [mG][mA][mG][mG][mA][m 577 1393 [mU][A][G][mG][U][G][C 961 1789 M0 U][mU][mU][C][A][C][U][U] ][A][A][A][mG][mU][G][ [mU][mG][mC][mA][mC][C] A][A][mA][U][mC][C][U] [mU][mA] [mC][mU][mU] 1174 siRNA186- [mU][mU][mU][mC][mA][m 578 1394 [mU][C][C][mU][C][A][A 962 1790 M0 C][mU][mU][U][G][C][A][C] ][G][G][U][mG][mC][A][ [mC][mU][mU][mG][mA][G] A][A][mG][U][mG][A][A] [mG][mA] [mA][mU][mU] 1177 siRNA187- [mC][mA][mC][mU][mU][m 579 1395 [mU][C][G][mU][C][C][U 963 1791 M0 U][mG][mC][A][C][C][U][U] ][C][A][A][mG][mG][U][ [mG][mA][mG][mG][mA][C] G][C][mA][A][mA][G][U] [mG][mA] [mG][mU][mU] 1178 siRNA188- [mA][mC][mU][mU][mU][m 580 1396 [mU][C][C][mG][U][C][C 964 1792 M0 G][mC][mA][C][C][U][U][G] ][U][C][A][mA][mG][G][ [mA][mG][mG][mA][mC][G] U][G][mC][A][mA][A][G] [mG][mA] [mU][mU][mU] 1189 siRNA189- [mU][mG][mA][mG][mG][m 581 1397 [mU][U][G][mU][U][G][U 965 1793 M0 A][mC][mG][G][U][U][C][C] ][A][G][G][mA][mA][C][ [mU][mA][mC][mA][mA][C] C][G][mU][C][mC][U][C] [mA][mA] [mA][mU][mU] 1191 siRNA190- [mA][mG][mG][mA][mC][m 582 1398 [mU][G][A][mU][G][U][U 966 1794 M0 G][mG][mU][U][C][C][U][A] ][G][U][A][mG][mG][A][ [mC][mA][mA][mC][mA][U] A][C][mC][G][mU][C][C] [mC][mA] [mU][mU][mU] 1194 siRNA191- [mA][mC][mG][mG][mU][m 583 1400 [mU][U][C][mG][G][A][U 967 1795 M0 U][mC][mC][U][A][C][A][A] ][G][U][U][mG][mU][A][ [mC][mA][mU][mC][mC][G] G][G][mA][A][mC][C][G] [mA][mA] [mU][mU][mU] 1196 siRNA192- [mG][mG][mU][mU][mC][m 584 1401 [mU][U][U][mU][C][G][G 968 1796 M0 C][mU][mA][C][A][A][C][A] ][A][U][G][mU][mU][G][ [mU][mC][mC][mG][mA][A] U][A][mG][G][mA][A][C] [mA][mA] [mC][mU][mU] 1200 siRNA193- [mC][mC][mU][mA][mC][m 585 1402 [mU][A][U][mC][U][U][U 969 1797 M0 A][mA][mC][A][U][C][C][G] ][U][C][G][mG][mA][U][ [mA][mA][mA][mA][mG][A] G][U][mU][G][mU][A][G] [mU][mA] [mG][mU][mU] 1201 siRNA194- [mC][mU][mA][mC][mA][m 586 1403 [mU][C][A][mU][C][U][U 970 1798 M0 A][mC][mA][U][C][C][G][A] ][U][U][C][mG][mG][A][ [mA][mA][mA][mG][mA][U] U][G][mU][U][mG][U][A] [mG][mA] [mG][mU][mU] 1203 siRNA195- [mA][mC][mA][mA][mC][m 587 1404 [mU][G][U][C][A][U][C 971 1799 M0 A][mU][mC][C][G][A][A][A] ][U][U][U][mU][mC][G][ [mA][mG][mA][mU][mG][A] G][A][mU][G][mU][U][G] [mC][mA] [mU][mU][mU] 1204 siRNA196- [mC][mA][mA][mC][mA][m 588 1405 [mU][U][G][mU][C][A][U 972 1800 M0 U][mC][mC][G][A][A][A][A] ][C][U][U][mU][mU][C][ [mG][mA][mU][mG][mA][C] G][G][mA][U][mG][U][U] [mA][mA] [mG][mU][mU] 1205 siRNA197- [mA][mA][mC][mA][mU][m 589 1406 [mU][A][U][mG][U][C][A 973 1801 M0 C][mC][mG][A][A][A][A][G] ][U][C][U][mU][mU][U][ [mA][mU][mG][mA][mC][A] C][G][mG][A][mU][G][U] [mU][mA] [mU][mU][mU] 1207 siRNA198- [mC][mA][mU][mC][mC][m 590 1407 [mU][U][G][mA][U][G][U] 974 1802 M0 G][mA][mA][A][A][G][A][U] ][C][A][U][mC][mU][U][ [mG][mA][mC][mA][mU][C] U][U][mC][G][mG][A][U] ][mA][mA] [mG][mU][mU] 1209 siRNA199- [mU][mC][mC][mG][mA][m 591 1408 [mU][U][A][mU][G][A][U 975 1803 M0 A][mA][mA][G][A][U][G][A ][G][U][C][mA][mU][C][ ][mC][mA][mU][mC][mA][U U][U][mU][U][mC][G][G] ][mA][mA] [mA][mU][mU] 1212 siRNA200- [mG][mA][mA][mA][mA][m 592 1410 [mU][A][G][mC][U][A][U 976 1804 M0 G][mA][mU][G][A][C][A][U] ][G][A][U][mG][mU][C][ [mC][mA][mU][mA][mG][C] A][U][mC][U][mU][U][U] [mU][mA] [mC][mU][mU] 1214 siRNA201- [mA][mA][mA][mG][mA][m 593 1411 [mU][A][G][mA][G][C][U 977 1805 M0 U][mG][mA][C][A][U][C][A] ][A][U][G][mA][mU][G][ [mU][mA][mG][mC][mU][C] U][C][mA][U][mC][U][U] [mU][mA] [mU][mU][mU] 1215 siRNA202- [mA][mA][mG][mA][mU][m 594 1412 [mU][A][A][mG][A][G][C 978 1806 M0 G][mA][mC][A][U][C][A][U] ][U][A][U][mG][mA][U][ [mA][mG][mC][mU][mC][U] G][U][mC][A][mU][C][U] [mU][mA] [mU][mU][mU] 1217 siRNA203- [mG][mA][mU][mG][mA][m 595 1413 [mU][U][A][mA][A][G][A 979 1807 M0 C][mA][mU][C][A][U][A][G] ][G][C][U][mA][mU][G][ [mC][mU][mC][mU][mU][U] A][U][mG][U][mC][A][U] [mA][mA] [mC][mU][mU] 1226 siRNA204- [mA][mU][mA][mG][mC][m 596 1415 [mU][A][A][mC][U][G][U 980 1808 M0 U][mC][mU][U][U][A][C][C] ][G][G][G][mU][mA][A][ [mC][mA][mC][mA][mG][U] A][G][mA][G][mC][U][A] [mU][mA] [mU][mU][mU] 1227 siRNA205- [mU][mA][mG][mC][mU][m 597 1416 ][U][G][G][mG][mU][A][ 981 1809 M0 C][mU][mU][U][A][C][C][C] ][U][G][G][mG][mA][A][ [mA][mC][mA][mG][mU][U] A][A][mG][A][mG][C][U] [mA][mA] [mA][mU][mU] 1228 siRNA206- [mA][mG][mC][mU][mC][m 598 1419 [mU][U][U][mA][A][C][U 982 1810 M0 U][mU][mU][A][C][C][C][A] ][G][U][G][mG][mG][U][ [mC][mA][mG][mU][mU][A] A][A][mA][G][mA][G][C] [mA][mA] [mU][mU][mU] 1229 siRNA207- [mG][mC][mU][mC][mU][m 599 1420 [mU][A][U][mU][A][A][C 983 1811 M0 U][mU][mA][C][C][C][A][C] ][U][G][U][mG][mG][G][ [mA][mG][mU][mU][mA][A] U][A][mA][A][mG][A][G] [mU][mA] [mC][mU][mU] 1230 siRNA208- [mC][mU][mC][mU][mU][m 600 1421 [mU][C][A][mU][U][A][A 984 1812 M0 U][mA][mC][C][C][A][C][A] ][C][U][G][mU][mG][G][ [mG][mU][mU][mA][mA][U] G][U][mA][A][mA][G][A] [mG][mA] [mG][mU][mU] 1231 siRNA209- [mU][mC][mU][mU][mU][m 601 1422 [mU][G][C][mA][U][U][A 985 1813 M0 A][mC][mC][C][A][C][A][G] ][A][C][U][mG][mU][G][ [mU][mU][mA][mA][mU][G] G][G][mU][A][mA][A][G] [mC][mA] [mA][mU][mU] 1233 siRNA210- [mU][mU][mU][mA][mC][m 602 1423 [mU][G][U][mG][C][A][U 986 1814 M0 C][mC][mA][C][A][G][U][U] ][U][A][A][mC][mU][G][ [mA][mA][mU][mG][mC][A] U][G][mG][G][mU][A][A] [mC][mA] [mA][mU][mU] 1236 siRNA211- [mA][mC][mC][mC][mA][m 603 1424 [mU][U][A][mA][G][U][G 987 1815 M0 C][mA][mG][U][U][A][A][U] ][C][A][U][mU][mA][A][ [mG][mC][mA][mC][mU][U] C][U][mG][U][mG][G][G] [mA][mA] [mU][mU][mU] 1237 siRNA212- [mC][mC][mC][mA][mC][m 604 1426 [mU][C][U][mA][A][G][U 988 1816 M0 A][mG][mU][U][A][A][U][G] ][G][C][A][mU][mU][A][ ][mC][mA][mC][mU][mU][A ][G][C][A][mU][mU][A][ ][mG][mA] [mG][mU][mU] 1238 siRNA213- [mC][mC][mA][mC][mA][m 605 1427 [mU][U][C][mU][A][A][G 989 1817 M0 G][mU][mU][A][A][U][G][C] ][U][G][C][mA][mU][U][ [mA][mC][mU][mU][mA][G] ][A][A][C][mG][mU][G][ [mA][mA] [mG][mU][mU] 1239 siRNA214- [mC][mA][mC][mA][mG][m 606 1428 [mU][A][U][mC][U][A][A 990 1818 M0 U][mU][mA][A][U][G][C][A] ][G][U][G][mC][mA][U][ [mC][mU][mU][mA][mG][A] U][A][mA][C][mU][G][U] [mU][mA] [mG][mU][mU] 1240 siRNA215- [mA][mC][mA][mG][mU][m 607 1429 [mU][G][A][mU][C][U][A 991 1819 M0 U][mA][mA][U][G][C][A][C] ][A][G][U][mG][mC][A][ [mU][mU][mA][mG][mA][U] U][U][mA][A][mC][U][G] [mC][mA] [mU][mU][mU] 1241 siRNA216- [mC][mA][mG][mU][mU][m 608 1430 [mU][G][G][mA][U][C][U 992 1820 M0 U][mA][mA][U][G][C][A][C] ][A][A][G][mU][mG][C][ [mU][mA][mG][mA][mU][C] A][U][mU][A][mA][C][U] [mC][mA] [mG][mU][mU] 1242 siRNA217- [mA][mG][mU][mU][mA][m 609 1431 [mU][U][G][mG][A][U][C 993 1821 M0 A][mU][mG][C][A][C][U][U] ][U][A][A][mG][mU][G][ [mA][mG][mA][mU][mC][C] C][A][mU][U][mA][A][C] [mA][mA] [mU][mU][mU] 1248 siRNA218- [mU][mG][mC][mA][mC][m 610 1432 [mU][G][A][mU][U][U][C 994 1822 M0 U][mU][mA][G][A][U][C][C] ][U][G][G][mA][mU][C][ [mA][mG][mA][mA][mA][U] U][A][mA][G][mU][G][C] [mC][mA [mA][mU][mU] 1249 siRNA219- [mG][mC][mA][mC][mU][m 611 1433 [mU][A][G][mA][U][U][U 995 1823 M0 U][mA][mG][A][U][C][C][A] ][C][U][G][mG][mA][U][ [mG][mA][mA][mA][mU][C] C][U][mA][A][mG][U][G] [mU][mA] [mC][mU][mU 1261 siRNA220- [mA][mG][mA][mA][mA][m 612 1434 [mU][A][A][mG][G][G][U 996 1824 M0 U][mC][mU][A][C][C][C][A] ][C][U][G][mG][mG][U][ [mG][mA][mC][mC][mC][U] A][G][mA][U][mU][U][C] [mU][mA] [mU][mU][mU] 1283 siRNA221- [mA][mC][mU][mU][mU][m 613 1435 [mU][U][A][mC][C][U][A 997 1825 M0 U][mA][mA][A][U][A][U][G ][U][C][A][mU][mA][U][ ][mA][mU][mA][mG][mG][U U][U][mA][A][mA][A][G] ][mA][mA] [mU][mU][mU] 1285 siRNA222- [mU][mU][mU][mU][mA][m 614 1436 [mU][G][A][mU][A][C][C 998 1826 M0 A][mA][mU][A][U][G][A][U ][U][A][U][mC][mA][U][ ][mA][mG][mG][mU][mA][U A][U][mU][U][mA][A][A] ][mC][mA] [mA][mU][mU] 1286 siRNA223- [mU][mU][mU][mA][mA][m 615 1437 [mU][A][G][mA][U][A][C 999 1827 M0 A][mU][mA][U][G][A][U][A ][C][U][A][mU][mC][A][ ][mG][mG][mU][mA][mU][C U][A][mU][U][mU][A][A] ][mU][mA] [mA][mU][mU] 1288 siRNA224- [mU][mA][mA][mA][mU][m 616 1438 [mU][C][A][mA][G][A][U 1000 1828 M0 A][mU][mG][A][U][A][G][G ][A][C][C][mU][mA][U][ ][mU][mA][mU][mC][mU][U C][A][mU][A][mU][U][U] ][mG][mA] [mA][mU][mU] 1290 siRNA225- [mA][mA][mU][mA][mU][m 617 1439 [mU][A][U][mC][A][A][G 1001 1829 M0 G][mA][mU][A][G][G][U][A ][A][U][A][mC][mC][U][ ][mU][mC][mU][mU][mG][A A][U][mC][A][mU][A][U] [mU][mA] [mU][mU][mU] 1291 siRNA226- [mA][mU][mA][mU][mG][m 618 1440 [mU][C][A][mU][C][A][A 1002 1830 M0 A][mU][mA][G][G][U][A][U ][G][A][U][mA][mC][C][ ][mC][mU][mU][mG][mA][U U][A][mU][C][mA][U][A] ][mG][mA] [mU][mU][mU] 1292 siRNA227- [mU][mA][mU][mG][mA][m 619 1442 [mU][U][C][mA][U][C][A 1003 1831 M0 U][mA][mG][G][U][A][U][C] ][A][G][A][mU][mA][C][ [m][mU][mG][mA][mU][G] C][U][mA][U][mC][A][U] [mA][mA] [mA][mU][mU] 1293 siRNA228- [mA][mU][mG][mA][mU][m 620 1443 [mU][U][U][mC][A][U][C 1004 1832 M0 A][mG][mG][U][A][U][C][U] ][A][A][G][mA][mU][A][ [mU][mG][mA][mU][mG][A] C][C][mU][A][mU][C][A] [mA][mA] [mU][mU][mU] 1296 siRNA229- [mA][mU][mA][mG][mG][m 621 1444 [mU][G][U][mU][U][U][C 1005 1833 M0 U][mA][mU][C][U][U][G][A] ][A][U][C][mA][mA][G][ [mU][mG][mA][mA][mA][A] A][U][mA][C][mC][U][A] [mC][mA] [mU][mU][mU] 1297 siRNA230- [mU][mA][mG][mG][mU][m 622 1445 [mU][C][G][mU][U][U][U 1006 1834 M0 A][mU][mC][U][U][G][A][U] ][C][A][U][mC][mA][A][ [mG][mA][mA][mA][mA][C] G][A][mU][A][mC][C][U] [mG][mA] [mA][mU][mU] 1298 siRNA231- [mA][mG][mG][mU][mA][m 623 1446 [mU][C][C][mG][U][U][U 1007 1835 M0 U][mC][mU][U][G][A][U][G] ][U][C][U][mU][mC][A][ [mA][mA][mA][mA][mC][G] A][G][mA][U][mA][C][C] [mG][mA] [mU][mU][mU] 1299 siRNA232- [mG][mG][mU][mA][mU][m 624 1448 [mU][C][C][mC][G][U][U 1008 1836 M0 C][mU][mU][G][A][U][G][A] ][U][U][C][mA][mU][C][ [mA][mA][mA][mC][mG][G] A][A][mG][A][mU][A][C] [mG][mA] [mC][mU][mU] 1300 siRNA233- [mG][mU][mA][mU][mC][m 625 1449 [mU][U][C][mC][C][G][U 1009 1837 M0 U][mU][mG][A][U][G][A][A ][U][U][U][mC][mA][U][ ][mA][mA][mC][mG][mG][G C][A][mA][G][mA][U][A] ][mA][mA] [mC][mU][mU] 1301 siRNA234- [mU][mA][mU][mC][mU][m 626 1450 [mU][U][U][mC][C][C][G 1010 1838 M0 U][mG][mA][U][G][A][A][A ][U][U][U][mU][mC][A][ ][mA][mC][mG][mG][mG][A U][C][mA][A][mG][A][U] ][mA][mA] [mA][mU][mU] 1302 siRNA235- [mA][mU][mC][mU][mU][m 627 1451 [mU][C][U][mU][C][C][C 1011 1839 M0 G][mA][mU][G][A][A][A][A ][G][U][U][mU][mU][C][ ][mC][mG][mG][mG][mA][A A][U][mC][A][mA][G][A] ][mG][mA] [mU][mU][mU] 1303 siRNA236- [mU][mC][mU][mU][mG][m 628 1452 [mU][U][C][mU][U][U][C 1012 1840 M0 A][mU][mG][A][A][A][A][C] ][C][G][U][mU][mU][U][ [mG][mG][mG][mA][mA][G] C][A][mU][C][mA][A][G] [mA][mA] [mA][mU][mU] 1306 siRNA237- [mU][mG][mA][mU][mG][m 629 1453 [mU][U][U][mG][U][C][U 1013 1841 M0 A][mA][mA][A][C][G][G][G] [U][C][C][mC][mG][U][ [mA][mA][mG][mA][mC][A] U][U][mU][C][mA][U][C] [mA][mA] [mA][mU][mU] 1307 siRNA238- [mG][mA][mU][mG][mA][m 630 1454 [mU][U][U][mU][G][U][C 1014 1842 M0 A][mA][mA][C][G][G][G][A] ][U][U][C][mC][mC][G][ [mA][mG][mA][mC][mA][A] U][U][mU][U][mC][A][U] [mA][mA] [mC][mU][mU] 1311 siRNA239- [mA][mA][mA][mA][mC][m 631 1455 [mU][A][G][mU][C][U][U 1015 1843 M0 G][mG][mG][A][A][G][A][C] ][U][G][U][mC][mU][U][ [mA][mA][mA][mG][mA][C] C][C][mC][G][mU][U][U] [mU][mA] [mU][mU][mU] 1313 siRNA240- [mA][mA][mC][mG][mG][m 632 1456 [mU][G][U][mA][G][U][C 1016 1844 M0 G][mA][mA][G][A][C][A][A] ][U][U][U][mG][mU][C][ [mA][mG][mA][mC][mU][A] U][U][mC][C][mC][G][U] [mC][mA] [mU][mU][mU] 1314 siRNA241- [mA][mC][mG][mG][mG][m 633 1457 [mU][G][G][mU][A][G][U 1017 1845 M0 A][mA][mG][A][C][A][A][A] ][C][U][U][mU][mG][U][ [mG][mA][mC][mU][mA][C] C][U][mU][C][mC][C][G] [mC][mA] [mU][mU][mU] 1353 siRNA242- [mA][mG][mU][mU][mA][m 634 1458 [mU][C][A][mU][G][U][A 1018 1846 M0 A][mA][mG][U][A][U][U][A] ][G][U][A][mA][mU][A][ ][mC][mU][mA][mC][mA][U C][U][mU][U][mA][A][C] ][mG][mA] [mU][mU][mU] 1354 siRNA243- [mG][mU][mU][mA][mA][m 635 1459 [mU][G][C][mA][U][G][U 1019 1847 M0 A][mG][mU][A][U][U][A][C] ][A][G][U][mA][mA][U][ [mU][mA][mC][mA][mU][G] A][C][mU][U][mU][A][A] [mC][mA] [mC][mU][mU] 1358 siRNA244- [mA][mA][mG][mU][mA][m 636 1460 [mU][A][A][mG][G][G][C 1020 1848 M0 U][mU][mA][C][U][A][C][A] ][A][U][G][mU][mA][G][ [mU][mG][mC][mC][mC][U] U][A][mA][U][mA][C][U] [mU][mA] [mU][mU][mU] 1360 siRNA245- [mG][mU][mA][mU][mU][m 637 1461 [mU][C][A][mA][A][G][G 1021 1849 M0 A][mC][mU][A][C][A][U][G] ][G][C][A][mU][mG][U][ [mC][mC][mC][mU][mU][U] A][G][mU][A][mA][U][A] [mG][mA] [mC][mU][mU] 1361 siRNA246- [mU][mA][mU][mU][mA][m 638 1462 [mU][C][C][mA][A][A][G 1022 1850 M0 C][mU][mA][C][A][U][G][C] ][G][G][C][mA][mU][G][ [mC][mC][mU][mU][mU][G] U][A][mG][U][mA][A][U] [mG][mA] [mA][mU][mU] 1363 siRNA247- [mU][mU][mA][mC][mU][m 639 1463 [mU][A][U][mC][C][A][A 1023 1851 M0 A][mC][mA][U][G][C][C][C] ][A][G][G][mG][mC][A][ [mU][mU][mU][mG][mG][A] U][G][mU][A][mG][U][A] [mU][mA] [mA][mU][mU] 1364 siRNA248- [mU][mA][mC][mU][mA][m 640 1464 [mU][G][A][mU][C][C][A 1024 1852 M0 C][mA][mU][G][C][C][C][U] ][A][A][G][mG][mG][C][ [mU][mU][mG][mG][mA][U] A][U][mG][U][mA][G][U] [mC][mA] [mA][mU][mU] 1382 siRNA249- [mU][mC][mG][mG][mG][m 641 1465 [mU][G][G][mA][C][A][U 1025 1853 M0 A][mG][mC][U][A][C][A][A] ][A][U][U][mG][mU][A][ [mU][mA][mU][mG][mU][C] G][C][mU][C][mC][C][G] [mC][mA] [mA][mU][mU] 1386 siRNA250- [mG][mA][mG][mC][mU][m 642 1466 [mU][U][C][mC][A][G][G 1026 1854 M0 A][mC][mA][A][U][A][U][G] ][A][C][A][mU][mA][U][ [mU][mC][mC][mU][mG][G] U][G][mU][A][mG][C][U] [mA][mA] [mC][mU][mU] 1388 siRNA251- [mG][mC][mU][mA][mC][m 643 1468 [mU][C][U][mU][C][C][A 1027 1855 M0 A][mA][mU][A][U][G][U][C] ][G][G][A][mC][mA][U][ [mC][mU][mG][mG][mA][A] A][U][mU][G][mU][A][G] [mG][mA] [mC][mU][mU] 1389 siRNA252- [mC][mU][mA][mC][mA][m 644 1469 [mU][U][C][mU][U][C][C 1028 1856 M0 A][mU][mA][U][G][U][C][C] ][A][G][G][mA][mC][A][ [mU][mG][mG][mA][mA][G] U][A][mU][U][mG][U][A] [mA][mA] [mG][mU][mU] 1413 siRNA253- [mU][mC][mG][mC][mU][m 645 1470 [mU][C][U][mU][G][A][U 1029 1857 M0 A][mU][mC][C][A][C][G][A] ][U][U][C][mG][mU][G][ [mA][mA][mU][mC][mA][A] [mU][C][U][mU][G][A][U [mG][mA] [mA][mU][mU] 1415 siRNA254- [mG][mC][mU][mA][mU][m 646 1471 [mU][U][G][mC][U][U][G 1030 1858 M0 C][mC][mA][C][G][A][A][A] ][A][U][U][mU][mC][G][ [mU][mC][mA][mA][mG][C] U][G][mG][A][mU][A][G] [mA][mA] [mC][mU][mU] 1416 siRNA255- [mC][mU][mA][mU][mC][m 647 1472 [mU][U][U][mG][C][U][U 1031 1859 M0 C][mA][mC][G][A][A][A][U] ][G][A][U][mU][mU][C][ [mC][mA][mA][mG][mC][A] G][U][mG][G][mA][U][A] [mA][mA] [mG][mU][mU] 1417 siRNA256- [mU][mA][mU][mC][mC][m 648 1473 [mU][A][U][mU][G][C][U 1032 1860 M0 A][mC][mG][A][A][A][U][C] ][U][G][A][mU][mU][U][ [mA][mA][mG][mC][mA][A] C][G][mU][G][mG][A][U] [mU][mA] [mA][mU][mU] 1424 siRNA257- [mG][mA][mA][mA][mU][m 649 1475 [mU][A][U][mC][A][A][A 1033 1861 M0 C][mA][mA][G][C][A][A][U] ][A][A][U][mU][mG][C][ [mU][mU][mU][mU][mG][A] U][U][mG][A][mU][U][U] [mU][mA] [mC][mU][mU] 1425 siRNA258- [mA][mA][mA][mU][mC][m 650 1476 [mU][A][A][mU][C][A][A 1034 1862 M0 A][mA][mG][C][A][A][U][U] ][A][A][A][mU][mU][G][ [mU][mU][mU][mG][mA][U] C][U][mU][G][mA][U][U] [mU][mA] [mU][mU][mU] 1431 siRNA259- [mA][mG][mC][mA][mA][m 651 1477 [mU][C][A][mU][C][A][G 1035 1863 M0 U][mU][mU][U][U][G][A][U ][A][A][U][mC][mA][A][ ][mU][mC][mU][mG][mA][U A][A][mA][U][mU][G][C] ][mG][mA] [mU][mU][mU] 1433 siRNA260- [mC][mA][mA][mU][mU][m 652 1478 [mU][A][G][mC][A][U][C 1036 1864 M0 U][mU][mU][G][A][U][U][C] ][A][G][A][mA][mU][C][ [mU][mG][mA][mU][mG][C] A][A][mA][A][mA][U][U] [mU][mA] [mG][mU][mU] 1506 siRNA261- [mA][mC][mC][mA][mG][m 653 1480 [mU][C][A][mA][G][C][C 1037 1865 M0 U][mC][mC][C][G][G][G][C] ][U][G][C][mC][mC][G][ [mA][mG][mG][mC][mU][U] G][G][mA][C][mU][G][G] [mG][mA] [mU][mU][mU] 1507 siRNA262- [mC][mC][mA][mG][mU][m 654 1481 [mU][C][C][mA][A][G][C 1038 1866 M0 C][mC][mC][G][G][G][C][A] ][C][U][G][mC][mC][C][ [mG][mG][mC][mU][mU][G] G][G][mG][A][mC][U][G] [mG][mA] [mG][mU][mU] 1510 siRNA263- [mG][mU][mC][mC][mC][m 655 1482 [mU][U][G][mC][C][C][A 1039 1867 M0 G][mG][mG][C][A][G][G][C] ][A][G][C][mC][mU][G][ [mU][mU][mG][mG][mG][C] C][C][mC][G][mG][G][A] [mA][mA] [mC][mU][mU] 1511 siRNA264- [mU][mC][mC][mC][mG][m 656 1483 [mU][A][U][mG][C][C][C 1040 1868 M0 G][mG][mC][A][G][G][C][U] ][A][A][G][mC][mC][U][ [mU][mG][mG][mG][mC][A] C][C][mC][C][mG][G][G] [mU][mA] [mA][mU][mU] 1513 siRNA265- [mC][mC][mG][mG][mG][m 657 1484 [mU][A][A][mA][U][G][C 1041 1869 M0 C][mA][mG][G][C][U][U][G] ][C][C][A][mA][mG][C][ [mG][mG][mC][mA][mU][U] C][U][mG][C][mC][C][G] [mU][mA] [mG][mU][mU] 1518 siRNA266- [mC][mA][mG][mG][mC][m 658 1485 [mU][C][G][mG][C][A][A 1042 1870 M0 U][mU][mG][G][G][C][A][U] ][A][A][U][mG][mC][C][ [mU][mU][mU][mG][mC][C] C][A][mA][G][mC][C][U] [mG][mA] [mG][mU][mU] 1555 siRNA267- [mA][mU][mU][mU][mA][m 659 1486 [mU][G][C][mU][U][G][A 1043 1871 M0 A][mA][mU][A][U][A][A][A ][A][U][U][mU][mA][U][ ][mU][mU][mC][mA][mA][G A][U][mU][U][mA][A][A] ][mC][mA] [mU][mU][mU] 1556 siRNA268- [mU][mU][mU][mA][mA][m 660 1487 [mU][U][G][mC][U][U][G 1044 1872 M0 A][mU][mA][U][A][A][A][U ][A][A][U][mU][mU][A][ ][mU][mC][mA][mA][mG][C U][A][mU][U][mU][A][A] ][mA][mA] [mA][mU][mU] 1557 siRNA269- [mU][mU][mA][mA][mA][m 661 1488 [mU][A][U][mG][C][U][U 1045 1873 M0 U][mA][mU][A][A][A][U][U ][A][A][A][mU][mU][U][ ][mC][mA][mA][mG][mC][A A][U][mA][U][mU][U][A] ][mU][mA] [mA][mU][mU] 1558 siRNA270- [mU][mA][mA][mA][mU][m 662 1489 [mU][A][A][mU][G][C][U 1046 1874 M0 A][mU][mA][A][A][U][U][C] ][U][G][A][mA][mU][U][ [mA][mA][mG][mC][mA][U] U][A][mU][A][mU][U][U] [mU][mA] [mA][mU][mU] 1559 siRNA271- [mA][mA][mA][mU][mA][m 663 1490 [mU][A][A][mA][U][G][C 1047 1875 M0 U][mA][mA][A][U][U][C][A] ][U][U][G][mA][mA][U][ [mA][mG][mC][mA][mU][U] U][U][mA][U][mA][U][U] [mU][mA] [mU][mU][mU] 1561 siRNA272- [mA][mU][mA][mU][mA][m 664 1491 [mU][A][C][mA][A][A][U 1048 1876 M0 A][mA][mU][U][C][A][A][G] ][G][C][U][mU][mG][A][ [mC][mA][mU][mU][mU][G] A][U][mU][U][mA][U][A] [mU][mA] [mU][mU][mU] 1593 siRNA273- [mG][mG][mA][mA][mU][m 665 1493 [mU][A][U][mC][U][A][G 1049 1877 M0 A][mA][mG][A][G][G][A][C] ][U][G][U][mC][mC][U][ [mA][mC][mU][mA][mG][A] C][U][mU][A][mU][U][C] [mU][mA] [mC][mU][mU] 1594 siRNA274- [mG][mA][mA][mU][mA][m 666 1494 [mU][C][A][mU][C][U][A 1050 1878 M0 A][mG][mA][G][G][A][C][A] ][G][U][G][mU][mC][C][ [mC][mU][mA][mG][mA][U] U][C][mU][U][mA][U][U] [mG][mA] [mC][mU][mU] 1597 siRNA275- [mU][mA][mA][mG][mA][m 667 1495 [mU][U][A][mU][C][A][U 1051 1879 M0 G][mG][mA][C][A][C][U][A] ][C][U][A][mG][mU][G][ [mG][mA][mU][mG][mA][U] U][C][mC][U][mC][U][U] [mA][mA] [mA][mU][mU] 1599 siRNA276- [mA][mG][mA][mG][mG][m 668 1496 [mU][A][A][mU][A][U][C 1052 1880 M0 A][mC][mA][C][U][A][G][A] ][A][U][C][mU][mA][G][ [mU][mG][mA][mU][mA][U] U][G][mU][C][mC][U][C] [mU][mA] [mU][mU][mU] 1601 siRNA277- [mA][mG][mG][mA][mC][m 669 1497 [mU][G][U][mA][A][U][A 1053 1881 M0 A][mC][mU][A][G][A][U][G] ][U][C][A][mU][mC][U][ [mA][mU][mA][mU][mU][A] U][G][mU][C][mC][U][C] [mC][mA] [mU][mU][mU] 1605 siRNA278- [mC][mA][mC][mU][mA][m 670 1498 [mU][U][C][mC][U][G][U 1054 1882 M0 G][mA][mU][G][A][U][A][U ][A][A][U][mA][mU][C][ ][mU][mA][mC][mA][mG][G A][U][mC][U][mA][G][U] ][mA][mA] [mG][mU][mU] 1606 siRNA279- [mA][mC][mU][mA][mG][m 671 1499 [mU][G][U][mC][C][U][G 1055 1883 M0 A][mU][mG][A][U][A][U][U ][U][A][A][mU][mA][U][ ][mA][mC][mA][mG][mG][A C][A][mU][C][mU][A][G] ][mC][mA] [mU][mU][mU] 1609 siRNA280- [mA][mG][mA][mU][mG][m 672 1500 [mU][G][C][mA][G][U][C 1056 1884 M0 A][mU][mA][U][U][A][C][A] ][C][U][G][mU][mA][A][ [mG][mG][mA][mC][mU][G] U][A][mU][C][mA][U][C] [mC][mA] [mU][mU][mU] 1610 siRNA281- [mG][mA][mU][mG][mA][m 673 1501 [mU][U][G][mC][A][G][U 1057 1885 M0 U][mA][mU][U][A][C][A][G] ][C][C][U][mG][mU][A][ [mG][mA][mC][mU][mG][C] A][U][mA][U][mC][A][U] [mA][mA] [mC][mU][mU] 1614 siRNA282- [mA][mU][mA][mU][mU][m 674 1502 [mU][G][U][mU][C][U][G 1058 1886 M0 A][mC][mA][G][G][A][C][U] ][C][A][G][mU][mC][C][ [mG][mC][mA][mG][mA][A] U][G][mU][A][mA][U][A] [mC][mA] [mU][mU][mU] 1638 siRNA283- [mU][mC][mA][mC][mC][m 675 1503 [mU][C][C][mA][A][A][G 1059 1887 M0 A][mC][mA][C][A][G][U][C] ][G][G][A][mC][mU][G][ [mC][mC][mU][mU][mU][G] U][G][mU][G][mG][U][G] [mG][mA] [mA][mU][mU] 1660 siRNA284- [mA][mA][mA][mU][mG][m 676 1504 [mU][U][A][mC][C][A][C 1060 1888 M0 C][mA][mU][U][U][A][G][U] ][C][A][C][mU][mA][A][ [mG][mG][mU][mG][mG][U] A][U][mG][C][mA][U][U] [mA][mA] [mU][mU][mU] 1661 siRNA285- [mA][mA][mU][mG][mC][m 677 1505 [mU][C][U][mA][C][C][A 1061 1889 M0 A][mU][mU][U][A][G][U][G ][C][C][A][mC][mU][A][ ][mG][mU][mG][mG][mU][A A][A][mU][G][mC][A][U] ][mG][mA] [mU][mU][mU] 1662 siRNA286- [mA][mU][mG][mC][mA][m 678 1506 [mU][U][C][mU][A][C][C 1062 1890 M0 U][mU][mU][A][G][U][G][G ][A][C][C][mA][mC][U][ ][mU][mG][mG][mU][mA][G A][A][mA][U][mG][C][A] [mA][mA] [mU][mU][mU] 1663 siRNA287- [mU][mG][mC][mA][mU][m 679 1507 [mU][U][U][mC][U][A][C 1063 1891 M0 U][mU][mA][G][U][G][G][U ][C][A][C][mC][mA][C][ ][mG][mG][mU][mA][mG][A U][A][mA][A][mU][G][C] ][mA][mA] [mA][mU][mU] 1669 siRNA288- [mU][mA][mG][mU][mG][m 680 1508 [mU][A][A][mU][C][A][U 1064 1892 M0 G][mU][mG][G][U][A][G][A ][U][U][C][mU][mA][C][ ][mA][mA][mU][mG][mA][U C][A][mC][C][mA][C][U] ][mU][mA] [mA][mU][mU] 1671 siRNA289- [mG][mU][mG][mG][mU][m 681 1509 [mU][U][G][mA][A][U][C 1065 1893 M0 G][mG][mU][A][G][A][A][A ][A][U][U][mU][mC][U][ ][mU][mG][mA][mU][mU][C A][C][mC][A][mC][C][A] ][mA][mA] [mC][mU][mU] 1673 siRNA290- [mG][mG][mU][mG][mG][m 682 1510 [mU][G][G][mU][G][A][A 1066 1894 M0 U][mA][mG][A][A][A][U][G ][U][C][A][mU][mU][U][ ][mA][mU][mU][mC][mA][C C][U][mA][C][mC][A][C] ][mC][mA] [mC][mU][mU] 1676 siRNA291- [mG][mG][mU][mA][mG][m 683 1511 [mU][C][C][mU][G][G][U 1067 1895 M0 A][mA][mA][U][G][A][U][U ][G][A][A][mU][mC][A][ ][mC][mA][mC][mC][mA][G U][U][mU][C][mU][A][C] ][mG][mA] [mC][mU][mU] 1683 siRNA292- [mA][mU][mG][mA][mU][m 684 1512 [mU][C][A][mU][U][G][G 1068 1896 M0 U][mC][mA][C][C][A][G][G] ][A][C][C][mU][mG][G][ [mU][mC][mC][mA][mA][U] U][G][mA][A][mU][C][A] [mG][mA] [mU][mU][mU] 1686 siRNA293- [mA][mU][mU][mC][mA][m 685 1513 [mU][C][A][mA][C][A][U 1069 1897 M0 C][mC][mA][G][G][U][C][C] ][U][G][G][mA][mC][C][ [mA][mA][mU][mG][mU][U] U][G][mG][U][mG][A][A] [mG][mA] [mU][mU][mU] 1689 siRNA294- [mC][mA][mC][mC][mA][m 686 1514 [mU][G][A][mA][C][A][A 1070 1898 M0 G][mG][mU][C][C][A][A][U] ][C][A][U][mU][mG][G][ [mG][mU][mU][mG][mU][U] [mU][G][A][mA][C][A][A [mC][mA] [mG][mU][mU] 1690 siRNA295- [mA][mC][mC][mA][mG][m 687 1515 [mU][U][G][mA][A][C][A 1071 1899 M0 G][mU][mC][C][A][A][U][G] ][A][C][A][mU][mU][G][ [mU][mU][mG][mU][mU][C] G][A][mC][C][mU][G][G] [mA][mA] [mU][mU][mU] 1692 siRNA296- [mC][mA][mG][mG][mU][m 688 1516 [mU][G][G][mU][G][A][A 1072 1900 M0 C][mC][mA][A][U][G][U][U] ][C][A][A][mC][mA][U][ [mG][mU][mU][mC][mA][C] U][G][mG][A][mC][C][U] [mC][mA] [mG][mU][mU] 1712 siRNA297- [mA][mG][mU][mG][mC][m 689 1517 [mU][A][A][mG][A][U][U 1073 1901 M0 U][mU][mG][C][U][U][G][U] ][C][A][C][mA][mA][G][ [mG][mA][mA][mU][mC][U] C][A][mA][G][mC][A][C] [mU][mA] [mU][mU][mU] 1713 siRNA298- [mG][mU][mG][mC][mU][m 690 1518 [mU][U][A][mA][G][A][U 1074 1902 M0 U][mG][mC][U][U][G][U][G] ][U][C][A][mC][mA][A][ [mA][mA][mU][C][mU][U] G][C][mA][A][mG][C][A] [mA][mA] [mC][mU][mU] 1769 siRNA299- [mU][mC][mU][mG][mC][m 691 1519 [mU][U][A][mG][U][U][C 1075 1903 M0 U][mA][mG][U][G][A][A][A ][U][U][U][mU][mC][A][ ][mA][mG][mA][mA][mC][U C][U][mA][G][mC][A][G] ][mA][mA] [mA][mU][mU] 1770 siRNA300- [mC][mU][mG][mC][mU][m 692 1520 [mU][C][U][mA][G][U][U 1076 1904 M0 A][mG][mU][G][A][A][A][A ][C][U][U][mU][mU][C][ ][mG][mA][mA][mC][mU][A A][C][mU][A][mG][C][A] ][mG][mA] [mG][mU][mU] 1824 siRNA301- [mU][mA][mA][mG][mU][m 693 1521 [mU][U][A][mU][G][A][A 1077 1905 M0 C][mC][mA][U][G][A][A][U] ][C][A][U][mU][mC][A][ [mG][mU][mU][mC][mA][U] U][G][mG][A][mC][U][U] [mA][mA] [mA][mU][mU 1825 siRNA302- [mA][mA][mG][mU][mC][m 694 1522 [mU][A][U][mA][U][G][A 1078 1906 M0 C][mA][mU][G][A][A][U][G] ][A][C][A][mU][mU][C][ [mU][mU][mC][mA][mU][A] A][U][mG][G][mA][C][U] [mU][mA] [mU][mU][mU] 1826 siRNA303- [mA][mG][mU][mC][mC][m 695 1523 [mU][U][A][mU][A][U][G 1079 1907 M0 A][mU][mG][A][A][U][G][U ][A][A][C][mA][mU][U][ ][mU][mC][mA][mU][mA][U C][A][mU][G][mG][A][C] ][mA][mA] [mU][mU][mU] 1827 siRNA304- [mG][mU][mC][mC][mA][m 696 1524 [mU][C][U][mA][U][A][U 1080 1908 M0 U][mG][mA][A][U][G][U][U ][G][A][A][mC][mA][U][ ][mC][mA][mU][mA][mU][A U][C][mA][U][mG][G][A] ][mG][mA] [mC][mU][mU] 1828 siRNA305- [mU][mC][mC][mA][mU][m 697 1525 [mU][G][C][mU][A][U][A 1081 1909 M0 G][mA][mA][U][G][U][U][C] ][U][G][A][mA][mC][A][ [mA][mU][mA][mU][mA][G] U][U][mC][A][mU][G][G] [mC][mA] [mA][mU][mU] 1829 siRNA306- [mC][mC][mA][mU][mG][m 698 1526 [mU][G][G][mC][U][A][U 1082 1910 M0 A][mA][mU][G][U][U][C][A] ][A][U][G][mA][mA][C][ [m][mA][mU][mA][mG][C] A][U][mU][C][mA][U][G] [mC][mA] [mG][mU][mU] 1830 siRNA307- [mC][mA][mU][mG][mA][m 699 1527 [mU][U][G][mG][C][U][A 1083 1911 M0 A][mU][mG][U][U][C][A][U] ][U][A][U][mG][mA][A][ [mA][mU][mA][mG][mC][C] C][A][mU][U][mC][A][U] [mA][mA] [mG][mU][mU] 1895 siRNA308- [mU][mU][mU][mU][mU][m 700 1528 [mU][U][A][mU][C][U][U 1084 1912 M0 U][mU][mC][A][A][A][A][U] ][C][A][U][mU][mU][U][ [mG][mA][mA][mG][mA][U] G][A][mA][A][mA][A][A] [mA][mA] [mA][mU][mU] 2052 siRNA309- [mU][mG][mU][mA][mU][m 701 1529 [mU][A][A][mU][C][U][G 1085 1913 M0 U][mC][mU][A][A][U][U][G] ][C][C][A][mA][mU][U][ [mG][mC][mA][mG][mA][U] A][G][mA][A][mU][A][C] [mU][mA] [mA][mU][mU] 2053 siRNA310- [mG][mU][mA][mU][mU][m 702 1530 [mU][C][A][mA][U][C][U 1086 1914 M0 C][mU][mA][A][U][U][G][G] ][G][C][C][mA][mA][U][ [mC][mA][mG][mA][mU][U] U][A][mG][A][mA][U][A] [mG][mA] [mC][mU][mU] 2075 siRNA311- [mU][mU][mU][mU][mC][m 703 1531 [mU][A][A][mG][C][A][G 1087 1915 M0 C][mU][mA][A][G][G][A][A] ][U][U][U][mC][mC][U][ [mA][mC][mU][mG][mC][U] U][A][mG][G][mA][A][A] [mU][mA] [mA][mU][mU] 2127 siRNA312- [mA][mA][mA][mU][mG][m 704 1532 [mU][G][A][mA][C][G][U 1088 1916 M0 U][mU][mC][A][A][A][U][U] ][G][A][A][mU][mU][U][ [mC][mA][mC][mG][mU][U] G][A][mA][C][mA][U][U] [mC][mA] [mU][mU][mU] 2128 siRNA313- [mA][mA][mU][mG][mU][m 705 1533 [mU][A][G][mA][A][C][G 1089 1917 M0 U][mC][mA][A][A][U][U][C] ][U][G][A][mA][mU][U][ [mA][mC][mG][mU][mU][C] U][G][mA][A][mC][A][U] [mU][mA] [mU][mU][mU] 2132 siRNA314- [mU][mU][mC][mA][mA][m 706 1534 [mU][C][A][mC][U][A][G 1090 1918 M0 A][mU][mU][C][A][C][G][U] ][A][A][C][mG][mU][G][ [mU][mC][mU][mA][mG][U] A][A][mU][U][mU][G][A] [mG][mA] [mA][mU][mU] 2133 siRNA315- [m][mC][mA][mA][mA][m 707 1535 [mU][U][C][mA][C][U][A 1091 1919 M0 U][mU][mC][A][C][G][U][U] ][G][A][A][mC][mG][U][ [mC][mU][mA][mG][mU][G] G][A][mA][U][mU][U][G] [mA][mA] [mA][mU][mU] 2134 siRNA316- [mC][mA][mA][mA][mU][m 708 1536 [mU][U][U][mC][A][C][U 1092 1920 M0 U][mC][mA][C][G][U][U][C] ][A][G][A][mA][mC][G][ [mU][mA][mG][mU][mG][A] U][G][mA][A][mU][U][U] [mA][mA] [mG][mU][mU] 2135 siRNA317- [mA][mA][mA][mU][mU][m 709 1537 [mU][U][U][mU][C][A][C 1093 1921 M0 C][mA][mC][G][U][U][C][U] ][U][A][G][mA][mA][C][ [mA][mG][mU][mG][mA][A] G][U][mG][A][mA][U][U] [mA][mA] [mU][mU][mU] 2136 siRNA318- [mA][mA][mU][mU][mC][m 710 1538 [mU][G][U][mU][U][C][A 1094 1922 M0 A][mC][mG][U][U][C][U][A] ][C][U][A][mG][mA][A][ [mG][mU][mG][mA][mA][A] C][G][mU][G][mA][A][U] [mC][mA] [mU][mU][mU] 2137 siRNA319- [mA][mU][mU][mC][mA][m 711 1539 [mU][A][G][mU][U][U][C 1095 1923 M0 C][mG][mU][U][C][U][A][G] ][A][C][U][mA][mG][A][ [m][mG][mA][mA][mA][C] A][C][mG][U][mG][A][A] [mU][mA] [mU][mU][mU] 2139 siRNA320- [m][mC][mA][mC][mG][m 712 1540 [mU][G][C][mA][G][U][U 1096 1924 M0 U][mU][mC][U][A][G][U][G] ][U][C][A][mC][mU][A][ [mA][mA][mA][mC][mU][G] G][A][mA][C][mG][U][G] [mC][mA] [mA][mU][mU] 2140 siRNA321- [mC][mA][mC][mG][mU][m 713 1541 [mU][U][G][mC][A][G][U 1097 1925 M0 U][mC][mU][A][G][U][G][A] ][U][U][C][mA][mC][U][ [mA][mA][mC][mU][mG][C] A][G][mA][A][mC][G][U] [mA][mA] [mG][mU][mU] 2144 siRNA322- [mU][mU][mC][mU][mA][m 714 1542 [mU][A][U][mA][A][U][G 1098 1926 M0 G][mU][mG][A][A][A][C][U] ][C][A][G][mU][mU][U][ [mG][mC][mA][mU][mU][A] C][A][mC][U][mA][G][A] [mU][mA] [mA][mU][mU] 2189 siRNA323- [mC][mG][mG][mG][mU][m 715 1543 [mU][A][U][mG][A][U][A 1099 1927 M0 G][mU][mG][A][U][C][A][U] ][U][A][U][mG][mA][U][ [mA][mU][mA][mU][mC][A] C][A][mC][A][mC][C][C] [mU][mA] [mG][mU][mU] 2190 siRNA324- [mG][mG][mG][mU][mG][m 716 1544 [mU][U][A][mU][G][A][U 1100 1928 M0 U][mG][mA][U][C][A][U][A] ][A][U][A][mU][mG][A][ [mU][mA][mU][mC][mA][U] U][C][mA][C][mA][C][C] [mA][mA] [mC][mU][mU] 2191 siRNA325- [mG][mG][mU][mG][mU][m 717 1545 [mU][U][U][mA][U][G][A 1101 1929 M0 G][mA][mU][C][A][U][A][U] ][U][A][U][mA][mU][G][ [mA][mU][mC][mA][mU][A] A][U][mC][A][mC][A][C] [mA][mA] [mC][mU][mU] 2192 siRNA326- [mG][mU][mG][mU][mG][m 718 1546 [mU][U][U][mU][A][U][G 1102 1930 M0 A][mU][mC][A][U][A][U][A] ][A][U][A][mU][mA][U][ [mU][mC][mA][mU][mA][A] G][A][mU][C][mA][C][A] [mA][mA] [mC][mU][mU] 2193 siRNA327- [mU][mG][mU][mG][mA][m 719 1547 [mU][C][U][mU][U][A][U 1103 1931 M0 U][mC][mA][U][A][U][A][U] ][G][A][U][mA][mU][A][ [mC][mA][mU][mA][mA][A] U][G][mA][U][mC][A][C] [mG][mA] [mA][mU][mU] 2194 siRNA328- [mG][mU][mG][mA][mU][m 720 1548 [mU][C][C][mU][U][U][A 1104 1932 M0 C][mA][mU][A][U][A][U][C] ][U][G][A][mU][mA][U][ [mA][mU][mA][mA][mA][G] A][U][mG][A][mU][C][A] [mG][mA] [mC][mU][mU] 2195 siRNA329- [mU][mG][mA][mU][mC][m 721 1549 [mU][U][C][mC][U][U][U 1105 1933 M0 A][mU][mA][U][A][U][C][A] ][A][U][G][mA][mU][A][ [mU][mA][mA][mA][mG][G] U][A][mU][G][mA][U][C] [mA][mA] [mA][mU][mU] 2199 siRNA330- [mC][mA][mU][mA][mU][m 722 1550 [mU][A][A][mU][A][U][C 1106 1934 M0 A][mU][mC][A][U][A][A][A] ][C][U][U][mU][mA][U][ [mG][mG][mA][mU][mA][U] G][A][mU][A][mU][A][U] [mU][mA] [mG][mU][mU] 2209 siRNA331- [mA][mA][mA][mG][mG][m 723 1551 [mU][A][A][mU][C][A][U 1107 1935 M0 A][mU][mA][U][U][U][C][A] ][U][U][G][mA][mA][A][ [mA][mA][mU][mG][mA][U] U][A][mU][C][mC][U][U] [mU][mA] [mU][mU][mU] 2210 siRNA332- [mA][mA][mG][mG][mA][m 724 1552 [mU][U][A][mA][U][C][A 1108 1936 M0 U][mA][mU][U][U][C][A][A] ][U][U][U][mG][mA][A][ [mA][mU][mG][mA][mU][U] A][U][mA][U][mC][C][U] [mA][mA] [mU][mU][mU] 2211 siRNA333- [mA][mG][mG][mA][mU][m 725 1553 [mU][A][U][mA][A][U][C 1109 1937 M0 A][mU][mU][U][C][A][A][A] ][A][U][U][mU][mG][A][ [mU][mG][mA][mU][mU][A] A][A][mU][A][mU][C][C] [mU][mA] [mU][mU][mU] 2215 siRNA334- [mU][mA][mU][mU][mU][m 726 1554 [mU][A][A][mU][C][A][U 1110 1938 M0 C][mA][mA][A][U][G][A][U] ][A][A][U][mC][mA][U][ [mU][mA][mU][mG][mA][U] U][U][mG][A][mA][A][U] [mU][mA] [mA][mU][mU] 2216 siRNA335- [mA][mU][mU][mU][mC][m 727 1555 [mU][U][A][mA][U][C][A 1111 1939 M0 A][mA][mA][U][G][A][U][U ][U][A][A][mU][mC][A][ [mA][mU][mG][mA][mU][U U][U][mU][G][mA][A][A] ][mA][mA] [mU][mU][mU] 2217 siRNA336- [mU][mU][mU][mC][mA][m 728 1556 [mU][C][U][mA][A][U][C 1112 1940 M0 A][mA][mU][G][A][U][U][A ][A][U][A][mA][mU][C][ ][mU][mG][mA][mU][mU][A A][U][mU][U][mG][A][A] ][mG][mA] [mA][mU][mU] 2218 siRNA337- [mU][mU][mC][mA][mA][m 729 1557 [mU][A][C][mU][A][A][U 1113 1941 M0 A][mU][mG][A][U][U][A][U ][C][A][U][mA][mA][U][ ][mG][mA][mU][mU][mA][G C][A][mU][U][mU][G][A] ][mU][mA] [mA][mU][mU] 2220 siRNA338- [mC][mA][mA][mA][mU][m 730 1558 [mU][U][A][mA][C][U][A 1114 1942 M0 G][mA][mU][U][A][U][G][A ][A][U][C][mA][mU][A][ ][mU][mU][mA][mG][mU][U ][A][U][C][mA][mU][A][ ][mA][mA] [mG][mU][mU] 2221 siRNA339- [mA][mA][mA][mU][mG][m 731 1559 [mU][A][U][mA][A][C][U 1115 1943 M0 A][mU][mU][A][U][G][A][U ][A][A][U][mC][mA][U][ ][mU][mA][mG][mU][mU][A A][A][mU][C][mA][U][U] ][mU][mA] [mU][mU][mU] 2223 siRNA340- [mA][mU][mG][mA][mU][m 732 1560 [mU][A][C][mA][U][A][A 1116 1944 M0 U][mA][mU][G][A][U][U][A ][C][U][A][mA][mU][C][ ][mG][mU][mU][mA][mU][G A][U][mA][A][mU][C][A] ][mU][mA] [mU][mU][mU] 2224 siRNA341- [mU][mG][mA][mU][mU][m 733 1561 [mU][G][A][mC][A][U][A 1117 1945 M0 A][mU][mG][A][U][U][A][G ][A][C][U][mA][mA][U][ ][mU][mU][mA][mU][mG][U C][A][mU][A][mA][U][C] ][mC][mA] [mA][mU][mU] 2225 siRNA342- [mG][mA][mU][mU][mA][m 734 1562 [mU][A][G][mA][C][A][U 1118 1946 M0 U][mG][mA][U][U][A][G][U ][A][A][C][mU][mA][A][ ][mU][mA][mU][mG][mU][C U][C][mA][U][mA][A][U] ][mU][mA] [mC][mU][mU] 2313 siRNA343- [mU][mU][mG][mA][mU][m 735 1563 [mU][U][A][mG][U][G][U 1119 1947 M0 U][mU][mC][C][C][A][A][A] ][U][U][U][mU][mG][G][ [mA][mA][mC][mA][mC][U] G][A][mA][A][mU][C][A] [mA][mA] [mA][mU][mU] 2316 siRNA344- [mA][mU][mU][mU][mC][m 736 1564 [mU][C][U][mU][U][A][G 1120 1948 M0 C][mC][mA][A][A][A][A][C] ][U][G][U][mU][mU][U][ [mA][mC][mU][mA][mA][A] U][G][mG][G][mA][A][A] [mG][mA] [mU][mU][mU] 2317 siRNA345- [mU][mU][mU][mC][mC][m 737 1565 [mU][C][C][mU][U][U][A 1121 1949 M0 C][mA][mA][A][A][A][C][A] ][G][U][G][mU][mU][U][ [mC][mU][mA][mA][mA][G] U][U][mG][G][mG][A][A] [mG][mA] [mA][mU][mU] 2319 siRNA346- [mU][mC][mC][mC][mA][m 738 1566 [mU][C][A][mC][C][U][U 1122 1950 M0 A][mA][mA][A][C][A][C][U] ][U][A][G][mU][mG][U][ [mA][mA][mA][mG][mG][U] U][U][mU][U][mG][G][G] [mG][mA] [mA][mU][mU] 2320 siRNA347- [mC][mC][mC][mA][mA][m 739 1567 [mU][C][C][mA][C][C][U 1123 1951 M0 A][mA][mA][C][A][C][U][A] ][U][U][A][mG][mU][G][ [mA][mA][mG][mG][mU][G] U][U][mU][U][mU][G][G] [mG][mA] [mG][mU][mU] 2321 siRNA348- [mC][mC][mA][mA][mA][m 740 1568 [mU][A][C][C][A][C][C 1124 1952 M0 A][mA][mC][A][C][U][A][A] ][U][U][U][mA][mG][U][ [mA][mG][mG][mU][mG][G] G][U][mU][U][mU][U][G] [mU][mA [mG][mU][mU] 2353 siRNA349- [mU][mC][mA][mU][mG][m 741 1569 [mU][A][A][mC][A][A][U 1125 1953 M0 U][mU][mU][U][A][A][C][U] ][A][A][G][mU][mU][A][ [mU][mA][mU][mU][mG][U] A][A][mA][C][mA][U][G] [mU][mA] [mA][mU][mU] 2355 siRNA350- [mA][mU][mG][mU][mU][m 742 1570 [mU][G][C][mA][A][C][A 1126 1954 M0 U][mU][mA][A][C][U][U][A] ][A][U][A][mA][mG][U][ [mU][mU][mG][mU][mU][G] U][A][mA][A][mA][C][A] [mC][mA] [mU][mU][mU] 2356 siRNA351- [mU][mG][mU][mU][mU][m 743 1571 [mU][A][G][mC][A][A][C 1127 1955 M0 U][mA][mA][C][U][U][A][U] ][A][A][U][mA][mA][G][ [mU][mG][mU][mU][mG][C] U][U][mA][A][mA][A][C] [mU][mA] [mA][mU][mU] 2362 siRNA352- [mA][mA][mC][mU][mU][m 744 1572 [mU][G][U][mU][U][U][C 1128 1956 M0 A][mU][mU][G][U][U][G][C] ][A][G][C][mA][mA][C][ [mU][mG][mA][mA][mA][A] A][A][mU][A][mA][G][U] [mC][mA] [mU][mU][mU] 2364 siRNA353- [mC][mU][mU][mA][mU][m 745 1573 [mU][G][A][mG][U][U][U 1129 1957 M0 U][mG][mU][U][G][C][U][G] ][U][C][A][mG][mC][A][ [mA][mA][mA][mA][mC][U] A][C][mA][A][mU][A][A] [mC][mA] [mG][mU][mU] 2365 siRNA354- [mU][mU][mA][mU][mU][m 746 1574 [mU][A][G][mA][G][U][U 1130 1958 M0 G][mU][mU][G][C][U][G][A] ][U][U][C][mA][mG][C][ [mA][mA][mA][mC][mU][C] A][A][mC][A][mA][U][A] [mU][mA] [mA][mU][mU] 2366 siRNA355- [mU][mA][mU][mU][mG][m 747 1575 [mU][U][A][mG][A][G][U 1131 1959 M0 U][mU][mG][C][U][G][A][A] ][U][U][U][mC][mA][G][ [mA][mA][mC][mU][mC][U] C][A][mA][C][mA][A][U] [mA][mA] [mA][mU][mU] 2370 siRNA356- [mG][mU][mU][mG][mC][m 748 1576 [mU][G][A][mC][A][U][A 1132 1960 M0 U][mG][mA][A][A][A][C][U] ][G][A][G][mU][mU][U][ [mC][mU][mA][mU][mG][U] U][C][mA][G][mC][A][A] [mC][mA] [mC][mU][mU] 2503 siRNA357- [mA][mA][mA][mA][mA][m 749 1577 [mU][C][A][mC][A][U][A 1133 1961 M0 U][mG][mU][A][G][C][U][U] ][A][A][A][mG][mC][U][ [mU][mU][mA][mU][mG][U] A][C][mA][U][mU][U][U] [mG][mA] [mU][mU][mU] 2562 siRNA358- [mG][mG][mA][mA][mG][m 750 1578 [mU][G][U][mU][U][C][A 1134 1962 M0 C][mU][mU][U][G][G][U][U] ][U][A][A][mC][mC][A][ [mA][mU][mG][mA][mA][A] A][A][mAG][C][mU][U][C] [mC][mA] [mC][mU][mU] 2563 siRNA359- [mG][mA][mA][mG][mC][m 751 1579 [mU][U][G][mU][U][U][C 1135 1963 M0 U][mU][mU][G][G][U][U][A ][A][U][A][mA][mC][C][ ][mU][mG][mA][mA][mA][C A][A][mA][G][mC][U][U] ][mA][mA] [mC][mU][mU] 2620 siRNA360- [mA][mU][mU][mU][mA][m 752 1580 [mU][G][C][mG][A][U][A 1136 1964 M0 A][mA][mU][G][C][U][U][U] ][A][A][A][mA][mG][C][ [mU][mU][mA][mU][mC][G] A][U][mU][U][mA][A][A] [mC][mA] [mU][mU][mU] 2621 siRNA361- [mU][mU][mU][mA][mA][m 753 1581 [mU][A][G][mC][G][A][U 1137 1965 M0 A][mU][mG][C][U][U][U][U] ][A][A][A][mA][mA][G][ [mU][mA][mU][mC][mG][C] C][A][mU][U][mU][A][A] [mU][mA] [mA][mU][mU] 2622 siRNA362- [mU][mU][mA][mA][mA][m 754 1582 [mU][U][A][mG][C][G][A 1138 1966 M0 U][mG][mC][U][U][U][U][U] ][U][A][A][mA][mA][A][ [mA][mU][mC][mG][mC][U] G][C][mA][U][mU][U][A] [mA][mA] [mA][mU][mU] 2623 siRNA363- [mU][mA][mA][mA][mU][m 755 1583 [mU][U][U][mA][G][C][G 1139 1967 M0 G][mC][mU][U][U][U][U][A] ][A][A][A][mA][mA][A][ [mU][mC][mG][mC][mU][A] A][G][mC][A][mU][U][U] [mA][mA] [mA][mU][mU] 2624 siRNA364- [mA][mA][mA][mU][mG][m 756 1584 [mU][U][U][mU][A][G][C 1140 1968 M0 C][mU][mU][U][U][U][A][U] ][G][A][U][mA][mA][A][ [mC][mG][mC][mU][mA][A] A][A][mG][C][mA][U][U] [mA][mA] [mU][mU][mU] 2625 siRNA365- [mA][mA][mU][mG][mC][m 757 1585 [mU][A][U][mU][U][A][G 1141 1969 M0 U][mU][mU][U][U][A][U][C] ][C][G][A][mU][mA][A][ [mG][mC][mU][mA][mA][A] A][A][mA][G][mC][A][U] [mU][mA] [mU][mU][mU] 2629 siRNA366- [mC][mU][mU][mU][mU][m 758 1586 [mU][A][G][mU][C][A][U 1142 1970 M0 U][mA][mU][C][G][C][U][A] ][U][U][A][mG][mC][G][ [mA][mA][mU][mG][mA][C] A][U][mA][A][mA][A][A] [mU][mA [mG][mU][mU] 2630 siRNA367- [mU][mU][mU][mU][mU][m 759 1587 [mU][A][A][mG][U][C][A 1143 1971 M0 A][mU][mC][G][C][U][A][A] ][U][U][U][mA][mG][C][ [mA][mU][mG][mA][mC][U] G][A][mU][A][mA][A][A] [mU][mA] [mA][mU][mU] 2632 siRNA368- [mU][mU][mU][mA][mU][m 760 1588 [mU][G][C][mA][A][G][U 1144 1972 M0 C][mG][mC][U][A][A][A][U] ][C][A][U][mU][mU][A][ [mG][mA][mC][mU][mU][G] G][C][mG][A][mU][A][A] [mC][mA] [mA][mU][mU] 2633 siRNA369- [mU][mU][mA][mU][mC][m 761 1589 [mU][U][G][mC][A][A][G 1145 1973 M0 G][mC][mU][A][A][A][U][G] ][U][C][A][mU][mU][U][ [mA][mC][mU][mU][mG][C] A][G][mC][G][mA][U][A] [mA][mA] [mA][mU][mU] 2635 siRNA370- [mA][mU][mC][mG][mC][m 762 1590 [mU][U][C][mU][G][C][A 1146 1974 M0 U][mA][mA][A][U][G][A][C] ][A][G][U][mC][mA][U][ [mU][mU][mG][mC][mA][G] U][U][mA][G][mC][G][A] [mA][mA] [mU][mU][mU] 2639 siRNA371- [mC][mU][mA][mA][mA][m 763 1591 [mU][U][U][mC][A][U][C 1147 1975 M0 U][mG][mA][C][U][U][G][C] ][U][G][C][mA][mA][G][ [mA][mG][mA][mU][mG][A] U][C][mA][U][mU][U][A] [mA][mA] [mG][mU][mU] 2679 siRNA372- [mU][mG][mU][mU][mU][m 764 1592 [mU][U][U][mG][U][A][C 1148 1976 M0 A][mA][mA][U][G][C][U][G] ][A][C][A][mG][mC][A][ [mU][mG][mU][mA][mC][A] U][U][mU][A][mA][A][C] [mA][mA] [mA][mU][mU] 2682 siRNA373- [mU][mU][mA][mA][mA][m 765 1593 [mU][U][U][mG][U][U][G 1149 1977 M0 U][mG][mC][U][G][U][G][U] ][U][A][C][mA][mC][A][ [mA][mC][mA][mA][mC][A] G][C][mA][U][mU][U][A] [mA][mA [mA][mU][mU] 2683 siRNA374- [[mU][mA][mA][mA][mU][m 766 1594 [mU][A][U][mU][G][U][U 1150 1978 M0 G][mC][mU][G][U][G][U][A] ][G][U][A][mC][mA][C][ [mC][mA][mA][mC][mA][A] A][G][mC][A][mU][U][U] [mU][mA] [mA][mU][mU] 2687 siRNA375- [mU][mG][mC][mU][mG][m 767 1595 [mU][A][A][mG][C][A][U 1151 1979 M0 U][mG][mU][A][C][A][A][C] ][U][G][U][mU][mG][U][ [mA][mA][mU][mG][mC][U] A][C][mA][C][mA][G][C] [mU][mA] [mA][mU][mU] 2690 siRNA376- [mU][mG][mU][mG][mU][m 768 1596 [mU][U][C][mA][A][A][G 1152 1980 M0 A][mC][mA][A][C][A][A][U] ][C][A][U][mU][mG][U][ [mG][mC][mU][mU][mU][G] U][G][mU][A][mC][A][C] [mA][mA] [mA][mU][mU] 2691 siRNA377- [mG][mU][mG][mU][mA][m 769 1597 [mU][A][U][mC][A][A][A 1153 1981 M0 C][mA][mA][C][A][A][U][G] ][G][C][A][mU][mU][G][ [mC][mU][mU][mU][mG][A] U][U][mG][U][mA][C][A] [mU][mA] [mC][mU][mU] 2692 siRNA378- [mU][mG][mU][mA][mC][m 770 1598 [mU][U][A][mU][C][A][A 1154 1982 M0 A][mA][mC][A][A][U][G][C] ][A][G][C][mA][mU][U][ [mU][mU][mU][mG][mA][U] G][U][mU][G][mU][A][C] [mA][mA] [mA][mU][mU] 2833 siRNA379- [mG][mA][mU][mA][mA][m 771 1599 [mU][U][G][mA][A][U][C 1155 1983 M0 U][mU][mU][U][G][A][A][A ][A][U][U][mU][mC][A][ ][mU][mG][mA][mU][mU][C A][A][mA][U][mU][A][U] ][mA][mA] [mC][mU][mU] 2834 siRNA380- [mA][mU][mA][mA][mU][m 772 1600 [mU][A][U][mG][A][A][U 1156 1984 M0 U][mU][mU][G][A][A][A][U ][C][A][U][mU][mU][C][ ][mG][mA][mU][mU][mC][A A][A][mA][A][mU][U][A] ][mU][mA] [mU][mU][mU] 2835 siRNA381- [mU][mA][mA][mU][mU][m 773 1601 [mU][G][A][mU][G][A][A 1157 1985 M0 U][mU][mG][A][A][A][U][G ][U][C][A][mU][mU][U][ ][mA][mU][mU][mC][mA][U C][A][mA][A][mA][U][U] ][mC][mA] [mA][mU][mU] 2838 siRNA382- [mU][mU][mU][mU][mG][m 774 1602 [mU][A][A][mA][G][A][U 1158 1986 M0 A][mA][mA][U][G][A][U][U ][G][A][A][mU][mC][A][ ][mC][mA][mU][mC][mU][U U][U][mU][C][mA][A][A] ][mU][mA] [mA][mU][mU] 2839 siRNA383- [mU][mU][mU][mG][mA][m 775 1603 [mU][G][A][mA][A][G][A 1159 1987 M0 A][mA][mU][G][A][U][U][C ][U][G][A][mA][mU][C][ [mA][mU][mC][mU][mU][U] A][U][mU][U][mC][A][A] [mC][mA] [mA][mU][mU] 2858 siRNA384- [mC][mA][mG][mA][mA][m 776 1604 [mU][G][A][mU][U][C][A 1160 1988 M0 A][mU][mA][A][A][A][G][U ][U][A][C][mU][mU][U][ ][mA][mU][mG][mA][mA][U U][A][mU][U][mU][C][U] ][mC][mA] [mG][mU][mU] *siRNA#in Table 5B correspond to the siRNA#in Table 5A, but with M0added to indicate modification pattern. M0 refers to the modification pattern of the siRNAs in Table 5B. In the sense strands, the nucleosides at positions 9-13 and 19 are ribonucleosides and the nucleosides at the rest of the positions are 2-O-Me modified nucleosides. In the antisense strands, the nucleosides at positions 2, 3, 5-10, 13-15, 17, 19, and 20 are 2-hydroxy nucleosides and the nucleosides at the rest of the positions are 2-O-Me modified nucleosides. Each uracil base (U) in any one of the sequences provided in Table 5B may independently and optionally be replaced with a thymine base (T).

Example 3: In Vitro Screening of siRNA Agents

(507) Initially, a library of 384 siRNAs as listed in Table 5B with the generic 21/23-mer partially modified design were prioritized for synthesis for a primary in-vitro screen. For this initial set of constructs, a combination of 2-OH (RNA) and 2O-Me RNA nucleosides were used. The chosen modification pattern is minimally modified and is expected to maintain the intrinsic potency of a given siRNA sequence.

(508) To determine knockdown efficiency of the synthesized siRNAs, a stable cell line overexpressing human CYP7A1 was generated. The whole library was then tested for their mRNA knockdown efficiency in vitro under transfection conditions. Two different concentrations (0.1 nM and 1 nM) were tested and siRNA that achieved 50%, or greater knockdown at the lowest concentration were selected.

(509) Generation of CYP7A1 Over-Expressing Cells

(510) U2OS cells were obtained from ATCC (Manassas, VA). Cells were thawed and sub-cultured according to manufacturer's recommendations. A stable over-expressing CYP7A11 U2OS cell line was generated by cloning hCyp7a1 DNA into a lentivirus expression vector and then co-transfecting along with lentivirus packaging plasmids into U2OS cells. After several weeks of growth under antibiotic selection, a stable pool was identified and expanded after evaluating mRNA expression of CYP7A1 by RT-PCR.

(511) In Vitro RNAi Activity Procedure

(512) U2OS cells overexpressing CYP7A1 mRNA were plated in a 96-well plate at 10,000 cells/well containing antibiotic-free medium and placed at 37 C. in an atmosphere with 5% CO2 in a humidified incubator. Within 24 hours after seeding, cells were transfected with biological duplicates for each treatment. Two concentrations (1 nM and 0.1 nM) of cyp7a1 siRNA as well as control siRNAs (ON-TARGETplus Human CYP7A1 control pool, Horizon Discovery) were applied to the cells following the manufacturer's recommendation (Thermofisher) for Lipofectamine RNAiMax. Cells were returned to the incubator for 24 hours. Cell lysates were prepared 24 hour post-transfection by removing medium and washing with cold PBS and the addition of lysis buffer (following the manufacturer's recommendation for Cells-to-CT 1-step TaqMan Kit, Thermofisher). Reverse transcription was performed on cell lysates which were then used for qPCR. qPCR was performed in duplicate for each sample in a 10 ul reaction mix, multiplexing the probe for CYP7A1 with the housekeeping gene probe (GAPDH): Nuclease-free water 5.5 ul+qRT-PCR Master Mix 2.5 ul+TaqMan Primer-1, 0.5 ul+TaqMan Primer-2, 0.5 ul.

(513) For each well, the target mRNA level was normalized to the respective GAPDH mRNA level. The activity of a given siRNA was expressed as percent mRNA concentration of the respective target (normalized to GAPDH mRNA) in treated cells, relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across mock control wells. Table 6 summarizes the in vitro screening results.

(514) TABLE-US-00043 TABLE 6 In vitro Screening of siRNAs % mRNA % mRNA Start_19mer siRNA#* remaining_1 nM KD_1 nM remaining_0.1 nM KD_0.1 nM 34 siRNA1-M0 97.0 3.0 91.3 8.7 40 siRNA2-M0 95.3 4.7 79.0 21.0 41 siRNA3-M0 84.4 15.6 81.5 18.5 42 siRNA4-M0 87.3 12.7 78.3 21.7 43 siRNA5-M0 86.1 13.9 83.0 17.0 45 siRNA6-M0 90.0 10.0 75.8 24.2 105 siRNA7-M0 48.9 51.1 87.9 12.1 110 siRNA8-M0 46.6 53.4 81.8 18.2 111 siRNA9-M0 40.5 59.5 80.8 19.2 112 siRNA10-M0 21.6 78.4 73.6 26.4 113 siRNA11-M0 86.5 13.5 89.0 11.0 115 siRNA12-M0 14.3 85.7 48.6 51.4 116 siRNA13-M0 33.1 66.9 70.6 29.4 117 siRNA14-M0 80.2 19.8 91.2 8.8 118 siRNA15-M0 31.4 68.6 55.5 44.5 139 siRNA16-M0 53.3 46.7 100.7 0.7 141 siRNA17-M0 55.4 44.6 88.7 11.3 142 siRNA18-M0 57.4 42.6 81.1 18.9 143 siRNA19-M0 34.1 65.9 69.3 30.7 181 siRNA20-M0 54.3 45.7 96.6 3.4 182 siRNA21-M0 63.2 36.8 85.8 14.2 183 siRNA22-M0 68.6 31.4 94.7 5.3 201 siRNA23-M0 29.0 71.0 73.8 26.2 203 siRNA24-M0 28.3 71.7 66.3 33.7 210 siRNA25-M0 99.2 0.8 96.1 3.9 214 siRNA26-M0 41.0 59.0 55.5 44.5 223 siRNA27-M0 18.3 81.7 18.0 82.0 225 siRNA28-M0 81.2 18.8 84.3 15.7 229 siRNA29-M0 62.3 37.7 90.3 9.7 230 siRNA30-M0 32.4 67.6 74.2 25.8 236 siRNA31-M0 28.5 71.5 78.7 21.3 238 siRNA32-M0 64.3 35.7 84.8 15.2 242 siRNA33-M0 45.9 54.1 76.3 23.7 243 siRNA34-M0 41.5 58.5 86.1 13.9 244 siRNA35-M0 80.3 19.7 70.6 29.4 245 siRNA36-M0 38.8 61.2 53.7 46.3 246 siRNA37-M0 81.0 19.0 75.5 24.5 251 siRNA38-M0 29.6 70.4 50.1 49.9 253 siRNA39-M0 68.2 31.8 199.0 99.0 254 siRNA40-M0 18.7 81.3 70.7 29.3 262 siRNA41-M0 37.7 62.3 62.7 37.3 264 siRNA42-M0 48.7 51.3 87.2 12.8 266 siRNA43-M0 66.8 33.2 72.8 27.2 267 siRNA44-M0 75.4 24.6 90.9 9.1 268 siRNA45-M0 17.6 82.4 56.3 43.7 291 siRNA46-M0 53.0 47.0 74.6 25.4 292 siRNA47-M0 17.4 82.6 36.7 63.3 294 siRNA48-M0 73.0 27.0 90.1 9.9 300 siRNA49-M0 37.2 62.8 58.8 41.2 302 siRNA50-M0 32.2 67.8 69.4 30.6 303 siRNA51-M0 23.6 76.4 50.3 49.7 304 siRNA52-M0 22.7 77.3 67.5 32.5 305 siRNA53-M0 58.9 41.1 83.0 17.0 309 siRNA54-M0 33.2 66.8 76.9 23.1 310 siRNA55-M0 79.3 20.7 74.7 25.3 311 siRNA56-M0 87.0 13.0 80.3 19.7 312 siRNA57-M0 65.3 34.7 94.3 5.7 313 siRNA58-M0 63.3 36.7 88.1 11.9 315 siRNA59-M0 78.7 21.3 82.2 17.8 319 siRNA60-M0 68.6 31.4 76.6 23.4 360 siRNA61-M0 50.5 49.5 75.9 24.1 363 siRNA62-M0 52.4 47.6 86.7 13.3 372 siRNA63-M0 26.8 73.2 79.6 20.4 375 siRNA64-M0 45.3 54.7 109.1 9.1 377 siRNA65-M0 62.0 38.0 90.3 9.7 390 siRNA66-M0 59.6 40.4 62.0 38.0 392 siRNA67-M0 78.8 21.2 99.8 0.2 394 siRNA68-M0 43.1 56.9 90.5 9.5 474 siRNA69-M0 53.5 46.5 89.6 10.4 475 siRNA70-M0 70.0 30.0 80.9 19.1 476 siRNA71-M0 64.7 35.3 80.0 20.0 477 siRNA72-M0 23.2 76.8 48.9 51.1 478 siRNA73-M0 25.9 74.1 46.0 54.0 482 siRNA74-M0 61.0 39.0 82.6 17.4 484 siRNA75-M0 66.9 33.1 76.9 23.1 486 siRNA76-M0 61.2 38.8 87.4 12.6 488 siRNA77-M0 62.0 38.0 86.3 13.7 501 siRNA78-M0 51.1 48.9 73.7 26.3 503 siRNA79-M0 28.5 71.5 76.7 23.3 505 siRNA80-M0 37.2 62.8 74.7 25.3 506 siRNA81-M0 15.8 84.2 52.8 47.2 507 siRNA82-M0 83.3 16.7 83.8 16.2 509 siRNA83-M0 75.5 24.5 103.0 3.0 510 siRNA84-M0 70.6 29.4 108.8 8.8 511 siRNA85-M0 166.5 66.5 86.8 13.2 512 siRNA86-M0 60.3 39.7 96.0 4.0 513 siRNA87-M0 71.0 29.0 82.7 17.3 514 siRNA88-M0 65.6 34.4 58.5 41.5 539 siRNA89-M0 85.3 14.7 77.3 22.7 552 siRNA90-M0 46.8 53.2 68.3 31.7 562 siRNA91-M0 82.4 17.6 87.1 12.9 563 siRNA92-M0 60.7 39.3 93.3 6.7 564 siRNA93-M0 36.6 63.4 71.0 29.0 585 siRNA94-M0 68.5 31.5 99.6 0.4 587 siRNA95-M0 63.2 36.8 93.8 6.2 588 siRNA96-M0 59.7 40.3 96.2 3.8 589 siRNA97-M0 29.5 70.5 73.1 26.9 593 siRNA98-M0 58.5 41.5 99.3 0.7 594 siRNA99-M0 70.2 29.8 70.8 29.2 595 siRNA100-M0 15.0 85.0 46.2 53.8 602 siRNA101-M0 17.7 82.3 52.6 47.4 605 siRNA102-M0 61.8 38.2 88.4 11.6 606 siRNA103-M0 19.1 80.9 62.5 37.5 607 siRNA104-M0 70.8 29.2 65.4 34.6 608 siRNA105-M0 50.4 49.6 82.4 17.6 609 siRNA106-M0 36.2 63.8 63.7 36.3 610 siRNA107-M0 61.5 38.5 69.4 30.6 612 siRNA108-M0 30.1 69.9 68.4 31.6 613 siRNA109-M0 22.6 77.4 65.7 34.3 653 siRNA110-M0 20.4 79.6 70.4 29.6 663 siRNA111-M0 19.1 80.9 65.1 34.9 665 siRNA112-M0 32.2 67.8 55.1 44.9 666 siRNA113-M0 34.6 65.4 58.6 41.4 667 siRNA114-M0 34.2 65.8 83.3 16.7 669 siRNA115-M0 75.7 24.3 96.6 3.4 670 siRNA116-M0 26.2 73.8 65.4 34.6 671 siRNA117-M0 60.5 39.5 71.7 28.3 673 siRNA118-M0 9.7 90.3 38.7 61.3 679 siRNA119-M0 55.5 44.5 84.2 15.8 712 siRNA120-M0 65.2 34.8 72.5 27.5 771 siRNA121-M0 82.5 17.5 77.6 22.4 781 siRNA122-M0 20.7 79.3 52.3 47.7 788 siRNA123-M0 77.9 22.1 94.1 5.9 841 siRNA124-M0 13.0 87.0 51.1 48.9 844 siRNA125-M0 7.6 92.4 28.0 72.0 846 siRNA126-M0 61.9 38.1 84.7 15.3 850 siRNA127-M0 88.0 12.0 85.0 15.0 851 siRNA128-M0 72.2 27.8 85.3 14.7 852 siRNA129-M0 48.8 51.2 73.3 26.7 861 siRNA130-M0 84.9 15.1 72.1 27.9 862 siRNA131-M0 50.4 49.6 79.9 20.1 901 siRNA132-M0 98.1 1.9 79.6 20.4 903 siRNA133-M0 99.1 0.9 93.7 6.3 904 siRNA134-M0 79.7 20.3 75.1 24.9 905 siRNA135-M0 84.8 15.2 100.5 0.5 906 siRNA136-M0 70.7 29.3 86.1 13.9 912 siRNA137-M0 35.4 64.6 90.7 9.3 915 siRNA138-M0 43.9 56.1 86.4 13.6 916 siRNA139-M0 63.9 36.1 91.5 8.5 917 siRNA140-M0 41.6 58.4 80.4 19.6 920 siRNA141-M0 20.2 79.8 74.2 25.8 921 siRNA142-M0 104.4 4.4 74.8 25.2 950 siRNA143-M0 67.8 32.2 55.3 44.7 954 siRNA144-M0 28.9 71.1 79.0 21.0 955 siRNA145-M0 61.3 38.7 71.7 28.3 956 siRNA146-M0 70.6 29.4 74.0 26.0 957 siRNA147-M0 93.4 6.6 82.9 17.1 980 siRNA148-M0 63.1 36.9 96.1 3.9 982 siRNA149-M0 40.3 59.7 72.4 27.6 983 siRNA150-M0 72.8 27.2 80.2 19.8 988 siRNA151-M0 46.7 53.3 84.6 15.4 989 siRNA152-M0 18.4 81.6 60.0 40.0 992 siRNA153-M0 43.2 56.8 63.3 36.7 993 siRNA154-M0 47.7 52.3 81.5 18.5 994 siRNA155-M0 53.3 46.7 64.3 35.7 995 siRNA156-M0 24.7 75.3 77.8 22.2 996 siRNA157-M0 17.3 82.7 73.5 26.5 1005 siRNA158-M0 10.9 89.1 49.9 50.1 1006 siRNA159-M0 49.9 50.1 80.6 19.4 1007 siRNA160-M0 28.5 71.5 71.7 28.3 1011 siRNA161-M0 20.7 79.3 33.4 66.6 1012 siRNA162-M0 42.4 57.6 84.6 15.4 1013 siRNA163-M0 81.6 18.4 83.4 16.6 1018 siRNA164-M0 84.5 15.5 89.5 10.5 1019 siRNA165-M0 24.9 75.1 55.2 44.8 1020 siRNA166-M0 25.7 74.3 76.2 23.8 1021 siRNA167-M0 67.7 32.3 83.9 16.1 1026 siRNA168-M0 55.9 44.1 76.2 23.8 1027 siRNA169-M0 75.7 24.3 71.9 28.1 1029 siRNA170-M0 85.4 14.6 100.6 0.6 1036 siRNA171-M0 39.8 60.2 68.1 31.9 1039 siRNA172-M0 9.0 91.0 31.4 68.6 1042 siRNA173-M0 49.2 50.8 86.8 13.2 1084 siRNA174-M0 10.5 89.5 33.0 67.0 1087 siRNA175-M0 39.2 60.8 73.1 26.9 1134 siRNA176-M0 81.8 18.2 87.0 13.0 1151 siRNA177-M0 67.9 32.1 88.7 11.3 1152 siRNA178-M0 41.0 59.0 90.1 9.9 1155 siRNA179-M0 46.8 53.2 71.1 28.9 1158 siRNA180-M0 62.8 37.2 81.5 18.5 1160 siRNA181-M0 87.0 13.0 90.4 9.6 1162 siRNA182-M0 28.9 71.1 94.0 6.0 1165 siRNA183-M0 73.3 26.7 71.3 28.7 1168 siRNA184-M0 29.1 70.9 76.2 23.8 1169 siRNA185-M0 19.9 80.1 76.9 23.1 1174 siRNA186-M0 42.3 57.7 69.9 30.1 1177 siRNA187-M0 32.2 67.8 83.9 16.1 1178 siRNA188-M0 49.5 50.5 101.8 1.8 1189 siRNA189-M0 76.9 23.1 76.3 23.7 1191 siRNA190-M0 19.6 80.4 33.9 66.1 1194 siRNA191-M0 63.8 36.2 94.1 5.9 1196 siRNA192-M0 85.1 14.9 83.6 16.4 1200 siRNA193-M0 48.4 51.6 78.3 21.7 1201 siRNA194-M0 25.3 74.7 60.4 39.6 1203 siRNA195-M0 63.4 36.6 92.5 7.5 1204 siRNA196-M0 192.8 92.8 147.7 47.7 1205 siRNA197-M0 17.4 82.6 58.1 41.9 1207 siRNA198-M0 40.2 59.8 82.9 17.1 1209 siRNA199-M0 14.3 85.7 50.9 49.1 1212 siRNA200-M0 19.7 80.3 67.2 32.8 1214 siRNA201-M0 26.4 73.6 81.1 18.9 1215 siRNA202-M0 23.8 76.2 60.0 40.0 1217 siRNA203-M0 14.6 85.4 44.6 55.4 1226 siRNA204-M0 69.0 31.0 92.1 7.9 1227 siRNA205-M0 10.9 89.1 32.1 67.9 1228 siRNA206-M0 9.2 90.8 44.2 55.8 1229 siRNA207-M0 23.4 76.6 78.7 21.3 1230 siRNA208-M0 16.8 83.2 70.6 29.4 1231 siRNA209-M0 18.5 81.5 72.0 28.0 1233 siRNA210-M0 15.8 84.2 71.0 29.0 1236 siRNA211-M0 73.4 26.6 80.9 19.1 1237 siRNA212-M0 24.5 75.5 44.1 55.9 1238 siRNA213-M0 36.5 63.5 98.3 1.7 1239 siRNA214-M0 41.1 58.9 79.0 21.0 1240 siRNA215-M0 16.5 83.5 65.4 34.6 1241 siRNA216-M0 18.8 81.2 55.0 45.0 1242 siRNA217-M0 13.0 87.0 53.6 46.4 1248 siRNA218-M0 42.3 57.7 57.4 42.6 1249 siRNA219-M0 37.1 62.9 67.3 32.7 1261 siRNA220-M0 30.8 69.2 77.4 22.6 1283 siRNA221-M0 52.0 48.0 88.6 11.4 1285 siRNA222-M0 91.2 8.8 88.3 11.7 1286 siRNA223-M0 54.9 45.1 88.2 11.8 1288 siRNA224-M0 49.7 50.3 79.4 20.6 1290 siRNA225-M0 26.9 73.1 77.2 22.8 1291 siRNA226-M0 42.0 58.0 47.1 52.9 1292 siRNA227-M0 38.2 61.8 72.9 27.1 1293 siRNA228-M0 31.7 68.3 71.0 29.0 1296 siRNA229-M0 33.3 66.7 72.3 27.7 1297 siRNA230-M0 34.0 66.0 78.5 21.5 1298 siRNA231-M0 18.8 81.2 39.3 60.7 1299 siRNA232-M0 40.9 59.1 76.8 23.2 1300 siRNA233-M0 131.7 31.7 83.1 16.9 1301 siRNA234-M0 31.8 68.2 74.9 25.1 1302 siRNA235-M0 61.4 38.6 80.7 19.3 1303 siRNA236-M0 31.1 68.9 69.7 30.3 1306 siRNA237-M0 24.4 75.6 67.8 32.2 1307 siRNA238-M0 39.1 60.9 84.7 15.3 1311 siRNA239-M0 42.0 58.0 65.8 34.2 1313 siRNA240-M0 55.4 44.6 87.5 12.5 1314 siRNA241-M0 24.0 76.0 80.3 19.7 1353 siRNA242-M0 19.9 80.1 68.9 31.1 1354 siRNA243-M0 54.1 45.9 74.2 25.8 1358 siRNA244-M0 77.0 23.0 84.7 15.3 1360 siRNA245-M0 23.7 76.3 61.7 38.3 1361 siRNA246-M0 48.1 51.9 82.5 17.5 1363 siRNA247-M0 26.4 73.6 83.7 16.3 1364 siRNA248-M0 24.1 75.9 67.2 32.8 1382 siRNA249-M0 56.8 43.2 96.7 3.3 1386 siRNA250-M0 16.1 83.9 44.4 55.6 1388 siRNA251-M0 21.5 78.5 64.7 35.3 1389 siRNA252-M0 28.5 71.5 83.3 16.7 1413 siRNA253-M0 35.5 64.5 77.6 22.4 1415 siRNA254-M0 34.9 65.1 76.4 23.6 1416 siRNA255-M0 61.0 39.0 75.9 24.1 1417 siRNA256-M0 14.6 85.4 29.7 70.3 1424 siRNA257-M0 25.8 74.2 76.1 23.9 1425 siRNA258-M0 61.4 38.6 95.1 4.9 1431 siRNA259-M0 18.1 81.9 61.2 38.8 1433 siRNA260-M0 13.9 86.1 52.7 47.3 1506 siRNA261-M0 75.2 24.8 97.4 2.6 1507 siRNA262-M0 68.1 31.9 87.1 12.9 1510 siRNA263-M0 94.6 5.4 88.2 11.8 1511 siRNA264-M0 220.8 120.8 89.1 10.9 1513 siRNA265-M0 69.5 30.5 80.5 19.5 1518 siRNA266-M0 73.7 26.3 87.5 12.5 1555 siRNA267-M0 77.4 22.6 83.5 16.5 1556 siRNA268-M0 61.1 38.9 72.1 27.9 1557 siRNA269-M0 66.4 33.6 66.5 33.5 1558 siRNA270-M0 42.5 57.5 79.8 20.2 1559 siRNA271-M0 75.3 24.7 81.0 19.0 1561 siRNA272-M0 20.7 79.3 39.8 60.2 1593 siRNA273-M0 99.5 0.5 95.6 4.4 1594 siRNA274-M0 81.6 18.4 77.0 23.0 1597 siRNA275-M0 70.3 29.7 99.0 1.0 1599 siRNA276-M0 89.3 10.7 74.0 26.0 1601 siRNA277-M0 86.2 13.8 69.5 30.5 1605 siRNA278-M0 83.1 16.9 84.7 15.3 1606 siRNA279-M0 87.5 12.5 74.1 25.9 1609 siRNA280-M0 92.3 7.7 70.9 29.1 1610 siRNA281-M0 77.7 22.3 81.0 19.0 1614 siRNA282-M0 76.2 23.8 76.5 23.5 1638 siRNA283-M0 91.0 9.0 87.1 12.9 1660 siRNA284-M0 83.7 16.3 87.9 12.1 1661 siRNA285-M0 88.0 12.0 74.9 25.1 1662 siRNA286-M0 95.5 4.5 84.6 15.4 1663 siRNA287-M0 100.1 0.1 88.9 11.1 1669 siRNA288-M0 84.0 16.0 85.6 14.4 1671 siRNA289-M0 81.0 19.0 76.3 23.7 1673 siRNA290-M0 66.1 33.9 84.0 16.0 1676 siRNA291-M0 70.8 29.2 60.1 39.9 1683 siRNA292-M0 93.8 6.2 87.5 12.5 1686 siRNA293-M0 89.6 10.4 79.9 20.1 1689 siRNA294-M0 72.5 27.5 86.6 13.4 1690 siRNA295-M0 78.9 21.1 84.9 15.1 1692 siRNA296-M0 82.3 17.7 83.8 16.2 1712 siRNA297-M0 85.0 15.0 67.3 32.7 1713 siRNA298-M0 81.0 19.0 82.0 18.0 1769 siRNA299-M0 89.8 10.2 72.7 27.3 1770 siRNA300-M0 84.2 15.8 91.3 8.7 1824 siRNA301-M0 95.1 4.9 88.3 11.7 1825 siRNA302-M0 82.2 17.8 82.2 17.8 1826 siRNA303-M0 94.2 5.8 97.6 2.4 1827 siRNA304-M0 90.7 9.3 70.4 29.6 1828 siRNA305-M0 73.2 26.8 97.4 2.6 1829 siRNA306-M0 84.5 15.5 82.5 17.5 1830 siRNA307-M0 87.6 12.4 72.7 27.3 1895 siRNA308-M0 73.2 26.8 93.4 6.6 2052 siRNA309-M0 89.0 11.0 71.4 28.6 2053 siRNA310-M0 82.2 17.8 82.6 17.4 2075 siRNA311-M0 87.6 12.4 94.8 5.2 2127 siRNA312-M0 70.3 29.7 83.7 16.3 2128 siRNA313-M0 88.9 11.1 75.2 24.8 2132 siRNA314-M0 81.8 18.2 96.2 3.8 2133 siRNA315-M0 76.5 23.5 83.6 16.4 2134 siRNA316-M0 77.5 22.5 102.2 2.2 2135 siRNA317-M0 215.9 115.9 84.5 15.5 2136 siRNA318-M0 74.7 25.3 91.1 8.9 2137 siRNA319-M0 77.0 23.0 77.9 22.1 2139 siRNA320-M0 79.1 20.9 89.0 11.0 2140 siRNA321-M0 91.0 9.0 87.2 12.8 2144 siRNA322-M0 92.0 8.0 109.0 9.0 2189 siRNA323-M0 91.3 8.7 75.1 24.9 2190 siRNA324-M0 66.9 33.1 95.5 4.5 2191 siRNA325-M0 90.3 9.7 76.3 23.7 2192 siRNA326-M0 80.4 19.6 89.2 10.8 2193 siRNA327-M0 68.6 31.4 90.3 9.7 2194 siRNA328-M0 87.0 13.0 67.6 32.4 2195 siRNA329-M0 70.1 29.9 98.0 2.0 2199 siRNA330-M0 80.0 20.0 74.5 25.5 2209 siRNA331-M0 67.3 32.7 94.6 5.4 2210 siRNA332-M0 91.9 8.1 72.9 27.1 2211 siRNA333-M0 78.6 21.4 75.7 24.3 2215 siRNA334-M0 86.8 13.2 73.1 26.9 2216 siRNA335-M0 70.6 29.4 98.7 1.3 2217 siRNA336-M0 89.4 10.6 77.7 22.3 2218 siRNA337-M0 78.0 22.0 103.6 3.6 2220 siRNA338-M0 77.2 22.8 107.3 7.3 2221 siRNA339-M0 76.6 23.4 87.1 12.9 2223 siRNA340-M0 78.4 21.6 84.5 15.5 2224 siRNA341-M0 88.3 11.7 76.5 23.5 2225 siRNA342-M0 73.1 26.9 64.5 35.5 2313 siRNA343-M0 100.1 0.1 78.6 21.4 2316 siRNA344-M0 78.0 22.0 80.5 19.5 2317 siRNA345-M0 91.2 8.8 94.7 5.3 2319 siRNA346-M0 87.0 13.0 76.9 23.1 2320 siRNA347-M0 67.4 32.6 90.1 9.9 2321 siRNA348-M0 82.4 17.6 93.8 6.2 2353 siRNA349-M0 87.9 12.1 71.9 28.1 2355 siRNA350-M0 92.7 7.3 73.7 26.3 2356 siRNA351-M0 89.1 10.9 70.4 29.6 2362 siRNA352-M0 78.1 21.9 78.9 21.1 2364 siRNA353-M0 71.1 28.9 108.2 8.2 2365 siRNA354-M0 78.5 21.5 86.1 13.9 2366 siRNA355-M0 80.7 19.3 96.5 3.5 2370 siRNA356-M0 80.5 19.5 85.4 14.6 2503 siRNA357-M0 71.6 28.4 72.1 27.9 2562 siRNA358-M0 87.5 12.5 79.4 20.6 2563 siRNA359-M0 96.3 3.7 78.3 21.7 2620 siRNA360-M0 87.4 12.6 73.9 26.1 2621 siRNA361-M0 66.9 33.1 90.9 9.1 2622 siRNA362-M0 83.8 16.2 79.2 20.8 2623 siRNA363-M0 84.1 15.9 76.1 23.9 2624 siRNA364-M0 83.8 16.2 88.9 11.1 2625 siRNA365-M0 78.8 21.2 81.5 18.5 2629 siRNA366-M0 71.5 28.5 87.7 12.3 2630 siRNA367-M0 96.6 3.4 87.7 12.3 2632 siRNA368-M0 96.0 4.0 85.8 14.2 2633 siRNA369-M0 80.4 19.6 92.6 7.4 2635 siRNA370-M0 185.9 85.9 80.3 19.7 2639 siRNA371-M0 97.2 2.8 75.4 24.6 2679 siRNA372-M0 69.8 30.2 97.7 2.3 2682 siRNA373-M0 92.1 7.9 84.9 15.1 2683 siRNA374-M0 78.0 22.0 86.0 14.0 2687 siRNA375-M0 79.6 20.4 79.1 20.9 2690 siRNA376-M0 79.8 20.2 81.5 18.5 2691 siRNA377-M0 99.8 0.2 79.3 20.7 2692 siRNA378-M0 78.3 21.7 79.0 21.0 2833 siRNA379-M0 78.2 21.8 82.1 17.9 2834 siRNA380-M0 193.4 93.4 88.9 11.1 2835 siRNA381-M0 94.4 5.6 82.8 17.2 2838 siRNA382-M0 81.5 18.5 158.5 58.5 2839 siRNA383-M0 76.9 23.1 80.2 19.8 2858 siRNA384-M0 85.5 14.5 75.3 24.7 *siRNA# in Table 6 correspond to the siRNA# in Table 5B

Example 4: In Vivo Screening of siRNA Agents in Mouse

(515) Initial Screening

(516) Based in the in vitro screening results, 30 target sequences were selected for further study. siRNAs were then prepared as fully chemically modified siRNA conjugated to a targeting moiety comprising a structure of Formula (Z.sup.6). A combination of 2-O-methyl-RNA phosphoramidites and 2-Deoxy-2-fluoro-RNA phosphoramidites were used to obtain siRNA for in vivo study (FIG. 1, Table 7B). Monomer phosphoramidites were used to incorporate the targeting moiety at the 3-end of the sense strand. Table 7A provides the nucleobase sequences of the siRNAs. It is to be understood that any chemical modification pattern may be applied to the nucleobase sequences of the siRNAs in Table 7A. For the initial screening, modification pattern MOD 3 (see FIG. 1) was used. Table 7B below lists the exemplary siRNAs used for the initial in vivo screening in mouse.

(517) Chemically Modified Duplex siRNA Preparation

(518) Oligoribonucleotides were synthesized according to the phosphoramidite technology on solid phase employing a Mermade 12 synthesizer (LGC Bioautomation). Syntheses were performed on a solid support made of controlled pore glass (CPG). Specifically, oligonucleotides were assembled on a 500 universal solid support with a loading of 89.3 mol/g, purchased from Chemgenes. All 2-modified RNA phosphoramidites as well as ancillary reagents were purchased from commercially available resources. Specifically, the following 2-O-Methyl phosphoramidites were used: (5-O-dimethoxytrityl-N6-(benzoyl)-2-O-methyl-adenosine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5-O-dimethoxytrityl-N4-(acetyl)-2-O-methyl-cytidine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, (5-O-dimethoxytrityl-N2-(isobutyryl)-2-O-methyl-guanosine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5-O-dimethoxytrityl-2-O-methyl-uridine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2-Deoxy-2-fluoro-phosphoramidites carried the same protecting groups as the 2-O-methyl RNA amidites. For introduction of the targeting moiety, 1-O-GalNAc-5-O-dimethoxytrityl-2-O-methyl-ribose-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was used. All phosphoramidites were dissolved in anhydrous acetonitrile (100 mM) besides 5-O-dimethoxytrityl-2-O-methyl-uridine-3-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite which was dissolved in a 20% dimethylformamide/acetonitrile solution (100 mM). Molecular sieves (3 ) were added to all amidite solutions and reagent solutions 6 hours before the synthesis. 5-Ethyl thiotetrazole (ETT, 500 mM in acetonitrile) was used as an activator. Coupling times were 6 minutes. In order to introduce phosphorothioate linkages a 50 mM solution of 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes, Wimington, MA, USA) in anhydrous acetonitrile/pyridine (1:1 v/v) was employed. Oligonucleotides were synthesized with removal of the final DMT protecting group (DMT off).

(519) Cleavage and Deprotection of Support Bound Oligomer

(520) After finalization of the solid phase synthesis, oligonucleotides were cleaved from the solid support by the addition a mixture consisting of 5 wt % diethylamine in concentrated aqueous ammonia, both available from Sigma Aldrich). To achieve quantitative removal of all protecting groups, the solutions were incubated with shaking at 35 C. for 24 hours. The crude suspension was filtered through a 0.45 micron Millex PVDF filter, washed the solid support with additional water, then concentrated the filtrate in vacuo to remove the diethylamine and aqueous ammonia residues.

(521) Purification of Oligoribonucleotides

(522) Crude oligomers were purified by anionic exchange HPLC using SourceQ column (GE Healthcare, Freiburg, Germany) on an AKTA Purifier system (GE Healthcare, Freiburg, Germany). Buffer A was 20 mM sodium phosphate, pH 8 and contained 10% acetonitrile and buffer B contained 1M sodium bromide in buffer A. A flow rate of 10 mL/min and a gradient starting from 25% to 65% buffer B was employed. UV traces at 260 and 280 nm were recorded to monitor product elution. Appropriate fractions were pooled and analyzed via mass spec. Pooled oligonucleotides were desalted via size exclusion chromatography on an AKTA Purifier system (GE Healthcare, Freiburg, Germany) utilizing Milli-Q filtered water. Desalted oligonucleotides were then frozen over dry ice and lyophilized.

(523) Annealing of Oligoribonucleotides to Generate siRNA

(524) Complementary strands were dissolved in Milli-Q or DNAse/RNAse water, then mixed at an equimolar ratio. The mixtures were annealed using ThermoMixer C equipment (Eppendorf). It was shaked at 400 rpm and heated at 90 C. for 15 minutes, then slowly reduced to 15 C. An aliquot was sampled and analyzed using UPLC/MS (QTOF or Orbitrap) to confirm completion and proper of annealing process.

(525) TABLE-US-00044 TABLE7A NucleobasesequencesofexemplarysiRNAs SEQ SEQ ID ID Position siRNA#* SenseStrandSequence NO: Anti-SenseStrandSequence NO: 223 siRNA27 UCUUGAGUUCCUCAGAGCAAA 419 UUUGCUCUGAGGAACUCAAGAAG 1161 844 siRNA125 GCGCAUGUUUCUCAAUGACAA 517 UUGUCAUUGAGAAACAUGCGCAG 1162 1417 siRNA256 UAUCCACGAAAUCAAGCAAUA 648 UAUUGCUUGAUUUCGUGGAUAAG 1163 1039 siRNA172 AAAAGUCAGCUUGGAAGGCAA 564 UUGCCUUCCAAGCUGACUUUUAG 1164 1227 siRNA205 UAGCUCUUUACCCACAGUUAA 597 UUAACUGUGGGUAAAGAGCUAAG 1165 1084 siRNA174 ACUGAAUGACCUGCCAGUAUA 566 UAUACUGGCAGGUCAUUCAGUAG 1166 1011 siRNA161 UGAAAAGAACAUUAGAGAAUA 553 UAUUCUCUAAUGUUCUUUUCAAG 1167 1191 siRNA190 AGGACGGUUCCUACAACAUCA 582 UGAUGUUGUAGGAACCGUCCUAG 1168 292 siRNA47 UGUCCAUUUCAUCACAAAUCA 439 UGAUUUGUGAUGAAAUGGACAAG 1169 673 siRNA118 UCUAAACAAUCUUGACAACUA 510 UAGUUGUCAAGAUUGUUUAGAAG 1170 1298 siRNA231 AGGUAUCUUGAUGAAAACGGA 623 UCCGUUUUCAUCAAGAUACCUAG 1171 1561 siRNA272 AUAUAAAUUCAAGCAUUUGUA 664 UACAAAUGCUUGAAUUUAUAUAG 1172 1237 siRNA212 CCCACAGUUAAUGCACUUAGA 604 UCUAAGUGCAUUAACUGUGGGAG 1173 1228 siRNA206 AGCUCUUUACCCACAGUUAAA 598 UUUAACUGUGGGUAAAGAGCUAG 1174 1386 siRNA250 GAGCUACAAUAUGUCCUGGAA 642 UUCCAGGACAUAUUGUAGCUCAG 1175 1217 siRNA203 GAUGACAUCAUAGCUCUUUAA 595 UUAAAGAGCUAUGAUGUCAUCAG 1176 478 siRNA73 CUUGAAUUCCCUCACGGAAAA 465 UUUUCCGUGAGGGAAUUCAAGAG 1177 595 siRNA100 CCGAGUGAUGUUUGAAGCUGA 492 UCAGCUUCAAACAUCACUCGGAG 1178 1291 siRNA226 AUAUGAUAGGUAUCUUGAUGA 618 UCAUCAAGAUACCUAUCAUAUAG 1179 115 siRNA12 UUGUCUAUGGCUUAUUCUUGA 404 UCAAGAAUAAGCCAUAGACAAAG 1180 477 siRNA72 CCUUGAAUUCCCUCACGGAAA 464 UUUCCGUGAGGGAAUUCAAGGAG 1181 1005 siRNA158 AAGAAGUGAAAAGAACAUUAA 550 UUAAUGUUCUUUUCACUUCUUAG 1182 251 siRNA38 AAACAUGGUCAUGUUUUUACA 430 UGUAAAAACAUGACCAUGUUUAG 1183 303 siRNA51- UCACAAAUCCCUUGUCAUACA 443 UGUAUGACAAGGGAUUUGUGAAG 1184 1209 siRNA199 UCCGAAAAGAUGACAUCAUAA 591 UUAUGAUGUCAUCUUUUCGGAAG 1185 841 siRNA124 CCUGCGCAUGUUUCUCAAUGA 516 UCAUUGAGAAACAUGCGCAGGAG 1186 781 siRNA122 GGCAGAGAGCUUGAGGCACGA 514 UCGUGCCUCAAGCUCUCUGCCAG 1187 602 siRNA101 AUGUUUGAAGCUGGGUAUUUA 493 UAAAUACCCAGCUUCAAACAUAG 1188 1433 siRNA260 CAAUUUUUGAUUCUGAUGCUA 652 UAGCAUCAGAAUCAAAAAUUGAG 1189 506 siRNA81 GAAAACCUCCAACGUAUCAUA 473 UAUGAUACGUUGGAGGUUUUCAG 1190 *The after an siRNA#(e.g., siRNA#) indicates that the siRNA correspond to the same siRNA#in Table 5A, where the siRNA#and siRNA#has the same sense strand nucleobase sequence, but the siRNA#has the last two nucleobases of the antisense strand (counting 5- 3) changed fromUUto AG, relative to siRNA#. Each uracil base (U) in any one of the sequences provided in Table 7A may independently and optionally be replaced with a thymine base (T).

(526) TABLE-US-00045 TABLE7B ListofChemicallyModifiedDuplexesPreparedfortheinitialinvivoScreening SEQ SEQ IDNO IDNO of Nucleobase of Nucleobase modified SEQ modified SEQID sense ID antisense Position siRNA#* SenseStrandSequence NO: strand: Anti-SenseStrandSequence NO: strand: 223 siRNA27- [mUs][mCs][mU][mU][mG][mA] 419 1219 [mUs][fUs][fU][mG][fC][fU][fC] 1161 1989 M3{circumflex over ()} [mG][mU][fU][fC][fC][fU][mC] [fU][mG][fA][mG][mG][mA][fA] [mA][mG][mA][mG][mC][mA] [mC][mU][mC][mA][mA][mG] [mA][mAs] [mAs][mAs][mG] 844 siRNA125- [mGs][mCs][mG][mC][mA][mU] 517 1329 [mUs][fUs][fG][mU][fC][fA][fU] 1162 1993 M3 [mG][mU][fU][fU][fC][fU][mC] [fU][mG][fA][mG][mA][mA][fA] [mA][mA][mU][mG][mA][mC] [mC][mA][mU][mG][mC][mG] [mA][mAs] [mCs][mAs][mG] 1417 siRNA256- [mUs][mAs][mU][mC][mC][mA] 648 1474 [mUs][fAs][fU][mU][fG][fC][fU] 1163 1997 M3 [mC][mG][fA][fA][fA][fU][mC] [fU][mG][fA][mU][mU][mU][fC] [mA][mA][mG][mC][mA][mA] [mG][mU][mG][mG][mA][mU] [mU][mAs] [mAs][mAs][mG] 1039 siRNA172- [mAs][mAs][mA][mA][mG][mU] 564 1379 [mUs][fUs][fG][mC][fC][fU][fU] 1164 1998 M3 [mC][mA][fG][fC][fU][fU][mG] [fC][mC][fA][mA][mG][mC][fU] [mG][mA][mA][mG][mG][mC] [mG][mA][mC][mU][mU][mU] [mA][mAs] [mUs][mAs][mG] 1227 siRNA205- [mUs][mAs][mG][mC][mU][mC] 597 1417 [mUs][fUs][fA][mA][fC][fU][fG] 1165 1999 M3 [mU][mU][fU][fA][fC][fC][mC] [fU][mG][fG][mG][mU][mA][fA] [mA][mC][mA][mG][mU][mU] [mA][mG][mA][mG][mC][mU] [mA][mAs] [mAs][mAs][mG] 1084 siRNA174- [mAs][mCs][mU][mG][mA][mA] 566 1382 [mUs][fAs][fU][mA][fC][fU][fG] 1166 2003 M3 [mU][mG][fA][fC][fC][fU][mG] [fG][mC][fA][mG][mG][mU][fC] [mC][mC][mA][mG][mU][mA] [mA][mU][mU][mC][mA][mG] [mU][mAs] [mUs][mAs][mG] 1011 siRNA161- [mUs][mGs][mA][mA][mA][mA] 553 1367 [mUs][fAs][fU][mU][fC][fU][fC] 1167 2007 M3 [mG][mA][fA][fC][fA][fU][mU] [fU][mA][fA][mU][mG][mU][fU] [mA][mG][mA][mG][mA][mA] [mC][mU][mU][mU][mU][mC] [mU][mAs] [mAs][mAs][mG] 1191 siRNA190- [mAs][mGs][mG][mA][mC][mG] 582 1399 [mUs][fGs][fA][mU][fG][fU][fU] 1168 2008 M3 [mG][mU][fU][fC][fC][fU][mA] [fG][mU][fA][mG][mG][mA][fA] [mC][mA][mA][mC][mA][mU] [mC][mC][mG][mU][mC][mC] [mC][mAs] [mUs][mAs][mG] 292 siRNA47- [mUs][mGs][mU][mC][mC][mA] 439 1241 [mUs][fGs][fA][mU][fU][fU][fG] 1169 2009 M3 [mU][mU][fU][fC][fA][fU][mC] [fU][mG][fA][mU][mG][mA][fA] [mA][mC][mA][mA][mA][mU] [mA][mU][mG][mG][mA][mC] [mC][mAs] [mAs][mAs][mG] 673 siRNA118- [mUs][mCs][mU][mA][mA][mA] 510 1319 [mUs][fAs][fG][mU][fU][fG][fU] 1170 2013 M3 [mC][mA][fA][fU][fC][fU][mU] [fC][mA][fA][mG][mA][mU][fU [mG][mA][mC][mA][mA][mC] [mG][mU][mU][mU][mA][mG] [mU][mAs] [mAs][mAs][mG] 1298 siRNA231- [mAs][mGs][mG][mU][mA][mU] 623 1447 [mUs][fCs][fC][mG][fU][fU][fU] 1171 2014 M3 [mC][mU][fU][fG][fA][fU][mG] [fU][mC][fA][mU][mC][mA][fA] [mA][mA][mA][mA][mC][mG] [mG][mA][mU][mA][mC][mC] [mG][mAs] [mUs][mAs][mG] 1561 siRNA272- [mAs][mUs][mA][mU][mA][mA] 664 1492 [mUs][fAs][fC][mA][fA][fA][fU] 1172 2015 M3 [mA][mU][fU][fC][fA][fA][mG] [fG][mC][fU][mU][mG][mA][fA] [mC][mA][mU][mU][mU][mG] [mU][mU][mU][mA][mU][mA] [mU][mAs] [mUs][mAs][mG] 1237 siRNA212- [mCs][mCs][mC][mA][mC][mA] |604 1425 [mUs][fCs][fU][mA][fA][fG][fU] 1173 2017 M3 [mG][mU][fU][fA][fA][fU][mG] [fG][mC][fA][mU][mU][mA][fA] [mC][mA][mC][mU][mU][mA] [mC][mU][mG][mU][mG][mG] [mG][mAs] [mGs][mAs][mG] 1228 siRNA206- [mAs][mGs][mC][mU][mC][mU] 598 1418 [mUs][fUs][fU][mA][fA][fC][fU] 1174 2021 M3 [mU][mU][fA][fC][fC][fC][mA] [fG][mU][fG][mG][mG][mU][fA] [mC][mA][mG][mU][mU][mA] [mA][mA][mG][mA][mG][mC] [mA][mAs] [mUs][mAs][mG] 1386 siRNA250- [mGs][mAs][mG][mC][mU][mA] 642 1467 [mUs][fUs][fC][mC][fA][fG][fG] 1175 2024 M3 [mC][mA][fA][fU][fA][fU][mG] [fA][mC][fA][mU][mA][mU][fU [mU][mC][mC][mU][mG][mG] [mG][mU][mA][mG][mC][mU] [mA][mAs] [mCs][mAs][mG] 1217 siRNA203- [mGs][mAs][mU][mG][mA][mC] 595 1414 [mUs][fUs][fA][mA][fA][fG][fA] 1176 2025 M3 [mA][mU][fC][fA][fU][fA][mG] [fG][mC][fU][mA][mU][mG][fA] [mC][mU][mC][mU][mU][mU] [mU][mG][mU][mC][mA][mU] [mA][mAs] [mCs][mAs][mG] 478 siRNA73- [mCs][mUs][mU][mG][mA][mA] 465 1270 [mUs][fUs][fU][mU][fC][fC][fG] 1177 2026 M3 [mU][mU][fC][fC][fC][fU][mC] [fU][mG][fA][mG][mG][mG][fA] [mA][mC][mG][mG][mA][mA] [mA][mU][mU][mC][mA][mA] [mA][mAs] [mGs][mAs][mG] 595 siRNA100- [mCs][mCs][mG][mA][mG][mU] 492 1299 [mUs][fCs][fA][mG][fC][fU][fU] 1178 2027 M3 [mG][mA][fU][fG][fU][fU][mU] [fC][mA][fA][mA][mC][mA][fU] [mG][mA][mA][mG][mC][mU] [mC][mA][mC][mU][mC][mG] [mG][mAs] [mGs][mAs][mG] 1291 siRNA226- [mAs][mUs][mA][mU][mG][mA] 618 1441 [mUs][fCs][fA][mU][fC][fA][fA] 1179 2028 M3 [mU][mA][fG][fG][fU][fA][mU] [fG][mA][f][mA][mC][mC][fU] [mC][mU][mU][mG][mA][mU] [mA][mU][mC][mA][mU][mA] [mG][mAs] [mUs][mAs][mG] 115 siRNA12- [[mUs][mUs][mG][mU][mC][mU] 404 1202 [mUs][fCs][fA][mA][fG][fA][fA] 1180 2029 M3 [mA][mU][fG][fG][fC][fU][mU] [fU][mA][fA][mG][mC][mC][fA] [mA][mU][mU][mC][mU][mU] [mU][mA][mG][mA][mC][mA] [mG][mAs] [mAs][mAs][mG] 477 siRNA72- [mCs][mCs][mU][mU][mG][mA] 464 1267 [mUs][fUs][fU][mC][fC][fG][fU] 1181 2034 M3 [mA][mU][fU][fC][fC][fC][mU] [fG][mA][fG][mG][mG][mA][fA] [mC][mA][mC][mG][mG][mA] [mU][mU][mC][mA][mA][mG] [mA][mAs] [mGs][mAs][mG] 1005 siRNA158- [mAs][mAs][mG][mA][mA][mG] 550 1363 [mUs][fUs][fA][mA][fU][fG][fU] 1182 2037 M3 [mU][mG][fA][fA][fA][fA][mG] [fU][mC][fU][mU][mU][mU][fC] [mA][mA][mC][mA][mU][mU] [mA][mC][mU][mU][mC][mU] [mA][mAs] [mUs][mAs][mG] 251 siRNA38- [mAs][mAs][mA][mC][mA][mU] 430 1231 [mUs][fGs][fU][mA][fA][fA][fA] 1183 2038 M3 [mG][mG][fU][fC][fA][fU][mG] [fA][mC][fA][mU][mG][mA][fC] [mU][mU][mU][mU][mU][mA] [mC][mA][mU][mG][mU][mU] [mC][mAs] [mUs][mAs][mG] 303 siRNA51- [mUs][mCs][mA][mC][mA][mA] 443 1246 [mUs][fGs][fU][mA][fU][fG][fA] 1184 2039 M3 [mA][mU][fC][fC][fC][fU][mU] [fC][mA][fA][mG][mG][mG][fA] [mG][mU][mC][mA][mU][mA] [mU][mU][mU][mG][mU][mG] [mC][mAs] [mAs][mAs][mG] 1209 siRNA199- [mUs][mCs][mC][mG][mA][mA] 591 1409 [mUs][fUs][fA][mU][fG][fA][fU] 1185 2043 M3 [mA][mA][fG][fA][fU][fG][mA] [fG][mU][fC][mA][mU][mC][fU] [mC][mA][mU][mC][mA][mU] [mU][mU][mU][mC][mG][mG] [mA][mAs] [mAs][mAs][mG] 841 siRNA124- [mCs][mCs][mU][mG][mC][mG] 516 1327 [mUs][fCs][fA][mU][fU][fG][fA] 1186 2044 M3 [mC][mA][fU][fG][fU][fU][mU] [fG][mA][fA][mA][mC][mA][fU [mC][mU][mC][mA][mA][mU] [mG][mC][mG][mC][mA][mG] [mG][mAs] [mGs][mAs][mG] 781 siRNA122- [mGs][mGs][mC][mA][mG][mA] 514 1324 [mUs][fCs][fG][mU][fG][fC][fC] 1187 2045 M3 [mG][mA][fG][fC][fU][fU][mG] [fU][mC][fA][mA][mG][mC][fU] [mA][mG][mG][mC][mA][mC] [mC][mU][mC][mU][mG][mC] [mG][mAs] [mCs][mAs][mG] 602 siRNA101- [mAs][mUs][mG][mU][mU][mU] 493 1301 [mUs][fAs][fA][mA][fU][fA][fC] 1188 2046 M3 [mG][mA][fA][fG][fC][fU][mG] [fC][mC][fA][mG][mC][mU][fU] [mG][mG][mU][mA][mU][mU] [mC][mA][mA][mA][mC][mA] [mU][mAs] [mUs][mAs][mG] 1433 siRNA260- [mCs][mAs][mA][mU][mU][mU] 652 1479 [mUs][fAs][fG][mC][fA][fU][fC] 1189 2050 M3 [mU][mU][fG][fA][fU][fU][mC] [fA][mG][fA][mA][mU][mC][fA] [mU][mG][mA][mU][mG][mC] [mA][mA][mA][mA][mU][mU] [mU][mAs] [mGs][mAs][mG] 506 siRNA81- [mGs][mAs][mA][mA][mA][mC] 473 1279 [mUs][fAs][fU][mG][fA][fU][fA] 1190 2051 M3 [mC][mU][fC][fC][fA][fA][mC] [fC][mG][fU][mU][mG][mG][fA] [mG][mU][mA][mU][mC][mA] [mG][mG][mU][mU][mU][mU] [mU][mAs] [mCs][mAs][mG] *siRNA#in Table 7B correspond to the siRNA#in Table 7A, but with M3added to indicate modification pattern {circumflex over ()}M3 refers to modification pattern MOD3 as shown in FIG. 1; mA, mC, mG, and mU are 2-O-methyl modified adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro modified adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate internucleoside linkage. Each uracil base (U) in any one of the sequences provided in Table 7B may independently and optionally be replaced with a thymine base (T). All siRNAs tested in this experiment are conjugated at the 3terminal nucleoside of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6). It is to be understood that the sof the 3-terminal [mAs]of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3terminal nucleoside of the sense strand) of a targeting moiety comprising a structure of Formula (Z.sup.6).
AAV8-Human CYP7A1 Mouse Model

(527) All animal studies were approved by the Institutional Animal Care and Use Committee at Takeda Development Center Americas. Mice were housed in a pathogen-free animal facility at 22 C. under controlled 12-hour light/12-hour dark cycle. All mice were kept on standard chow diet (PicoLab Rodent Diet 20-5053). Male C57Bl/6J mice, 8-10 weeks of age, were purchased from Jackson Laboratories (Sacrament, CA, USA). On day 7, animals received a single intravenous dose of 110.sup.11 vector genome copies of adeno-associated virus (AAV8 serotype) containing the gene encoding for human CYP7A1. On day 0, animals received a single subcutaneous dose of conjugated siRNA (conjugated at the 3terminal nucleoside of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6)). On day 7, day 14, or day 28, 30 mg of liver tissue was collected and snap frozen for RNA isolation and quantitative polymerase chain reaction (qPCR).

(528) RNA Extraction from Liver Tissue and Real Time PCR

(529) Total RNA was isolated from 30 mg pieces of frozen liver tissues using MagMAX mirVana Total RNA isolation kit (ThermoFisher). Quantitative PCR was carried out in duplicate with 50 ng of total RNA in 10 uL volume per well in 384-well plates using QuantiTect Multiplex RT-PCR (Qiagen) master mix and predesigned TaqMan primer and probes (ThermoFisher). Human CYP7A1 (Catalog #Hs00928359_g1) was used for target gene knockdown and endogenous control 18S (4319413E) was used as the reference gene. Relative gene expression was determined by using the delta-delta CT method, also known as the 2.sup.ct method.

(530) Relative fold gene expression was calculated by the delta-delta Ct method, also known as the 2.sup.ct method. Fold change was multiplied by 100 to represent the data as percent (%) mRNA remaining. Results are presented in Table 8.

(531) Statistical Analysis

(532) Results were expressed as meanSEM. Statistical analysis was performed using one-way ANOVA or two-way ANOVA followed, if significant, by post-hoc Dunnett's test. Non parametric tests like Mann-Whitney were used when the N was too small, or data did not follow Gaussian distribution. The difference was considered significant when p<0.05.

(533) TABLE-US-00046 TABLE 8 Results of the Initial in vivo Screening in Mouse Mean % mRNA Mean % Remaining Knockdown hAAV (3 mg/kg hAAV (3 mg/kg Position siRNA#* dose level, day 7) dose level, day 7) 223 siRNA27-M3 26.2 73.8 844 siRNA125-M3 23.4 76.6 1417 siRNA256-M3 38.6 61.4 1039 siRNA172-M3 34.7 65.3 1227 siRNA205-M3 25.5 74.5 1084 siRNA174-M3 22 78 1011 siRNA161-M3 50.6 49.4 1191 siRNA190-M3 38.5 61.5 292 siRNA47-M3 34.6 65.4 673 siRNA118-M3 35.8 64.2 1298 siRNA231-M3 38.3 61.7 1561 siRNA272-M3 44.2 55.8 1237 siRNA212-M3 33.6 66.4 1228 siRNA206-M3 24 76 1386 siRNA250-M3 53.5 46.5 1217 siRNA203-M3 58 42 478 siRNA73-M3 48.7 51.3 595 siRNA100-M3 45.7 54.3 1291 siRNA226-M3 41.3 58.7 115 siRNA12-M3 29.4 70.6 477 siRNA72-M3 28.3 71.7 1005 siRNA158-M3 40.9 59.1 251 siRNA38-M3 42.8 57.2 303 siRNA51-M3 34.3 65.7 1209 siRNA199-M3 52 48 841 siRNA124-M3 35.2 64.8 781 siRNA122-M3 45.3 54.7 602 siRNA101-M3 35.3 64.7 1433 siRNA260-M3 56.2 43.8 506 siRNA81-M3 13.8 86.2 *The siRNA# in Table 8 corresponds to the siRNA# in Table 7B. All siRNAs tested in this experiment are conjugated at the 3terminal nucleoside of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6).
Second Screening

(534) Twelve-siRNAs were selected based on their RNAi activity for further optimization. All these siRNA showed greater than 60% knockdown in a AAV model. Three (3) new modification patterns (MOD 1, MOD 2, and MOD 4, FIG. 1) were applied to these sequences (in addition to the modification pattern, MOD 3, used for the initial screening) to obtain 48-unique siRNAs (shown in Table 9).

(535) TABLE-US-00047 TABLE9 ListofChemicallyModifiedDuplexesPreparedforthesecondinvivoScreening SEQID SEQID Nucleobase NOof NOof SEQ modified Nucleobase modified ID sense SEQID antisense Position siRNA# SenseStrandSequence NO: strand: Anti-SenseStrandSequence NO: strand: 223 siRNA27- [mUs][mCs][mU][mU][mG][mA] 419 1219 [mUs][fUs][fU][mG][fC][fU][fC][fU] 1161 1989 M3{circumflex over ()} [mG][mU][fU][fC][fC][fU][mC] [mG][fA][mG][mG][mA][fA][mC] [mA][mG][mA][mG][mC][mA][mA] [mU][mC][mA][mA][mG][mAs] [mAs] [mAs][mG] 844 siRNA125- [mGs][mCs][mG][mC][mA][mU] 517 1329 [mUs][fUs][fG][mU][fC][fA][fU][fU] 1162 1993 M3 [mG][mU][fU][fU][fC][fU][mC] [mG][fA][mG][mA][mA][fA][mC] [mA][mA][mU][mG][mA][mC][mA] [mA][mU][mG][mC][mG][mCs][mAs [mAs] [mG] 1227 siRNA205- [mUs][mAs][mG][mC][mU][mC] 597 1417 [mUs][fUs][fA][mA][fC][fU][fG][fU] 1165 1999 M3 [mU][mU][fU][fA][fC][fC][mC] [mG][fG][mG][mU][mA][fA][mA] [mA][mC][mA][mG][mU][mU][mA] [mG][mA][mG][mC][mU][mAs] [mAs] [mAs][mG] 1084 siRNA174- [mAs][mCs][mU][mG][mA][mA] 566 1382 [mUs][fAs][fU][mA][fC][fU][fG][fG] 1166 2003 M3 [mU][mG][fA][fC][fC][fU][mG] [mC][fA][mG][mG][mU][fC][mA] [mC][mC][mA][mG][mU][mA][mU] [mU][mU][mC][mA][mG][mUs] [mAs] [mAs][mG] 292 siRNA47- [mUs][mGs][mU][mC][mC][mA] 439 1241 [mUs][fGs][fA][mU][fU][fU][fG][fU] 1169 2009 M3 [mU][mU][fU][fC][fA][fU][mC] [mG][fA][mU][mG][mA][fA][mA] [mA][mC][mA][mA][mA][mU][mC] [mU][mG][mG][mA][mC][mAs] [mAs [mAs][mG] 1237 siRNA212- [mCs][mCs][mC][mA][mC][mA] 604 1425 [mUs][fCs][fU][mA][fA][fG][fU][fG] 1173 2017 M3 [mG][mU][fU][fA][fA][fU][mG] [mC][fA][mU][mU][mA][fA][mC] [mC][mA][mC][mU][mU][mA][mG] [mU][mG][mU][mG][mG][mGs] [mAs [mAs][mG] 1228 siRNA206- [mAs][mGs][mC][mU][mC][mU] 598 1418 [mUs][fUs][fU][mA][fA][fC][fU][fG] 1174 2021 M3 [mU][mU][fA][fC][fC][fC][mA] [mU][fG][mG][mG][mU][fA][mA] [mC][mA][mG][mU][mU][mA][mA] [mA][mG][mA][mG][mC][mUs] [mAs] [mAs][mG] 115 siRNA12- [mUs][mUs][mG][mU][mC][mU] 404 1202 [mUs][fCs][fA][mA][fG][fA][fA][fU] 1180 2029 M3 [mA][mU][fG][fG][fC][fU][mU] [mA][fA][mG][mC][mC][fA][mU] [mA][mU][mU][mC][mU][mU][mG] [mA][mG][mA][mC][mA][mAs] [mAs] [mAs][mG] 477 siRNA72- [mCs][mCs][mU][mU][mG][mA] 464 1267 [mUs][fUs][fU][mC][fC][fG][fU][fG] 1181 2034 M3 [mA][mU][fU][fC][fC][fC][mU] [mA][fG][mG][mG][mA][fA][mU] [mC][mA][mC][mG][mG][mA][mA] [mU][mC][mA][mA][mG][mGs] [mAs] [mAs][mG] 303 siRNA51- [[mUs][mCs][mA][mC][mA][mA] 443 1246 [mUs][fGs][fU][mA][fU][fG][fA][fC] 1184 2039 M3 [mA][mU][fC][fC][fC][fU][mU] [mA][fA][mG][mG][mG][fA][mU] [mG][mU][mC][mA][mU][mA][mC] [mU][mU][mG][m][mG][mAs] [mAs [mAs][mG] 602 siRNA101- [mAs][mUs][mG][mU][mU][mU] 493 1301 [mUs][fAs][fA][mA][fU][fA][fC][fC] 1188 2046 M3 [mG][mA][fA][fG][fC][fU][mG] [mC][fA][mG][mC][mU][fU][mC] [mG][mG][mU][mA][mU][mU][mU] [mA][mA][mA][mC][mA][mUs] [mAs] [mAs][mG] 506 siRNA81- [mGs][mAs][mA][mA][mA][mC] 473 1279 [mUs][fAs][fU][mG][fA][fU][fA][fC] 1190 2051 M3 [mC][mU][fC][fC][fA][fA][mC] [mG][fU][mU][mG][mG][fA][mG] [mG][mU][mA][mU][mC][mA][mU] [mG][mU][mU][mU][mU][mCs] [mAs] [mAs][mG] 844 siRNA125- [mGs][mCs][mG][mC][mA][mU] 517 1329 [mUs][fUs][fG][mU][mC][mA][fU] 1162 1994 M4 [mG][mU][fU][fU][fC][fU][mC] [mU][mG][fA][mG][mA][mA][fA] [mA][mA][mU][mG][mA][mC][mA] [mC][mA][mU][mG][mC][mG][mCs] [mAs] [mAs][mG] 1227 siRNA205- [mUs][mAs][mG][mC][mU][mC] 597 1417 [mUs][fUs][fA][mA][mC][mU][fG] 1165 2000 M4 [mU][mU][fU][fA][fC][fC][mC] [mU][mG][fG][mG][mU][mA][fA] [mA][mC][mA][mG][mU][mU][mA] [mA][mG][mA][mG][mC][mU][mAs] [mAs] [mAs][mG] 1084 siRNA174- [mAs][mCs][mU][mG][mA][mA] 566 1382 [mUs][fAs][fU][mA][mC][mU][fG] 1166 2004 M4 [mU][mG][fA][fC][fC][fU][mG] [mG][mC][fA][mG][mG][mU][fC][mA] [mC][mC][mA][mG][mU][mA][mU] [mU][mU][mC][mA][mG][mUs] [mAs] [mAs][mG] 292 siRNA47- [mUs][mGs][mU][mC][mC][mA] 439 1241 [mUs][fGs][fA][mU][mU][mU][fG] 1169 2010 M4 [mU][mU][fU][fC][fA][fU][mC] [mU][mG][fA][mU][mG][mA][fA] [mA][mC][mA][mA][mA][mU][mC] [mA][mU][mG][mG][mA][mC][mAs] [mAs] [mAs][mG] 1237 siRNA212- [mCs][mCs][mC][mA][mC][mA] 604 1425 [mUs][fCs][fU][mA][mA][mG][fU] 1173 2018 M4 [mG][mU][fU][fA][fA][fU][mG] [mG][mC][fA][mU][mU][mA][fA] [mC][mA][mC][mU][mU][mA][mG] [mC][mU][mG][mU][mG][mG][mGs] [mAs] [mAs][mG] 1228 siRNA206- [mAs][mGs][mC][mU][mC][mU] 598 1418 [mUs][fUs][fU][mA][mA][mC][fU] 1174 2022 M4 [mU][mU][fA][fC][fC][fC][mA] [mG][mU][fG][mG][mG][mU][fA] [mC][mA][mG][mU][mU][mA][mA] [mA][mA][mG][mA][mG][mC][mUs] [mAs] [mAs][mG] 115 siRNA12- [mUs][mUs][mG][mU][mC][mU] 404 1202 [mUs][fCs][fA][mA][mG][mA][fA] 1180 2030 M4 [mA][mU][fG][fG][fC][fU][mU] [mU][mA][fA][mG][mC][mC][fA] [mA][mU][mU][mC][mU][mU][mG] [mU][mA][mG][mA][mC][mA][mAs] [mAs] [mAs][mG] 477 siRNA72- [mCs][mCs][mU][mU][mG][mA] 464 1267 [mUs][fUs][fU][mC][mC][mG][fU] 1181 2035 M4 [mA][mU][f][fC][fC][fC][mU] [mG][mA][fG][mG][mG][mA][fA] [mC][mA][mC][mG][mG][mA][mA] [mU][mU][mC][mA][mA][mG][mGs] [mAs] [mAs][mG] 303 siRNA51- [mUs][mCs][mA][mC][mA][mA] 443 1246 [mUs][fGs][fU][mA][mU][mG][fA] 1184 2040 M4 [mA][mU][fC][fC][fC][fU][mU] [mC][mA][fA][mG][mG][mG][fA] [mG][mU][mC][mA][mU][mA][mC] [mU][mU][mU][mG][mU][mG][mAs] [mAs] [mAs][mG] 602 siRNA101- [mAs][mUs][mG][mU][mU][mU] 493 1301 [mUs][fAs][fA][mA][mU][mA][fC] 1188 2047 M4 [mG][mA][fA][fG][fC][fU][mG] [mC][mC][fA][mG][mC][mU][fU] [mG][mG][mU][mA][mU][mU][mU] [mC][mA][mA][mA][mC][mA][mUs] [mAs] [mAs][mG] 506 siRNA81- [mGs][mAs][mA][mA][mA][mC] 473 1279 [mUs][fAs][fU][mG][mA][mU][fA] 1190 2052 M4 [mC][mU][fC][fC][fA][fA][mC] [mC][mG][fU][mU][mG][mG][fA] [mG][mU][mA][mU][mC][mA][mU] [mG][mG][mU][mU][mU][mU][mCs] [mAs] [mAs][mG] 223 siRNA27- [mUs][mCs][mU][mU][mG][mA] 419 1219 [mUs][fUs][fU][mG][mC][mU][fC] 1161 1990 M4 [mG][mU][fU][fC][fC][fU][mC] [mU][mG][fA][mG][mG][mA][fA] [mA][mG][mA][mG][mC][mA][mA] [mC][mU][mC][mA][mA][mG][mAs] [mAs] [mAs][mG] 844 siRNA125- [mGs][mCs][mG][mC][mA][mU] 517 1329 [mUs][fUs][fG][mU][fC][mA][fU] 1162 1995 M1 [mG][mU][fU][fU][fC][fU][mC] [fU][mG][fA][mG][mA][mA][fA][mC] [mA][mA][mU][mG][mA][mC][mA] [mA][mU][mG][mC][mG][mCs] [mAs] [mAs][mG] 1227 siRNA205- [mUs][mAs][mG][mC][mU][mC] 597 1417 [mUs][fUs][fA][mA][fC][mU][fG] 1165 2001 M1 [mU][mU][fU][fA][fC][fC][mC] [fU][mG][fG][mG][mU][mA][fA] [mA][mC][mA][mG][mU][mU][mA] [mG][mA][mG][mC][mU][mAs] [mAs] [mAs][mG] 1084 siRNA174- [mAs][mCs][mU][mG][mA][mA] 566 1382 [mUs][fAs][fU][mA][fC][mU][fG] 1166 2005 M1 [mU][mG][fA][fC][fC][fU][mG] [fG][mC][fA][mG][mG][mU][fC] [mC][mC][mA][mG][mU][mA][mU] [mU][mU][mC][mA][mG][mUs] [mAs] [mAs][mG] 292 siRNA47- [mUs][mGs][mU][mC][mC][mA] 439 1241 [mUs][fGs][fA][mU][fU][mU][fG] 1169 2011 M1 [mU][mU][fU][fC][fA][fU][mC] [fU][mG][fA][mU][mG][mA][fA] [mA][mC][mA][mA][mA][mU][mC] [mU][mG][mG][mA][mC][mAs] [mAs] [mAs][mG] 1237 siRNA212- [mCs][mCs][mC][mA][mC][mA] 604 1425 [mUs][fCs][fU][mA][fA][mG][fU] 1173 2016 M1 [mG][mU][fU][fA][fA][fU][mG] [fG][mC][fA][mU][mU][mA][fA][mC] [mC][mA][mC][mU][mU][mA][mG] [mU][mG][mU][mG][mG][mGs] [mAs] [mAs][mG] 1228 siRNA206- [mAs][mGs][mC][mU][mC][mU] 598 1418 [mUs][fUs][fU][mA][fA][mC][fU] 1174 2020 M1 [mU][mU][fA][fC][fC][fC][mA] [fG][mU][fG][mG][mG][mU][fA] [mC][mA][mG][mU][mU][mA][mA] [mA][mG][mA][mG][mC][mUs] [mAs] [mAs][mG] 115 siRNA12- [mUs][mUs][mG][mU][mC][mU] 404 1202 [mUs][fCs][fA][mA][fG][mA][fA] 1180 2031 M1 [mA][mU][fG][fG][fC][fU][mU] [fU][mA][fA][mG][mC][mC][fA][mU] [mA][mU][mU][mC][mU][mU][mG] [mA][mG][mA][mC][mA][mAs] [mAs] [mAs][mG] 477 siRNA72- [mCs][mCs][mU][mU][mG][mA] 464 1267 [mUs][fUs][fU][mC][fC][mG][fU] 1181 2036 M1 [mA][mU][fU][fC][fC][fC][mU] [fG][mA][fG][mG][mG][mA][fA][mU] [mC][mA][mC][mG][mG][mA][mA] [mU][mC][mA][mA][mG][mGs] [mAs] [mAs][mG] 303 siRNA51- [mUs][mCs][mA][mC][mA][mA] 443 1246 [mUs][fGs][fU][mA][fU][mG][fA] 1184 2041 M1 [mA][mU][fC][fC][fC][fU][mU] [fC][mA][fA][mG][mG][mG][fA][mU] [mG][mU][mC][mA][mU][mA][mC] [mU][mU][mG][mU][mG][mAs] [mAs] [mAs][mG] 602 siRNA101- [mAs][mUs][mG][mU][mU][mU] 493 1301 [mUs][fAs][fA][mA][fU][mA][fC] 1188 2048 M1 [mG][mA][fA][fG][fC][fU][mG] [fC][mC][fA][mG][mC][mU][fU][mC] [mG][mG][mU][mA][mU][mU][mU] [mA][mA][mA][mC][mA][mUs] [mAs] [mAs][mG] 506 siRNA81- [mGs][mAs][mA][mA][mA][mC] 473 1279 [mUs][fAs][fU][mG][fA][mU][fA] 1190 2053 M1 [mC][mU][fC][fC][fA][fA][mC] [fC][mG][fU][mU][mG][mG][fA][mG] [mG][mU][mA][mU][mC][mA][mU] [mG][mU][mU][mU][mU][mCs] [mAs] [mAs][mG] 223 siRNA27- [mUs][mCs][mU][mU][mG][mA] 419 1219 [mUs][fUs][fU][mG][fC][mU][fC] 1161 1991 M1 [mG][mU][fU][fC][fC][fU][mC] [fU][mG][fA][mG][mG][mA][fA][mC] [mA][mG][mA][mG][mC][mA][mA] [mU][mC][mA][mA][mG][mAs] [mAs] [mAs][mG] 844 siRNA125- [mGs][mCs][mG][mC][mA][mU] 517 1329 [mUs][fUs][fG][mU][fC][fA][fU] 1162 1996 M2 [mG][mU][fU][fU][fC][fU][mC] [mU][mG][fA][mG][mA][mA][fA][mC] [mA][mA][mU][mG][mA][mC][mA] [mA][mU][mG][mC][mG][mCs] [mAs] [mAs][mG] 1227 siRNA205- [mUs][mAs][mG][mC][mU][mC] 597 1417 [mUs][fUs][fA][mA][fC][fU][fG] 1165 2002 M2 [mU][mU][fU][fA][fC][fC][mC] [mU][mG][fG][mG][mU][mA][fA] [mA][mC][mA][mG][mU][mU][mA] [mG][mA][mG][mC][mU][mAs] [mAs] [mAs][mG] 1084 siRNA174- [mAs][mCs][mU][mG][mA][mA] 566 1382 [mUs][fAs][fU][mA][fC][fU][fG] 1166 2006 M2 [mU][mG][fA][fC][fC][fU][mG] [mG][mC][fA][mG][mG][mU][fC] [mC][mC][mA][mG][mU][mA][mU] [mU][mU][mC][mA][mG][mUs] [mAs] [mAs][mG] 292 siRNA47- [mUs][mGs][mU][mC][mC][mA] 439 1241 [mUs][fGs][fA][mU][fU][fU][fG] 1169 2012 M2 [mU][mU][fU][fC][fA][fU][mC] [mU][mG][fA][mU][mG][mA][fA] [mA][mC][mA][mA][mA][mU][mC] [mU][mG][mG][mA][mC][mAs] [mAs] [mAs][mG] 1237 siRNA212- [mCs][mCs][mC][mA][mC][mA] 604 1425 [mUs][fCs][fU][mA][fA][fG][fU] 1173 2019 M2 [mG][mU][fU][fA][fA][fU][mG] [mG][mC][fA][mU][mU][mA][fA][mC] [mC][mA][mC][mU][mU][mA][mG] [mU][mG][mU][mG][mG][mGs] [mAs] [mAs][mG] 1228 siRNA206- [mAs][mGs][mC][mU][mC][mU] 598 1418 [mUs][fUs][fU][mA][fA][fC][fU] 1174 2023 M2 [mU][mU][fA][fC][fC][fC][mA] [mG][mU][fG][mG][mG][mU][fA] [mC][mA][mG][mU][mU][mA][mA] [mA][mG][mA][mG][mC][mUs] [mAs] [mAs][mG] 115 siRNA12- [mUs][mUs][mG][mU][mC][mU] 404 1202 [mUs][fCs][fA][mA][fG][fA][fA] 1180 2032 M2 [mA][mU][fG][fG][fC][fU][mU] [mU][mA][fA][mG][mC][mC][fA][mU] [mA][mU][mU][mC][mU][mU][mG] [mA][mG][mA][mC][mA][mAs] [mAs] [mAs][mG] 477 siRNA72- [mCs][mCs][mU][mU][mG][mA] 464 1267 [mUs][fUs][fU][mC][fC][fG][fU] 1181 2033 M2 [mA][mU][fU][fC][fC][fC][mU] [mG][mA][fG][mG][mG][mA][fA][mU] [mC][mA][mC][mG][mG][mA][mA] [mU][mC][mA][mA][mG][mGs] [mAs] [mAs][mG] 303 siRNA51- [mUs][mCs][mA][mC][mA][mA] 443 1246 [mUs][fGs][fU][mA][fU][fG][fA] 1184 2042 M2 [mA][mU][fC][fC][fC][fU][mU] [mC][mA][fA][mG][mG][mG][fA][mU] [mG][mU][mC][mA][mU][mA][mC] [mU][mU][mG][mU][mG][mAs] [mAs] [mAs][mG] 602 siRNA101- [mAs][mUs][mG][mU][mU][mU] 493 1301 [mUs][fAs][fA][mA][fU][fA][fC] 1188 2049 M2 [mG][mA][fA][fG][fC][fU][mG] [mC][mC][fA][mG][mC][mU][fU][mC] [mG][mG][mU][mA][mU][mU][mU] [mA][mA][mA][mC][mA][mUs] [mAs] [mAs][mG] 506 siRNA81- [mGs][mAs][mA][mA][mA][mC] 473 1279 [mUs][fAs][fU][mG][fA][fU][fA] 1190 2054 M2 [mC][mU][fC][fC][fA][fA][mC] [mC][mG][fU][mU][mG][mG][fA][mG] [mG][mU][mA][mU][mC][mA][mU] [mG][mU][mU][mU][mU][mCs] [mAs] [mAs][mG] 223 siRNA27- [mUs][mCs][mU][mU][mG][mA] 419 1219 [mUs][fUs][fU][mG][fC][fU][fC] 1161 1992 M2 [mG][mU][fU][fC][fC][fU][mC] [mU][mG][fA][mG][mG][mA][fA][mC] [mA][mG][mA][mG][mC][mA][mA] [mU][mC][mA][mA][mG][mAs] [mAs] [mAs][mG] *The siRNA#corresponds to the siRNA#in Table 7A with different M#to indicate different modification patterns applied to the same nucleobase sequences. All siRNAs tested in this experiment are conjugated at the 3terminal nucleoside of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6). It is to be understood that the sof the 3-terminal [mAs]of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3terminal nucleoside of the sense strand) of a targeting moiety comprising a structure of Formula (Z.sup.6). {circumflex over ()}M1, M2, M3, and M4 refer to modification patterns MOD1, MOD2, MOD3, and MOD4, respectively, as shown in FIG. 1; mA, mC, mG, and mU are 2-O-methyl modified adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro modified adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate internucleoside linkage. Each uracil base (U) in any one of the sequences provided in Table 9 may independently and optionally be replaced with a thymine base (T).

(536) Table 10 summarizes the second in vivo screening results.

(537) TABLE-US-00048 TABLE 10 Results of the Second in vivo Screening in Mouse Mean % mRNA Mean % mRNA Remaining hAAV Remaining hAAV (2 mg/kg dose (2 mg/kg dose siRNA # level, day 14) level, day 28) 844 siRNA125-M3 53.3 42.8 1227 siRNA205-M3 44.9 57.5 1084 SIRNA174-M3 93.6 56.8 292 siRNA-47-M3 63.2 40.2 1237 siRNA212-M3 38.1 31.8 1228 siRNA206-M3 49.8 39.2 115 siRNA12-M3 57.0 49.1 477 siRNA72-M3 44.2 19.7 303 siRNA51-M3 48.5 32.5 602 siRNA101-M3 71.4 62.8 506 siRNA81-M3 43.4 61.4 223 siRNA27-M3 40.1 37.5 844 siRNA125-M4 51.4 50.9 1227 siRNA205-M4 96.0 63.9 1084 siRNA174-M4 85.5 52.2 292 siRNA47-M4 39.0 37.7 1237 siRNA212-M4 46.9 23.4 1228 siRNA206-M4 61.7 70.3 115 siRNA12-M4 57.9 31.8 477 siRNA72-M4 61.3 39.5 303 siRNA51-M4 49.0 33.5 602 siRNA101-M4 76.7 69.4 506 siRNA81-M4 51.1 41.7 223 siRNA27-M4 69.5 51.8 844 siRNA125-M1 35.5 45.4 1227 siRNA205-M1 70.1 76.7 1084 siRNA174-M1 57.6 89.2 292 siRNA47-M1 49.3 63.5 1237 siRNA212-M1 36.9 35.3 1228 siRNA206-M1 72.8 88.2 115 siRNA12-M1 35.9 38.8 477 siRNA72-M1 31.3 28.3 303 siRNA51-M1 27.5 35.8 602 siRNA101-M1 57.3 85.3 506 siIRNA81-M1 46.4 52.8 223 siRNA27-M1 51.5 73.1 844 siRNA125-M2 32.8 50.2 1227 siRNA205-M2 76.5 86.1 1084 siRNA174-M2 77.0 90.7 292 siRNA47-M2 31.1 65.3 1237 siRNA212-M2 45.3 67.7 1228 siRNA206-M2 39.4 87.4 115 siRNA12-M2 29.3 42.6 477 siRNA72-M2 29.9 71.5 303 siRNA51-M2 28.3 43.7 602 siRNA101-M2 42.5 81.8 506 siRNA81-M2 53.1 53.9 223 siRNA27-M2 44.7 50.4 * The siRNA# corresponds to the siRNA# in Table 9. All siRNAs tested in this experiment are conjugated at the 3terminal nucleoside of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6).

Example 5: In Vivo Efficacy in ANIT Cholestasis Model

(538) Mouse cross-reactive sequence siRNA206-M1 was used to demonstrate efficacy in a mouse model of cholestasis. -naphthylisothiocyanate (ANIT) is a commonly used chemical to induce intrahepatic cholestasis modeling human disease. C57Bl/6 mice were administered siRNA206-M1 on day 1, 8, and 15. ANIT was administered at 60 mg/kg by oral gavage on day 18. Serum chemistry, gene expression, and 7-hydroxy-4-cholesten-3-one (C4) biomarker analyses were performed on day 21. Gene expression data was represented as fold change over vehicle group using the delta Ct method.

(539) The results showed that the inhibition of CYP7A1 expression in hepatocytes leads to reduction in cholestasis. As shown in Tables 11 and 12, treatment with siRNA206-M1 resulted in the reduction in serum bile acids and liver injury markers ALP, AST, ALT, and bilirubin, indicating the decrease in liver injury. Cholestasis induced the expression of pro-fibrotic genes col1a1, timp1, and ccl2 as well as bile acid transport genes ostb, ntcp, and bsep while treatment with siRNA206-M1 normalized expression of these genes to near healthy control levels. Reduction of circulating CYP7A1 biomarker, serum C4, indicated inhibition of the target gene.

(540) TABLE-US-00049 TABLE 11 Serum Chemistry of ANIT Cholestasis Mice under Treatment Bilirubin Bile Acids ALP (U/L) AST (U/L) ALT (U/L) (mg/dL) C4 (ng/mL) (umol/L) Group sem sem sem sem sem sem Vehicle 289.3 39.93 4512 1068 2432 818.8 19.78 2.17 60.0 14.13 1769 306.8 siRNA206- 38.5 8.36 178.8 52.0 142.5 47.22 0.28 0.75 15.66 2.3 21.97 8.50 M1 Healthy 23.6 5.87 79.8 18.2 27 2.83 0.16 0.02 41.2 5.6 8.7 0.96 control

(541) TABLE-US-00050 TABLE 12 Gene Express Profile of ANIT Cholestasis Mice under Treatment Group Col1a1 sem Timp1 sem Ccl2 sem Ostb sem Ntcp sem Bsep sem Vehicle 1.11 0.22 1.04 0.13 1.008 0.06 1.01 0.08 1.04 0.12 1.04 0.14 siRNA206- 0.50 0.21 0.19 0.11 0.12 0.05 0.04 0.03 2.94 0.32 0.78 0.08 M1 Healthy 0.09 0.02 0.005 0.0008 0.014 0.003 0.006 0.001 3.39 0.17 0.87 0.05 control

Example 6: In Vitro RNAi Activity in HepaRG Human Hepatic Cell Line

(542) To assess the efficacy and potency of sequences against the human CYP7A1 transcript, an in vitro dose response study was performed in the HepaRG human hepatic cell line. HepaRG cells are widely considered a robust surrogate for primary human hepatocytes due to similar expression profile, unlike other hepatic cell lines which have lost major liver-like functions.

(543) Four (4) siRNA constructs, conjugated siRNA212-M1, conjugated siRNA206-M1, conjugated siRNA72-M2, or conjugated siRNA12-M3 (shown in Table 13), were tested in HepaRG cells. HepaRG cells were plated in a 96-well plate at 60,000 cells/well containing antibiotic-free medium and placed at 37 C. in an atmosphere with 5% CO.sub.2 in a humidified incubator. Within 24 hours after seeding, cells were transfected with biological triplicates for each treatment. A 5-fold, 11-point dose response with a top dose of 1 M of CYP7A1 siRNA constructs were applied to the cells following the manufacturer's recommendation (Thermofisher) for Lipofectamine RNAiMax. Cells were returned to the incubator for 48 hours with a medium change at 24 hours. Cell lysates were prepared 48 hours post-transfection by removing medium and washing with cold PBS and the addition of lysis buffer (following the manufacturer's recommendation for Cells-to-CT 1-step TaqMan Kit, Thermofisher). Reverse transcription was performed on cell lysates which were then used for qPCR. qPCR was performed in duplicate for each sample in a 10 l reaction mix, multiplexing the probe for CYP7A1 with the housekeeping gene probe (GAPDH): Nuclease-free water 5.5 l+qRT-PCR Master Mix 2.5 l+TaqMan Primer-1, 0.5 l+TaqMan Primer-2, 0.5 l.

(544) For each well, the target mRNA level was normalized to the respective GAPDH mRNA level and relative expression was calculated based on mock transfection control wells. Dose response curves were generated in GraphPad Prism and used to calculate half-maximal inhibitory concentrations (IC50) and maximal gene knockdown of a given siRNA construct. Table 14 summarizes the in vitro screening results.

(545) TABLE-US-00051 TABLE13 ChemicallyModifiedDuplexesPreparedfortheinvitroHepaRGCellStudy SEQID SEQID NOof NOof Nucleobase modified Nucleobase modified SEQ sense SEQ antisense Position siRNA#* SenseStrandSequence IDNO: strand: Anti-SenseStrandSequence IDNO: strand: 1237 siRNA212- [mCs][mCs][mC][mA][mC] 604 1425 [mUs][fCs][fU][mA][fA][mG] 1173 2016 M1 [mA][mG][mU][fU][fA][fA] [fU][fG][mC][fA][mU][mU] [fU][mG][mC][mA][mC][mU] [mA][fA][mC][mU][mG][mU] [mU][mA][mG][mAs] [mG][mG][mGs][mAs][mG] 1228 siRNA206- [mAs][mGs][mC][mU][mC] 598 1418 [mUs][fUs][fU][mA][fA][mC] 1174 2020 M1 [mU][mU][mU][fA][fC][fC] [fU][fG][mU][fG][mG][mG] [fC][mA][mC][mA][mG][mU] [mU][fA][mA][mA][mG][mA] [mU][mA][mA][mAs] [mG][mC][mUs][mAs][mG] 477 siRNA72- [mCs][mCs][mU][mU][mG] 464 1267 [mUs][fUs][fU][mC][fC][fG] 1181 2033 M2 [mA][mA][mU][fU][fC][fC] [fU][mG][mA][fG][mG][mG] [fC][mU][mC][mA][mC][mG] [mA][fA][mU][mU][mC][mA] [mG][mA][mA][mAs] [mA][mG][mGs][mAs][mG] 115 siRNA12- [mUs][mUs][mG][mU][mC] 404 1202 [mUs][fCs][fA][mA][fG][fA] 1180 2029 M3 [mU][mA][mU][fG][fG][fC] [fA][fU][mA][fA][mG][mC][mC] [fU][mU][mA][mU][mU][mC] [fA][mU][mA][mG][mA][mC] [mU][mU][mG][mAs] [mA][mAs][mAs][mG] *The siRNA#corresponds to the siRNA#in Table 9. All siRNAs tested in this experiment are conjugated at the 3terminal nucleoside of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6). It is to be understood that the sof the 3-terminal [mAs]of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3terminal nucleoside of the sense strand) of a targeting moiety comprising a structure of Formula (Z.sup.6). Each uracil base (U) in any one of the sequences provided in Table 12 may independently and optionally be replaced with a thymine base (T).

(546) TABLE-US-00052 TABLE 14 In vitro Screening of siRNAs in HepaRG Human Hepatic Cell Line Maximum mRNA Position siRNA#* IC50 (nM) knockdown (%) 1237 siRNA212-M1 0.16 69 1228 siRNA206-M1 0.24 78 115 siRNA12-M3 0.34 72 477 siRNA72-M2 0.52 61 *The siRNA# corresponds to the siRNA# in Table 13. All siRNAs tested in this experiment are conjugated at the 3terminal nucleoside of the sense strand to a targeting moiety comprising a structure of Formula (Z.sup.6).

Example 7: In Vivo Study of siRNA Agents in Non-Human Primate (NHP)

(547) The same four (4) siRNAs from Example 6 were tested in a single injection, single dose level (20 mg/kg), 56-day duration study in cynomolgus monkeys (Macaca fascicularis). The research protocol and animal housing were approved by the IACUC of Huazhen Laboratory Animal Breeding Center, Guangzhou China. Briefly, healthy nave male cynomolgus monkeys 3-5 years of age were individually housed in a controlled environment with 12:12-h light:darkness cycle, controlled humidity range of 60-80% and temperature maintained in the range of 18-26 C. Animals were fed twice a day (BID) with a snack in between meals and had free access to drinking water. Animals were acclimated to all experimental procedures prior to study initiation. Vehicle, conjugated siRNA212-M1, conjugated siRNA206-M1, conjugated siRNA72-M2, or conjugated siRNA12-M3 (shown in Table 13) was administered in a single subcutaneous injection on day 0. Blood samples were collected at pre-dose day 7 and on days 7, 14, 21, 28, 35, 42, 49, and 56. All samples were analyzed for serum 7-hydroxy-4-cholesten-3-one (C4), total bile acid, and cholic acid levels by mass spectrometry. C4 data are represented as percent reduction relative to vehicle group while total bile acid and cholic acid are represented percent reduction relative to pre-treatment samples.

(548) Target engagement and pathway modulation were assessed by quantitation of serum C4 and the primary bile acid, cholic acid. Around 40-60% decrease in plasma C4 level was observed. Around 35-90% decrease in total bile acid, and around 40-97% decrease in cholic acid was observed.

ADDITIONAL EMBODIMENTS

(549) 1. An RNAi agent for inhibiting expression of Cytochrome P450 family 7 subfamily A member 1 (CYP7A1) in a cell, wherein the RNAi agent comprises a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises a region of complementarity of at least 15 nucleosides to a CYP7A1 target sequence in Table 2, wherein the region of complementarity comprises a nucleoside sequence that contains no more than 3 mismatches to the CYP7A1 target sequence, and wherein the sense strand is at least substantially complementary to the antisense strand.
2. An RNAi agent for inhibiting expression of Cytochrome P450 family 7 subfamily A member 1 (CYP7A1) in a cell, wherein the RNAi agent comprises a sense strand and an antisense strand forming a duplex region, wherein the antisense strand comprises a region of complementarity of at least 15 nucleosides to a CYP7A1 target sequence of nucleotides 113-133, 221-241, 249-269, 290-310, 301-321, 475-495, 476-496, 504-524, 593-613, 600-620, 671-691, 779-799, 839-859, 842-862, 1003-1023, 1009-1029, 1037-1057, 1082-1102, 1189-1209, 1207-1227, 1215-1235, 1225-1245, 1226-1246, 1235-1255, 1289-1309, 1296-1316, 1384-1404, 1415-1435, 1431-1451, or 1559-1579 of SEQ ID NO: 1, wherein the region of complementarity comprises a nucleoside sequence that contains no more than 3 mismatches to the CYP7A1 target sequence, and wherein the sense strand is at least substantially complementary to the antisense strand.
3. The RNAi agent of embodiment 1 or embodiment 2, wherein the antisense strand comprises at least 15 consecutive nucleobases of any one of SEQ ID NOs: 777-1190.
4. The RNAi agent of any one of embodiments 1-3, wherein the sense strand comprises at least 15 consecutive nucleobases of any one of SEQ ID NOs: 393-776.
5. The RNAi agent of any one of embodiments 1-4, wherein the sense strand is 21 nucleosides in length and the antisense strand is 23 nucleosides in length, optionally wherein the RNAi agent comprises a 3 overhang of at least 1 nucleoside on a least one strand.
6. The RNAi agent of any one of embodiments 1-4, wherein the antisense strand comprises nucleobases 2-21 (counting 5.fwdarw.3) of any one of SEQ ID NOs: 777-1190 and the sense strand comprises the nucleobase sequence of any one of SEQ ID NOs: 393-776.
7. The RNAi agent of any one of embodiments 1-6, wherein the antisense strand comprises the nucleobase sequence of any one of SEQ ID NOs: 777-1190 and the sense strand comprises the nucleobase sequence of any one of SEQ ID NOs: 393-776.
8. The RNAi agent of any one of embodiments 1-7, wherein the RNAi agent comprises the nucleobases sequences of an siRNA selected from siRNA1-siRNA384, siRNA12, siRNA27, siRNA38, siRNA47, siRNA51, siRNA72, siRNA73, siRNA81, siRNA100, siRNA101, siRNA118, siRNA122, siRNA124, siRNA125, siRNA158, siRNA161, siRNA172, siRNA174, siRNA190, siRNA199, siRNA203, siRNA205, siRNA206, siRNA212, siRNA226, siRNA231, siRNA250, siRNA256, siRNA260, and siRNA272.
9. The RNAi agent of any one of embodiments 1-7, wherein the antisense strand comprises a nucleobase sequence selected from (5.fwdarw.3):

(550) TABLE-US-00053 (SEQIDNO:1161) (i) UUUGCUCUGAGGAACUCAAGAAG (SEQIDNO:1162) (ii) UUGUCAUUGAGAAACAUGCGCAG (SEQIDNO:1165) (iii) UUAACUGUGGGUAAAGAGCUAAG (SEQIDNO:1166) (iv) UAUACUGGCAGGUCAUUCAGUAG (SEQIDNO:1169) (v) UGAUUUGUGAUGAAAUGGACAAG (SEQIDNO:1173) (vi) UCUAAGUGCAUUAACUGUGGGAG (SEQIDNO:1174) (vii) UUUAACUGUGGGUAAAGAGCUAG (SEQIDNO:1180) (viii) UCAAGAAUAAGCCAUAGACAAAG (SEQIDNO:1181) (ix) UUUCCGUGAGGGAAUUCAAGGAG (SEQIDNO:1184) (x) UGUAUGACAAGGGAUUUGUGAAG (SEQIDNO:1188) (xi) UAAAUACCCAGCUUCAAACAUAG; and (SEQIDNO:1190) (xii) UAUGAUACGUUGGAGGUUUUCAG.
10. The RNAi agent of embodiment 9, wherein the sense strand comprises a nucleobase sequence selected from (5.fwdarw.3):

(551) TABLE-US-00054 (SEQIDNO:419) (i) UCUUGAGUUCCUCAGAGCAAA; (SEQIDNO:517) (ii) GCGCAUGUUUCUCAAUGACAA; (SEQIDNO:597) (iii) UAGCUCUUUACCCACAGUUAA; (SEQIDNO:566) (iv) ACUGAAUGACCUGCCAGUAUA; (SEQIDNO:439) (v) UGUCCAUUUCAUCACAAAUCA; (SEQIDNO:604) (vi) CCCACAGUUAAUGCACUUAGA; (SEQIDNO:598) (vii) AGCUCUUUACCCACAGUUAAA; (SEQIDNO:404) (viii) UUGUCUAUGGCUUAUUCUUGA; (SEQIDNO:464) (ix) CCUUGAAUUCCCUCACGGAAA; (SEQIDNO:443) (x) UCACAAAUCCCUUGUCAUACA; (SEQIDNO:493) (xi) AUGUUUGAAGCUGGGUAUUUA; and (SEQIDNO:473) (xii) GAAAACCUCCAACGUAUCAUA.
11. The RNAi agent of any one of embodiments 1-10, wherein the RNAi agent comprises one or more modified nucleosides, optionally wherein each nucleoside of the antisense strand is a modified nucleoside and each nucleoside of the sense strand is a modified nucleoside.
12. The RNAi agent of embodiment 11, wherein the one or more modified nucleosides are 2 modified nucleosides.
13. The RNAi agent of embodiment 12, wherein the 2-modified nucleoside is selected from 2-fluoro (2-F), 2-O-methyl (2-O-Me), 2-O-methoxyethyl (2-MOE), 2-O-aminopropyl (2-O-AP), 2-O-dimethylaminoethyl (2-O-DMAOE), 2-O-dimethylaminopropyl (2-O-DMAP), 2-O-dimethylaminoethyloxyethyl (2-O-DMAEOE), or 2-ON-methylacetamido (2-O-NMA) modified nucleoside and combinations thereof, optionally wherein the 2-modified nucleoside is selected from a 2-O-methyl (2-O-Me) modified nucleoside, a 2-fluoro (2-F) modified nucleoside, and combinations thereof.
14. The RNAi agent of any one of embodiments 11-13, wherein each nucleoside of the antisense strand is selected from a 2-F modified nucleoside and a 2-O-Me modified nucleoside, and each nucleoside of the sense strand is a 2-modified nucleoside selected from a 2-F modified nucleoside and a 2-O-Me modified nucleoside.
15. The RNAi agent of any one of embodiments 11-14, wherein the nucleosides at one or more positions 9, 10, 11, and 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, optionally wherein the nucleosides at positions 9, 10, 11, and 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides.
16. The RNAi agent of any one of embodiments 11-15, wherein the nucleosides at one or more positions 1, 2, 3, 4, 5, 6, 7, 8 and 13, 14, 15, 16, 17, 18, 19, 20, 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides, optionally wherein the nucleosides at positions 1, 2, 3, 4, 5, 6, 7, 8 and 13, 14, 15, 16, 17, 18, 19, 20, 21 (counting 5.fwdarw.3) of the sense strand are 2-O-Me modified nucleosides.
17. The RNAi agent of any one of embodiments 11-16, wherein the nucleosides at one or more positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides.
18. The RNAi agent of any one of embodiments 11-17, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides.
19. The RNAi agent of any one of embodiments 11-17, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides.
20. The RNAi agent of any one of embodiments 11-17, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides.
21. The RNAi agent of any one of embodiments 11-17, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides.
22. The RNAi agent of any one of embodiments 11-21, wherein the nucleosides at one or more positions 1, 4, 5, 6, 8, 9, 11-13, and 15-23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides, optionally wherein the nucleosides at positions 1, 4, 9, 11-13, and 15-23 (counting 5.fwdarw.3) of the antisense strand are 2-O-Me modified nucleosides.
23. The RNAi agent of any one of embodiments 1-22, wherein the RNAi agent comprises one or more modified internucleoside linkages, optionally wherein the RNAi agent comprises one or more phosphorothioate internucleoside linkages in at least one strand.
24. The RNAi agent of any one of embodiments 1-23, wherein the RNAi agent comprises two phosphorothioate internucleoside linkages in the sense strand, optionally wherein the two phosphorothioate internucleoside linkages are the first two internucleoside linkages in the sense strand from 5.fwdarw.3.
25. The RNAi agent of any one of embodiments 1-24, wherein the RNAi agent comprises four phosphorothioate internucleoside linkages in the antisense strand, wherein the four phosphorothioate internucleoside linkages are the first two internucleoside linkages and the last two internucleoside linkages in the antisense strand from 5.fwdarw.3.
26. The RNAi agent of any one of embodiments 1-25, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.
27. The RNAi agent of any one of embodiments 1-25, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.
28. The RNAi agent of any one of embodiments 1-25, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 5, 6, 7, 8, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.
29. The RNAi agent of any one of embodiments 1-25, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the nucleosides at positions 9, 10, 11, 12 (counting 5.fwdarw.3) of the sense strand are 2-F modified nucleosides, wherein the nucleosides at positions 2, 3, 7, 10, and 14 (counting 5.fwdarw.3) of the antisense strand are 2-F modified nucleosides, wherein all nucleosides of the antisense strand and the sense strand that are not 2-F modified nucleosides are 2-O-Me modified nucleosides, and wherein the first two internucleoside linkages of the sense strand (from 5.fwdarw.3) and the first two and the last two internucleoside linkages of the antisense strand (from 5.fwdarw.3) are phosphorothioate internucleoside linkages.
30. The RNAi agent of embodiment 1 or embodiment 2, wherein the antisense strand and the sense strand comprise a structure as provided in Table 5B, Table 7B, or Table 9.
31. The RNAi agent of embodiment 1 or embodiment 2, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1173 and a structure (5.fwdarw.3) of [mUs][fCs][fU][mA][fA][mG][fU][fG][mC][fA][mU][mU][mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG] (SEQ ID NO: 2016), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
32. The RNAi agent of embodiment 31, wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 604 and a structure (5.fwdarw.3) of [mCs][mCs][mC][mA][mC][mA][mG][mU][fU][fA][fA][fU][mG][mC][mA][mC][mU][mU][mA][mG][mAs] (SEQ ID NO: 1425), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
33. The RNAi agent of embodiment 1 or embodiment 2, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1174 and a structure (5.fwdarw.3) of [mUs][fUs][fU][mA][fA][mC][fU][fG][mU][fG][mG][mG][mU][fA][mA][mA][mG][mA][mG][mC][mUs][mAs][mG] (SEQ ID NO: 2020), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
34. The RNAi agent of embodiment 33, wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 598 and a structure (5.fwdarw.3) of [mAs][mGs][mC][mU][mC][mU][mU][mU][fA][fC][fC][fC][mA][mC][mA][mG][mU][mU][mA][mA][mAs] (SEQ ID NO: 1418), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
35.0. The RNAi agent of embodiment 1 or embodiment 2, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) selected from the group consisting of

(552) TABLE-US-00055 (SEQIDNO:2055) [mUs][fCs][mA][fA][mG][fA][mA][fU][mA][fA][mG][mC] [mC][fA][mU][fA][mG][fA][mC][fA][mAs][mAs][mG], (SEQIDNO:2056) [mUs][fCs][fA][fA][mG][fA][mA][fU][mA][fA][mG][fC] [mC][fA][mU][fA][mG][fA][mC][fA][mAs][mAs][mG], (SEQIDNO:2057) [mUs][fCs][mA][mA][mG][fA][mA][fU][fA][mA][mG][mC] [mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG], (SEQIDNO:2058) [mUs][fCs][mA][mA][mG][fA][mA][mU][mA][mA][mG][mC] [mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG], and (SEQIDNO:2059) [mUs][fCs][mA][mA][mG][fA][gA][fU][fA][mA][mG][mC] [mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG],
wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; gA is GNA adenosine; and s is a phosphorothioate linkage.
35. The RNAi agent of embodiment 1 or embodiment 2, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][fA][mA][fG][fA][fA][fU][mA][fA][mG][mC][mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2029), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
36.0. The RNAi agent of embodiment 35.0, wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of

(553) TABLE-US-00056 (SEQIDNO:2060) [fUs][mUs][fG][mU][fC][mU][fA][mU][fG][fG][fC][mU] [fU][mA][fU][mU][fC][mU][fU][mG][fAs], (SEQIDNO:2062) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][mG][fC][mU] [fU][mA][fU][mU][mC][mU][mU][mG][mAs], and (SEQIDNO:2061) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][fG][fC][mU] [mU][mA][mU][mU][mC][mU][mU][mG][mAs],
wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage.
36. The RNAi agent of embodiment 35, wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of [mUs][mUs][mG][mU][mC][mU][mA][mU][fG][fG][fC][fU][mU][mA][mU][mU][mC][mU][mU][mG][mAs] (SEQ ID NO: 1202), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
37. The RNAi agent of embodiment 1 or embodiment 2, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1181 and a structure (5.fwdarw.3) of [mUs][fUs][fU][mC][fC][fG][fU][mG][mA][fG][mG][mG][mA][fA][mU][mU][mC][mA][mA][mG][mGs][mAs][mG] (SEQ ID NO: 2033), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
38. The RNAi agent of embodiment 37, wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 464 and a structure (5.fwdarw.3) of [mCs][mCs][mU][mU][mG][mA][mA][mU][fU][fC][fC][fC][mU][mC][mA][mC][mG][mG][mA][mA][mAs] (SEQ ID NO: 1267), wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, 2-O-methyl cytidine, 2-O-methyl guanosine, and 2-O-methyl uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, 2-fluoro cytidine, 2-fluoro guanosine, and 2-fluoro uridine, respectively; and s is a phosphorothioate linkage.
39. The RNAi agent of any one of embodiments 1-38, further comprising a targeting moiety.
40. The RNAi agent of embodiment 39, wherein the targeting moiety is conjugated to the 3 end of the sense strand of the RNAi agent.
41. The RNAi agent of embodiment 39 or embodiment 40, wherein the targeting moiety comprises N-acetyl-galactosamine (GalNAc), optionally wherein the targeting moiety comprises a GalNAc trimer.
42. The RNAi agent of any one of embodiments 39-41, wherein the targeting moiety comprises one or more instances of GalNAc attached through a monovalent, bivalent, trivalent, or tetravalent branched linker.
43. The RNAi agent of embodiment 39, wherein the targeting moiety is of formula:

(554) ##STR00151##
or a pharmaceutically acceptable salt thereof, wherein the

(555) ##STR00152##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand.
43.1. The RNAi agent of embodiment 39, wherein the targeting moiety is of formula:

(556) ##STR00153##
or a pharmaceutically acceptable salt thereof, wherein the

(557) ##STR00154##
indicates the attachment point that is covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand.
44. The RNAi agent of embodiment 43 or 43.1, wherein the resulting conjugate comprises a structure of:

(558) ##STR00155##
or a pharmaceutically acceptable salt thereof.
45. An RNAi agent comprisiing: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1173 and a strucutre (5.fwdarw.3) of [mUs][fCs][fU][mA][fA][mG][fU][fG][mC][fA][mU][mU][mA][fA][mC][mU][mG][mU][mG][mG][mGs][mAs][mG] (SEQ ID NO: 2016), wherein: an a sense strand comprising the nucleobase sequence of SEQ ID NO: 604 and a structure (5.fwdarw.3) of

(559) TABLE-US-00057 (SEQIDNO:1425) [mCs][mCs][mC][mA][mC][mA][mG][mU][fU][fA][fA][fU] [mG][mC][mA][mC][mU][mU][mA][mG][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(560) ##STR00156##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
46. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1174 and a structure (5.fwdarw.3) of [mUs][fUs][fU][mA][fA][mC][fU][fG][mU][fG][mG][mG][mU][fA][mA][mA][mG][mA][mG][mC][mUs][mAs][mG] (SEQ ID NO: 2020); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 598 and a structure (5.fwdarw.3) of

(561) TABLE-US-00058 (SEQIDNO:1418) [mAs][mGs][mC][mU][mC][mU][mU][mU][fA][fC][fC][fC] [mA][mC][mA][mG][mU][mU][mA][mA][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(562) ##STR00157##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
47. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][fA][mA][fG][fA][fA][fU][mA][fA][mG][mC][mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2029); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of

(563) TABLE-US-00059 (SEQIDNO:1202) [mUs][mUs][mG][mU][mC][mU][mA][mU][fG][fG][fC][fU] [mU][mA][mU][mU][mC][mU][mU][mG][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(564) ##STR00158##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
47.1. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][mA][fA][mG][fA][mA][fU][mA][fA][mG][mC][mC][fA][mU][fA][mG][fA][mC][fA][mAs][mAs][mG] (SEQ ID NO: 2055); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of

(565) TABLE-US-00060 (SEQIDNO:2060) [fUs][mUs][fG][mU][fC][mU][fA][mU][fG][fG][fC][mU] [fU][mA][fU][mU][fC][mU][fU][mG][fAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(566) ##STR00159##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
47.2. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.>3) of [mUs][fCs][fA][fA][mG][fA][mA][fU][mA][fA][mG][fC][mC][fA][mU][fA][mG][fA][mC][fA][mAs][mAs][mG] (SEQ ID NO: 2056); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of

(567) TABLE-US-00061 (SEQIDNO:2062) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][mG][fC][mU] [fU][mA][fU][mU][mC][mU][mU][mG][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(568) ##STR00160##
or a pharmaceutically accept-able salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
47.3. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][mA][mA][mG][fA][mA][fU][fA][mA][mG][mC][mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2057); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of

(569) TABLE-US-00062 (SEQIDNO:2061) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][fG][fC][mU] [mU][mA][mU][mU][mC][mU][mU][mG][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(570) ##STR00161##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
47.4. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][mA][mA][mG][fA][mA][mU][mA][mA][mG][mC][mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2058); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of

(571) TABLE-US-00063 (SEQIDNO:2061) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][fG][fC][mU] [mU][mA][mU][mU][mC][mU][mU][mG][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(572) ##STR00162##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
47.5. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][mA][mA][mG][fA][gA][fU][fA][mA][mG][mC][mC][fA][mU][fA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2059); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of

(573) TABLE-US-00064 (SEQIDNO:2061) [mUs][mUs][mG][mU][mC][mU][fA][mU][fG][fG][fC][mU] [mU][mA][mU][mU][mC][mU][mU][mG][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; gA is GNA adenosine; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(574) ##STR00163##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
48. An RNAi agent comprising: an antisense strand comprising the nucleobase sequence of SEQ ID NO: 1181 and a structure (5.fwdarw.3) of [mUs][fUs][fU][mC][fC][fG][fU][mG][mA][fG][mG][mG][mA][fA][mU][mU][mC][mA][mA][mG][mGs][mAs][mG] (SEQ ID NO: 2033); and a sense strand comprising the nucleobase sequence of SEQ ID NO: 464 and a structure (5.fwdarw.3) of

(575) TABLE-US-00065 (SEQIDNO:1267) [mCs][mCs][mU][mU][mG][mA][mA][mU][fU][fC][fC][fC] [mU][mC][mA][mC][mG][mG][mA][mA][mAs];
wherein mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage;
and wherein the 3 terminal nucleoside of the sense strand is covalently linked to a structure of Formula (Z.sup.6)

(576) ##STR00164##
or a pharmaceutically acceptable salt thereof, wherein the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of the first repeat unit of a targeting moiety (i.e., the repeat unit that is directly covalently linked to the 3-O of the sugar moiety of the 3 terminal nucleoside of the sense strand), wherein the targeting moiety comprises a structure of Formula (Z.sup.6).
49. A conjugate comprising a Cytochrome P450 family 7 subfamily A member 1 (CYP7A1) RNAi agent covalently linked to a targeting moiety, wherein the CYP7A1 RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand comprises the nucleobase sequence of SEQ ID NO: 1180 and a structure (5.fwdarw.3) of [mUs][fCs][fA][mA][fG][fA][fA][fU][mA][fA][mG][mC][mC][fA][mU][mA][mG][mA][mC][mA][mAs][mAs][mG] (SEQ ID NO: 2029); and wherein the sense strand comprises the nucleobase sequence of SEQ ID NO: 404 and a structure (5.fwdarw.3) of [mUs][mUs][mG][mU][mC][mU][mA][mU][fG][fG][fC][fU][mU][mA][mU][mU][mC][mU][mU][mG][mAs] (SEQ ID NO: 1202); wherein: mA, mC, mG, and mU are 2-O-methyl adenosine, cytidine, guanosine, and uridine, respectively; fA, fC, fG, and fU are 2-fluoro adenosine, cytidine, guanosine, and uridine, respectively; and s is a phosphorothioate linkage; wherein the targeting moiety is covalently linked to the 3 terminal nucleoside of the sense strand and the s of the 3-terminal [mAs] of the sense strand corresponds to the phosphorothioate linkage of a first repeat unit of the targeting moiety, wherein the conjugate comprises a structure of:

(577) ##STR00165##
or a pharmaceutically acceptable salt thereof.
50. A pharmaceutical composition for inhibiting expression of a gene encoding CYP7A1 comprising the RNAi agent of any one of embodiments 1-48 or the conjugate of embodiment 49.
51. A method of inhibiting expression of a CYP7A1 gene in a cell, the method comprising contacting the cell with the RNAi agent of any one of embodiments 1-48, the conjugate of embodiment 49, or the pharmaceutical composition of embodiment 50, thereby inhibiting expression of the CYP7A1 gene in the cell.
52. The method of embodiment 51, wherein the cell is within a subject, optionally wherein the subject is human.
53. The method of embodiment 52, wherein the subject has a CYP7A1 disease or CYP7A1-associated disease.
54. A method of treating a subject having a CYP7A1 disease or CYP7A1-associated disease, comprising administering to the subject a therapeutically effective amount of the RNAi agent of any one of embodiments 1-48, the conjugate of embodiment 49, or the pharmaceutical composition of embodiment 50, thereby treating the subject having the CYP7A1 disease or CYP7A1-associated disease.
55. A method of treating at least one symptom in a subject having a CYP7A1 disease or CYP7A1-associated disease, comprising administering to the subject an effective amount of the RNAi agent of any one of embodiments 1-48, the conjugate of embodiment 49, or the pharmaceutical composition of embodiment 50, thereby treating at least one symptom in the subject having the CYP7A1 disease or CYP7A1-associated disease.
56. The method of embodiment 54 or embodiment 55, wherein the disease is a CYP7A1 disease or CYP7A1-associated disease, optionally wherein the CYP7A1 disease or CYP7A1-associated disease is primary sclerosing cholangitis (PSC).
57. The method of any one of embodiments 54-56, wherein the subject is human.
58. The method of any one of embodiments 54-57, further comprising administering an agent for the treatment of a CYP7A1 disease or CYP7A1-associated disease.

EQUIVALENTS

(578) The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.