COMPOUND AND METHOD FOR TREATING MYOTONIC DYSTROPHY
20190264210 ยท 2019-08-29
Inventors
Cpc classification
C12N2810/40
CHEMISTRY; METALLURGY
A61P21/00
HUMAN NECESSITIES
C12N15/87
CHEMISTRY; METALLURGY
C07K2319/33
CHEMISTRY; METALLURGY
C12N2310/113
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Provided are 9-base morpholino antisense compounds targeted to polyCUG repeats in the 3UTR region of dystrophia myotonica protein kinase (DMPK) mRNA, and related methods for treating myotonic dystrophy DM1.
Claims
1. An antisense compound for treating myotonic dystrophy DM1, comprising a 9-base morpholino antisense oligonucleotide, where the 9 bases are complementary to polyCUG repeats in the 3UTR region of dystrophia myotonica protein kinase (DMPK) mRNA.
2. The antisense compound of claim 1, wherein the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
3. The antisense compound of claim 2, wherein at least one and up to about 1 per every 2 intersubunit linkage(s) contains a pendant cationic group.
4. The antisense compound of claim 3, wherein the cationic group comprises an optionally substituted piperazino group.
5. The antisense compound of claim 3, wherein the oligonucleotide is conjugated to a cell-penetrating peptide.
6. A method of treating myotonic dystrophy DM1 in a mammalian subject, comprising administering to the subject a 9-base morpholino antisense oligonucleotide, where the 9 bases are complementary to polyCUG repeats in the 3UTR region of dystrophia myotonica protein kinase (DMPK) mRNA, and repeating said administering at least once every one week to 3 months.
7. The method of claim 6, wherein the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO).
8. The method of claim 7, wherein at least one and up to about 1 per every 2 intersubunit linkage(s) contains a pendant cationic group.
9. The method of claim 8, wherein the cationic group comprises an optionally substituted piperazino group.
10. The method of claim 6, wherein the oligonucleotide is conjugated to a cell-penetrating peptide.
11. The method of claim 6, wherein said administering is by intravenous or subcutaneous injection to the subject, at a dose between 1-20 mg/kg body weight antisense compound.
12. The method of claim 6, wherein said administering is continued at regular intervals of every one to three months, and further includes monitoring the patient during the treatment period for improvement in skeletal or heart muscle performance.
13. The method of claim 6, wherein said administering is continued at regular intervals of every one to three months, and further includes monitoring the patient during the treatment period for improvement in heart conduction properties.
14. The method of claim 6, wherein said administering is continued at regular intervals of every one to three months, and further includes monitoring the patient during the treatment period for reduction in serum creatine kinase.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0013]
DETAILED DESCRIPTION
Definitions
[0014] The terms below, as used herein, have the following meanings, unless indicated otherwise:
[0015] The terms cell penetrating peptide or CPP are used interchangeably and refer to cationic cell penetrating peptides, also called transport peptides, carrier peptides, or peptide transduction domains. Examples of cell-penetrating peptides include arginine-rich peptides. The peptides, as shown herein, typically have the capability of inducing cell penetration within 100% of cells of a given cell culture population and allow macromolecular translocation within multiple tissues in vivo upon systemic administration.
[0016] The terms antisense oligomer or antisense oligonucleotide or oligonucleotide are used interchangeably and refer to a sequence of cyclic subunits, each bearing a base-pairing moiety, linked by intersubunit linkages that allow the base-pairing moieties to hybridize to a target sequence in a nucleic acid (typically an RNA) by Watson-Crick base pairing, to form a nucleic acid:oligomer heteroduplex within the target sequence. The cyclic subunits are based on ribose or another pentose sugar or, in a preferred embodiment, a morpholino group (see description of morpholino oligomers below). The oligomer may have exact or near sequence complementarity to the target sequence; variations in sequence near the termini of an oligomer are generally preferable to variations in the interior.
[0017] In one aspect of the invention, for the treatment of DM1, the antisense oligonucleotide is complementary to at least 8, optionally 9-12 or more contiguous bases in polyCUG repeats within the 3 UTR regions of the transcript for dystrophia myotonica protein kinase (DMPK) in muscle cells, and is designed to bind by hybridization to these repeats, blocking binding of splice-associated proteins, such as one or more muscleblind family proteins, e.g., MBNL1, or CUGBP to the transcript. The oligonucleotide may be said to be directed to or targeted against 3UTR polyCUG repeats with which it hybridizes. The target sequence may include a polyCUG region of at least 8 contiguous bases, preferably at least 9-25, and up to 40 bases or more.
[0018] In another aspect of the invention, for the treatment of DM2, the antisense oligonucleotide is complementary to at least 8, optionally 9-12 or more contiguous bases in polyCUG repeats within intron 1 of the pre-mRNA transcript for zinc finger protein 9 (ZNF9) in muscle cells, and is designed to bind by hybridization to these repeats, blocking binding of splice-associated proteins, such as one or more muscleblind family proteins, e.g., MBNL1, or CUGBP to the pre-mRNA transcript or the excised intron 1 resulting from ZNF9 pre-mRNA processing. The oligonucleotide may be said to be directed to or targeted against polyCCUG repeats with which it hybridizes. The target sequence may include a polyCCUG region of at least 8 contiguous bases, preferably at least 9-25, and up to 40 bases or more.
[0019] Specific embodiments include 9 base antisense oligomers such as PMO, PMO+, PPMO, or PPMO+ antisense oligonucleotides/compounds that are fully complementary to polyCUG repeats within the 3 UTR regions of the RNA transcript for DMPK, and antisense oligonucleotides/compounds that are fully complementary to polyCCUG repeats within intron 1 of the pre-mRNA transcript for ZNF9. Examples include the antisense oligomers of SEQ ID NOS:1, 5, 9, and 15-18 targeted to polyCUG repeats, and SEQ ID NOS: 19, 21, 23, 26-29 targeted to polyCCUG repeats.
[0020] The terms morpholino oligomer or PMO (phosphoramidate- or phosphorodiamidate morpholino oligomer) refer to an oligonucleotide composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages, one to three atoms long, preferably two atoms long, and preferably uncharged or cationic, joining the morpholino nitrogen of one subunit to a 5 exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide. See, for example, the structure in
[0021] An amino acid subunit or amino acid residue can refer to an -amino acid residue (COCHRNH) or a - or other amino acid residue (e.g. CO(CH.sub.2).sub.nCHRNH), where R is a side chain (which may include hydrogen) and n is 1 to 6, preferably 1 to 4.
[0022] The term naturally occurring amino acid refers to an amino acid present in proteins found in nature. The term non-natural amino acids refers to those amino acids not present in proteins found in nature, examples include beta-alanine (-Ala), 6-aminohexanoic acid (Ahx) and 6-aminopentanoic acid.
[0023] A marker compound refers to a detectable compound attached to a transport peptide for evaluation of transport of the resulting conjugate into a cell. The compound may be visually or spectrophotometrically detected, e.g. a fluorescent compound or fluorescently labeled compound, which may include a fluorescently labeled oligomer. Preferably, the marker compound is a labeled or unlabeled antisense oligomer. In this case, detection of transport involves detection of a product resulting from modulation of splicing and/or transcription of a nucleic acid by an antisense oligomeric compound. Exemplary methods, such as a splice correction assay or exon skipping assay, are described in Materials and Methods below.
[0024] An effective amount or therapeutically effective amount refers to an amount of therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.
[0025] Treatment of an individual (e.g. a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
[0026] The terms antisense compound or compound or conjugate compound include stand-alone antisense oligonucleotides (e.g., PMO, PMO+), and compounds formed by conjugating a cell-penetrating peptide (e.g., arginine-rich peptide) to an antisense oligonucleotide (e.g., PPMO, PPMO+). Examples of arginine-rich peptides include SEQ ID NOS:30-44, including the (RXRR(X/B)R).sub.2XB (SEQ ID NO:55) cell-penetrating peptides, which can be conjugated, for example, to an oligonucleotide targeted against a region of polyCUG or polyCCUG repeats.
[0027] Systemic administration of a compound refers to administration, such as intravenous (iv) subcutaneous (subQ), intramuscular (IM), and intraperitoneal (IP) that delivers the compound directly into the bloodstream.
[0028] A systemically administered antisense oligonucleotide can be targeted, for example, to heart muscle tissue by conjugation to the CPP (RXRRBR).sub.2 (SEQ ID NO:42) with an XB linkage, or other cell-penetrating peptide. In certain instances, the compound, when administered systemically to a DM1 subject in accordance with the method herein, produces a measurable improvement in heart muscle performance and/or improvement in conduction properties of the heart, as measured by known methods.
Structural Features of Transport Peptides
[0029] Exemplary cell-penetrating peptides that can employed in the invention include a class of a transport peptide having 8 to 30 amino acid residues in length and consisting of subsequences selected from the group consisting of RXR, RX, RB, and RBR; where R is arginine (which may include D-arginine, represented in the sequences herein by r), B is -alanine, and each X is independently C(O)(CHR.sup.1).sub.nNH, where n is 4-6 and each R.sup.1 is independently H or methyl, such that at most two R.sup.1's are methyl. Preferably, each R.sup.1 is hydrogen. These peptides have the generic formula (RXRR(B/X)R).sub.2XB (SEQ ID NO:55), where R is arginine; B is -alanine; and each X is C(O)(CH.sub.2).sub.nNH, where n is 4-6, preferably 6, and include both (RXRRBR).sub.2 (SEQ ID NO:42) with an XB linkage, and (RXRRXR).sub.2 (SEQ ID NO:40) with an XB linkage, and where R is arginine; B is -alanine; and each X is C(O)(CH.sub.2).sub.nNH, where n is 4-6. As discussed below, these peptides have been discovered to selectively target an oligonucleotide, including a PMO, to muscle tissue, including heart muscle tissue.
[0030] Table 1 below includes certain transport peptides in this class that have been evaluated, in conjugation with suitable antisense oligonucleotides, for their ability to selectively target various tissues, including heart and skeletal muscle. See, e.g., U.S. application Ser. No. 12/493,140, incorporated by reference in its entirety. The peptides have been evaluated for cellular uptake, as determined by flow cytometry; for antisense activity, as determined by a splice correction assay (Kang, Cho et al. 1998); and for cellular toxicity, as determined by MTT cell viability, propidium iodide membrane integrity and hemolysis assays, and microscopic imaging, and their uptake and functional activity in muscle tissue relative to a variety of non-muscle tissue were compared. The (RXRRXR).sub.2 peptide (SEQ ID NO:40) with an XB linkage was among the most active in antisense activity, as determined by the splice correction assay, both in the presence and absence of added serum. Both (RXRR(B/X)R).sub.2XB (SEQ ID NO:55) peptides were effective in selectively targeting oligonucleotides to heart and skeletal tissue, while showing relatively low-level targeting to a variety of other tissues, including mammary gland tissue, ovary/prostate (particularly (RXRRXR).sub.2 (SEQ ID NO:40) with an XB linkage), and brain. Embodiments of the present invention may employ any one or more of these cell-penetrating or arginine-rich peptides.
TABLE-US-00001 TABLE1 Cell-PenetratingPeptides SEQ Name ID (Designation) Sequence NO..sup.a rTAT RRRQRRKKR 30 Tat RKKRRQRRR 31 R.sub.9F.sub.2 RRRRRRRRRFF 32 R.sub.5F.sub.2R.sub.4 RRRRRFFRRRR 33 R.sub.4 RRRR 34 R.sub.5 RRRRR 35 R.sub.6 RRRRRR 36 R.sub.7 RRRRRRR 37 R.sub.8 RRRRRRRR 38 R.sub.9 RRRRRRRRR 39 (RAhxR).sub.4; RAhxRRAhxRRAhxRRAhxR 40 (P007) (RAhxR).sub.5; RAhxRRAhxRRAhxRRAhxRRAhxR 41 (CP04057) (RAhxRRBR).sub.2; RAhxRRBRRAhxRRBR 42 (CP06062) (RAR).sub.4F.sub.2 RARRARRARRARFFC 43 (RGR).sub.4F.sub.2 RGRRGRRGRRGRFFC 44 .sup.aSequences assigned to SEQ ID NOs do not include the linkage portion (e.g., C, G, Ahx, B, AhxB where Ahx and B refer to 6-aminohexanoic acid and beta-alanine, respectively).
Therapeutic Applications
[0031] The phosphorodiamidate morpholino oligomers (e.g., PMO, PMO+) and other antisense oligomers described herein are useful for treating myotonic dystrophy type 1 (DM1), and the conjugate compounds (e.g., PPMO, PPMO+) of the present invention are further useful for targeting and delivering these antisense oligomers across both the cell and nuclear membranes to the nucleus of muscle cells in skeletal and heart muscle tissue, by exposing the cell to an antisense oligomer or conjugate comprising the oligomer covalently linked to a carrier peptide, as described herein.
[0032] Treatment of Myotonic Dystrophy.
[0033] As the name of the disorder implies, the characteristic clinical manifestation in DM is myotonia (muscle hyperexcitability) and muscle degeneration. Affected individuals will also develop insulin resistance, cataracts, heart conduction defects, testicular atrophy, hypogammaglobulinemia and sleep disorders. Symptoms of DM can manifest in the adult or in childhood. The childhood onset form of the disease is often associated with mental retardation. In addition, there is a form of the disease referred to as congenital myotonic dystrophy. This latter form of the disease is frequently fatal and is seen almost exclusively in children born of mothers who themselves are mildly affected by the disease. In congenital DM the facial manifestations are distinctive due to bilateral facial palsy and marked jaw weakness. Many infants with congenital DM die due to respiratory insufficiency before a proper diagnosis of the disease is made.
[0034] DM1 initially involves the distal muscles of the extremities and only as the disease progresses do proximal muscles become affected. In addition, muscles of the head and neck are affected early in the course of the disease. Weakness in eyelid closure, limited extraocular movement and ptosis results from involvement of the extraocular muscles. Many individuals with DM1 exhibit a characteristic haggard appearance that is the result of atrophy of the masseters (large muscles that raise and lower the jaw), sternocleidomastoids (large, thick muscles that pass obliquely across each side of the neck and contribute to arm movement) and the temporalis muscle (muscle involved in chewing).
[0035] Treatment of DM1, in accordance with general embodiments of the invention, may comprise, for example: (i) administering to the subject with DM1, an antisense compound comprising an antisense oligonucleotide having 8-30 bases, with at least 8 contiguous bases being complementary to the polyCUG repeats in the 3UTR region of dystrophia myotonica protein kinase (DMPK) mRNA in DM1 and optionally conjugated to the oligonucleotide, a cell-penetrating peptide, and (ii) optionally repeating the compound administration at least once every one week to once every three months or longer. Examples of cell-penetrating peptides include the peptides of SEQ ID NOS:30-44. In specific embodiments, the cell-penetrating peptide may have the sequence (RXRR(B/X)R).sub.2XB (SEQ ID NO:55), where R is arginine; B is 3-alanine; and each X is C(O)(CH.sub.2).sub.nNH, where n is 4-6.
[0036] Treatment of DM1, in accordance with specific embodiments of the invention, may comprise: (i) administering to the subject with DM1 a 9-base morpholino antisense oligonucleotide, where the 9 bases are complementary to polyCUG repeats in the 3UTR region of dystrophia myotonica protein kinase (DMPK) mRNA, and (ii) optionally repeating said administering at least once every one week to 3 months. The morpholino antisense oligomer may be a phosphorodiamidate morpholino oligonucleotide (PMO), and/or it may be a PMO where at least one and up to about 1 per every 2 intersubunit linkage(s) contains a pendant cationic group, such as an optionally piperazino group (PMO+). The PMO or PMO+ compound may be optionally conjugated to a cell-penetrating peptide, such as an arginine-rich peptide (e.g., PPMO, PPMO+).
[0037] The compound is preferably administered by intravenous or subcutaneous injection to the subject, at a dose between 1-5 or 1-20 mg/kg body weight antisense compound, at a dosing schedule of once a month to once every 2-3 months. For subQ administration, the dose required may be roughly twice that for IV administration. During the course of treatment, the patient is monitored for improvement or stabilization of muscle performance, improvement in heart conduction properties and/or reduction in serum reduction in serum creatine kinase. Because myotonic dystrophy is a chronic disease, the treatment method will be applied over the subject's lifetime, with dose adjustments being made during the treatment period to achieve a desired level of muscle function and to accommodate patient growth.
[0038] The treatment methods offer a number of important advantages over earlier proposed antisense methods of treating DM1. First, targeting, uptake and antisense activity of the antisense compounds described herein into skeletal muscle, heart muscle, or both, is efficient. This allows effective treatment with relatively modest compound doses, e.g., in the range 1-5 mg/kg subject weight. Second, little or no compound toxicity has been observed, as evidenced, for example, by no microscopically observable increases in muscle damage, inflammatory cellular infiltrates, or necrotic fibers in muscles injected with PPMOs and/or PMOs. Finally, in certain instances, the effect of a single dose may be effective for up to three months or more, allowing the patient to be effectively treated by dosing at intervals of no less than one month, and up to 3 months or more between successive treatments.
Combination with Homing Peptides
[0039] The antisense oligonucleotides and conjugate compounds of the invention may be used in conjunction with homing peptides selective for the target tissue, to further enhance muscle-specific delivery. An example of this approach can be found in the application of muscle-binding peptides (Samoylova and Smith, 1999; Vodyanoy et al., U.S. Appn. Pubn. No. 20030640466) coupled to antisense oligomers designed to be therapeutic treatments for Duchenne muscular dystrophy (DMD) (Gebski, Mann et al. 2003; Alter, Lou et al. 2006) (PCT Pubn. No. WO2006000057). The heptapeptide sequence ASSLNIA (SEQ ID NO:45) has enhanced in vivo skeletal and cardiac muscle binding properties, as described by Samoylova and Smith. As a further example, a pancreas-homing peptide, CRVASVLPC (SEQ ID NO:56), mimics the natural prolactin receptor ligand (Kolonin, Sun et al. 2006).
[0040] An exemplary dual peptide molecule has a cell penetrating peptide to one terminus, e.g. at the 5 end of the antisense oligomer, as described herein, and a homing peptide coupled to the other terminus, i.e. the 3 terminus. The homing peptide localizes the peptide-conjugated PMO to the target tissue, where the cell-penetrating peptide moiety effects transport into the cells of the tissue.
[0041] Alternatively, a preferred exemplary dual peptide molecule would have both a homing peptide (HP) and cell-penetrating peptide (CPP) conjugated to one end, e.g. the 5 terminus of the antisense oligomer, in either a HP-CPP-PMO configuration or, more preferably, a CPP-HP-PMO configuration.
TABLE-US-00002 TABLE2 ExamplesofMuscle-specificHomingPeptides(HP) SEQ PeptideSequence ID TargetTissue (NH.sub.2toCOOH) NO. Skeletal ASSLNIA 45 Muscle-SMP1 SMP2 SLGSFP 46 SMP3 SGASAV 47 SMP4 GRSGAR 48 SMP5 TARGEHKEEELI 49 Cardiac WLSEAGPVVTVRALRGTGSW 50 Muscle-CMP1 CMP2 VTVRALRGTSW 51 CMP3 VVTVRALRGTGSW 52 CMP4 CRPPR 53 CMP5 SKTFNTHPQSTP 54 CRVASVLPC 56
Peptide-Antisense Oligomer Conjugate Compositions
Conjugates for Specific Muscle Treatments
[0042] Therapeutic conjugates comprising selected transport peptide sequences are also provided by the invention. These include conjugates comprising a carrier peptide (RXRR(B/X)R).sub.2XB (SEQ ID NO:55), as described herein, conjugated to an oligonucleotide, e.g., PMO, designed for therapeutic action within muscle tissue. Also included are conjugates comprising an oligonucleotide conjugated to any one of SEQ ID NOS:30-44.
[0043] The conjugates may further comprise a targeting moiety effective to bind to tissue specific receptors of a target tissue type, linked to the therapeutic compound or, preferably, to another terminus of the carrier peptide. In particularly preferred embodiments, a homing peptide such as described above is conjugated to therapeutic compound or to the cell-penetrating peptide.
[0044] For use in treating myotonic dystrophy DM1, the conjugate compound may comprise an antisense oligonucleotide, having 8-30 bases, preferably 9 bases, with at least 8 or 9 or more contiguous bases being complementary to the polyCUG repeats in the 3UTR region of dystrophia myotonica protein kinase (DMPK) mRNA, and conjugated to the oligonucleotide, a cell-penetrating peptide of any one of SEQ ID NOS:30-44, including, for example, a peptide having the sequence (RXRR(B/X)R).sub.2XB (SEQ ID NO:55), where R is arginine; B is -alanine; and each X is C(O)(CH.sub.2).sub.nNH, where n is 4-6. Such compounds are effective to selectively block the sequestration of muscleblind-like 1 protein (MBNL1) and/or CUGBP in heart and quadricep muscle in a myotonic dystrophy animal model.
Morpholino Oligomers Having Cationic and Other Intersubunit Linkages
[0045] In preferred embodiments, as noted above, the antisense oligomer is a phosphorodiamidate morpholino oligonucleotide (PMO). Certain PMOs may include between about 10-50% or 20-50% positively charged or cationic backbone linkages, as described below and further in WO/2008/036127, which is incorporated by reference.
[0046] Certain cationic PMOs (e.g., PMO+) include morpholino oligomers in which at least one intersubunit linkage between two consecutive morpholino ring structures contains a pendant cationic group. The pendant group bears a distal nitrogen atom that can bear a positive charge at neutral or near-neutral (e.g., physiological) pH. Examples are shown in
[0047] The intersubunit linkages in these oligomers are preferably phosphorus-containing linkages, having the structure:
##STR00001##
where [0048] W is S or 0, and is preferably O, [0049] X=NR.sup.1R.sup.2 or OR.sup.6, [0050] Y=O or NR.sup.7,
[0051] and each said linkage in the oligomer is selected from:
[0052] (a) uncharged linkage (a), where each of R.sup.1, R.sup.2, R.sup.6 and R.sup.7 is independently selected from hydrogen and lower alkyl;
[0053] (b1) cationic linkage (b1), where X=NR.sup.1R.sup.2 and Y=O, and NR.sup.1R.sup.2 represents an optionally substituted piperazino group, such that R.sup.1R.sup.2=CHRCHRN(R.sup.3)(R.sup.4)CHRCHR, where
[0054] each R is independently H or CH.sub.3,
[0055] R.sup.4 is H, CH.sub.3, or an electron pair, and
[0056] R.sup.3 is selected from H, lower alkyl, e.g. CH.sub.3, C(NH)NH.sub.2, Z-L-NHC(NH)NH.sub.2, and {C(O)CHRNH}.sub.mH, where: Z is C(O) or a direct bond, L is an optional linker up to 18 atoms in length, preferably up to 12 atoms, and more preferably up to 8 atoms in length, having bonds selected from alkyl, alkoxy, and alkylamino, R is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6, preferably 1 to 4;
[0057] (b2) cationic linkage (b2), where X=NRR.sup.2 and Y=O, R.sup.1=H or CH.sub.3, and R.sup.2=LNR.sup.3R.sup.4R.sup.5, where L, R.sup.3, and R.sup.4 are as defined above, and R.sup.5 is H, lower alkyl, or lower (alkoxy)alkyl; and
[0058] (b3) cationic linkage (b3), where Y=NR.sup.7 and X=OR.sup.6, and R.sup.7=LNR.sup.3R.sup.4R.sup.5, where L, R.sup.3, R.sup.4 and R.sup.5 are as defined above, and R.sup.6 is H or lower alkyl;
[0059] and at least one said linkage is selected from cationic linkages (b1), (b2), and (b3).
[0060] Preferably, the oligomer includes at least two consecutive linkages of type (a) (i.e. uncharged linkages). In further embodiments, at least 5% of the linkages in the oligomer are cationic linkages (i.e. type (b1), (b2), or (b3)); for example, 10% to 80%, 10% to 50%, or 10% to 35% of the linkages may be cationic linkages.
[0061] In one embodiment, at least one linkage is of type (b1), where, preferably, each R is H, R.sup.4 is H, CH.sub.3, or an electron pair, and R.sup.3 is selected from H, lower alkyl, e.g. CH.sub.3, C(NH)NH.sub.2, and C(O)-L-NHC(NH)NH.sub.2. The latter two embodiments of R.sup.3 provide a guanidino moiety, either attached directly to the piperazine ring, or pendant to a linker group L, respectively. For ease of synthesis, the variable Z in R.sup.3 is preferably C(O) (carbonyl), as shown.
[0062] The linker group L, as noted above, contains bonds in its backbone selected from alkyl (e.g. CH.sub.2CH.sub.2), alkoxy (CO), and alkylamino (e.g. CH.sub.2NH), with the proviso that the terminal atoms in L (e.g., those adjacent to carbonyl or nitrogen) are carbon atoms. Although branched linkages (e.g. CH.sub.2CHCH.sub.3) are possible, the linker is preferably unbranched. In one embodiment, the linker is a hydrocarbon linker. Such a linker may have the structure (CH.sub.2).sub.n, where n is 1-12, preferably 2-8, and more preferably 2-6.
[0063] The use of embodiments of linkage types (b1), (b2) and (b3) above to link morpholino subunits may be illustrated graphically as follows:
##STR00002##
[0064] Preferably, all cationic linkages in the oligomer are of the same type; i.e. all of type (b1), all of type (b2), or all of type (b3). The base-pairing moieties Pi may be the same or different, and are generally designed to provide a sequence which binds to a target nucleic acid.
[0065] In further embodiments, the cationic linkages are selected from linkages (b1) and (b1) as shown below, where (b1) is referred to herein as a Pip linkage and (b1) is referred to herein as a GuX linkage:
##STR00003##
[0066] In the structures above, W is S or O, and is preferably O; each of R.sup.1 and R.sup.2 is independently selected from hydrogen and lower alkyl, and is preferably methyl; and A represents hydrogen or a non-interfering substituent on one or more carbon atoms in (b1) and (b1). Preferably, the ring carbons in the piperazine ring are unsubstituted; however, they may include non-interfering substituents, such as methyl or fluorine. Preferably, at most one or two carbon atoms is so substituted.
[0067] In further embodiments, at least 10% of the linkages are of type (b1) or (b1); for example, 20% to 80%, 20% to 50%, or 20% to 30% of the linkages may be of type (b1) or (b1).
[0068] In other embodiments, the oligomer contains no linkages of the type (b1) above. Alternatively, the oligomer contains no linkages of type (b1) where each R is H, R.sup.3 is H or CH.sub.3, and R.sup.4 is H, CH.sub.3, or an electron pair.
[0069] Oligomers having any number of cationic linkages can be used, including fully cationic-linked oligomers. Preferably, however, the oligomers are partially charged, having, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 percent cationic linkages. In selected embodiments, about 10 to 80, 20 to 80, 20 to 60, 20 to 50, 20 to 40, or about 20 to 35 percent of the linkages are cationic.
[0070] In one embodiment, the cationic linkages are interspersed along the backbone. The partially charged oligomers preferably contain at least two consecutive uncharged linkages; that is, the oligomer preferably does not have a strictly alternating pattern along its entire length.
[0071] Also considered are oligomers having blocks of cationic linkages and blocks of uncharged linkages; for example, a central block of uncharged linkages may be flanked by blocks of cationic linkages, or vice versa. In one embodiment, the oligomer has approximately equal-length 5, 3 and center regions, and the percentage of cationic linkages in the center region is greater than about 50%, preferably greater than about 70%.
[0072] Oligomers for use in antisense applications generally range in length from about 10 to about 40 subunits, more preferably about 15 to 25 subunits. For example, a cationic oligomer having 19-20 subunits, a useful length for an antisense oligomer, may ideally have two to seven, e.g. four to six, or three to five, cationic linkages, and the remainder uncharged linkages. An oligomer having 14-15 subunits may ideally have two to five, e.g. 3 or 4, cationic linkages and the remainder uncharged linkages. Specific examples include a 9 subunit oligomer with about 1, 2, or 3 cationic linkages, and the remainder uncharged linkages.
[0073] Each morpholino ring structure supports a base pairing moiety, to form a sequence of base pairing moieties which is typically designed to hybridize to a selected antisense target in a cell or in a subject being treated. The base pairing moiety may be a purine or pyrimidine found in native DNA or RNA (A, G, C, T, or U) or an analog, such as hypoxanthine (the base component of the nucleoside inosine) or 5-methyl cytosine.
[0074] As noted above, the substantially uncharged oligonucleotide may be modified to include one or more charged linkages, e.g. up to about 1 per every 2-5 uncharged linkages, typically 3-5 per every 10 uncharged linkages. Optimal improvement in antisense activity is seen where up to about half of the backbone linkages are cationic. Some, but not maximum enhancement is typically seen with a small number e.g., 10-20% cationic linkages; where the number of cationic linkages exceeds 50-60%, the sequence specificity of the antisense binding to its target may be compromised or lost.
[0075] The enhancement seen with added cationic backbone charges may, in some case, be further enhanced by distributing the bulk of the charges close of the center-region backbone linkages of the antisense oligonucleotide, e.g., in a 20-mer oligonucleotide with 8 cationic backbone linkages, having 70%-100% of these charged linkages localized in the 10 centermost linkages.
Other Oligomer Types
[0076] Delivery of alternative antisense chemistries can also benefit from the disclosed carrier peptide. Specific examples of other antisense compounds useful in this invention include those in which at least one, or all, of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, phosphorothioates, or phosphoramidates. Also included are molecules wherein at least one, or all, of the nucleotides contains a 2 lower alkyl moiety (e.g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, or isopropyl).
[0077] In other oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are modified. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-phosphate backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
[0078] Modified oligonucleotides may be classified as chimeric, e.g., containing at least one region wherein the oligonucleotide is modified so as to confer increased resistance to nuclease degradation or increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid.
EXAMPLES
[0079] The following examples are intended to illustrate but not to limit the invention.
Example 1. PMO, PMO+, PPMO and PPMO+ Consisting of (CAG)n Repeats Reverse Molecular and Physiological Manifestations of DM1 in a Mouse Model
[0080] To determine whether antisense compositions described herein (e.g., SEQ ID NOs: 1-18) can influence in vivo expanded CUG (CUGexp) repeat interactions with MBNL1 splicing factor, their effects can be examined in a transgenic mouse model of DM1. The antisense oligonucleotides and conjugates shown in Table A below can be manufactured according to routine techniques and then tested in this transgenic mouse model of DM1.
TABLE-US-00003 TABLEA PMO,PMO+,PPMO,andPPMO+ agentstargetedtopolyCUGrepeats inthe3UTRregionofdystrophiamyotonicaproteinkinase(DMPK). SampleName Sequence 5End 3End CAG9mer CAGCAGCAG(SEQIDNO:1) EG3 H CAG9mer-B CAGCAGCAG(SEQIDNO:1) EG3 CP06062 CAG9mer-R9F2 CAGCAGCAG(SEQIDNO:1) EG3 R9F2 CAG9mer-rTat CAGCAGCAG(SEQIDNO:1) EG3 rTat CAG12mer CAGCAGCAGCAG(SEQIDNO:2) EG3 H CAG12mer-B CAGCAGCAGCAG(SEQIDNO:2) EG3 CP06062 CAG15mer CAGCAGCAGCAGCAG(SEQIDNO:3) EG3 H CAG15mer-B CAGCAGCAGCAGCAG(SEQIDNO:3) EG3 CP06062 CAG18mer CAGCAGCAGCAGCAGCAG(SEQIDNO:4) EG3 H CAG18mer-B CAGCAGCAGCAGCAGCAG(SEQIDNO:4) EG3 CP06062 AGC9mer AGCAGCAGC(SEQIDNO:5) EG3 H AGC9mer-B AGCAGCAGC(SEQIDNO:5) EG3 CP06062 AGC12mer AGCAGCAGCAGC(SEQIDNO:6) EG3 H AGC12mer-B AGCAGCAGCAGC(SEQIDNO:6) EG3 CP06062 AGC15mer AGCAGCAGCAGCAGC(SEQIDNO:7) EG3 H AGC15mer-B AGCAGCAGCAGCAGC(SEQIDNO:7) EG3 CP06062 AGC18mer AGCAGCAGCAGCAGCAGC(SEQIDNO:8) EG3 H AGC18mer-B AGCAGCAGCAGCAGCAGC(SEQIDNO:8) EG3 CP06062 GCA9mer GCAGCAGCA(SEQIDNO:9) EG3 H GCA9mer-B GCAGCAGCA(SEQIDNO:9) EG3 CP06062 GCA12mer GCAGCAGCAGCA(SEQIDNO:10) EG3 H GCA12mer-B GCAGCAGCAGCA(SEQIDNO:10) EG3 CP06062 GCA15mer GCAGCAGCAGCAGCA(SEQIDNO:11) EG3 H GCA15mer-B GCAGCAGCAGCAGCA(SEQIDNO:11) EG3 CP06062 GCA18mer GCAGCAGCAGCAGCAGCA(SEQIDNO:12) EG3 H GCA18mer-B GCAGCAGCAGCAGCAGCA(SEQIDNO:12) EG3 CP06062 AGC25mer AGCAGCAGCAGCAGCAGCAGCAGCA EG3 H (SEQIDNO:13) AGC25mer-B AGCAGCAGCAGCAGCAGCAGCAGCA EG3 CP06062 (SEQIDNO:13) CAG25mer CAGCAGCAGCAGCAGCAGCAGCAGC EG3 H (SEQIDNO:14) CAG25mer-B CAGCAGCAGCAGCAGCAGCAGCAGC EG3 CP06062 (SEQIDNO:14) CAG25mer-R9F2 CAGCAGCAGCAGCAGCAGCAGCAGC EG3 R9F2 (SEQIDNO:14) CAG25mer-rTat CAGCAGCAGCAGCAGCAGCAGCAGC EG3 rTat (SEQIDNO:14) CAG9mer+ C+AGC+AGC+AG(SEQIDNO:15) EG3 H CAG9mer+B C+AGC+AGC+AG(SEQIDNO:15) EG3 CP06062 CAG9mer+ C+AGCAGCAG(SEQIDNO:16) EG3 H CAG9mer+ CAGCAGC+AG(SEQ1DNO:17) EG3 H CAG9mer+ CAGC+AGCAG(SEQIDNO:18) EG3 H * The linkage(s) between the oligonucleotide and the cell-penetrating peptide can included a variety of linkages, but preferred linkages are C, AhxB, G, and B.
[0081] HSA.sup.LR transgenic mice express human skeletal actin transcripts that have (CUG)250 inserted in the 3 UTR (Mankodi, Logigian et al. 2000). These mice accumulate CUGexp RNA and MBNL1 protein in nuclear foci in skeletal muscle, a process that depends on CUGexp-MBNL1 interaction (Dansithong, Paul et al. 2005). The effect of antisense compositions of the present invention can be examined in their ability to block foci development in muscle cells. PMO and PPMO can be delivered intravenously or intraperitoneally at doses ranging from 30 to 600 micrograms. Muscle tissue can be examined 1-3 weeks later by fluorescence in situ hybridization using probes that hybridize to the CUG repeat or to sequences flanking the repeat. Activity of any given compound can be measured by the magnitude of reduction of nuclear foci and redistribution of MBNL1 from a punctate pattern to diffuse localization in the nucleus.
[0082] Compositions of the present invention can also reverse the biochemical consequences of MBNL1 sequestration. Accumulation of CUGexp RNA-MBNL1 complexes in the foci results also in aberrant mis-splicing of several genes, namely, CIC-1, Serca-1, m-Titin, Tnnt3 and Zasp genes (Mulders, van den Broek et al. 2009). HSA.sup.LR transgenic mice show alternative splicing changes similar to those observed in human DM1 (Wheeler, Sobczak et al. 2009). DM1-affected, aberrantly spliced exons can be examined in mice treated with compositions of the invention to determine whether alternative splicing is corrected at three weeks following injection of compositions of the present invention. Effects of PMO or PPMO treatment on aberrant splicing can be expected to persist for at least fourteen weeks.
[0083] It is also expected that compositions of the present invention can rescue the physiological effects of DM1 (myotonia) and can be examined by measuring the expression and function of chloride channel 1 (ClC-1) which is inactivated by mis-splicing in DM1 and the HSA.sup.LR mouse model. Myotonia can be measured through determination of delayed muscle relaxation and repetitive action potentials and are expected to improve in HSA.sup.LR mice treated with compositions of the present invention.
Example 2
PMO, PPMOplus, PPMO AND PMO-X Consisting of (CCAG)n Repeats Reverse Molecular and Physiological Manifestations of DM2
[0084] To determine whether antisense compositions described herein (e.g., SEQ ID NOs: 19-29) can influence in vivo expanded CCUG (CCUGexp) repeat interactions with MBNL1 splicing factor, their effects can be examined in an analogous transgenic mouse model to that described above for DM1. The antisense oligonucleotides and conjugates shown in Table B below can be manufactured according to routine techniques and then tested in this transgenic mouse model of DM2. The expected experimental outcomes are similar to those described for DM1 in Example 1.
TABLE-US-00004 TABLEB PMO,PMO+,PPMO,andPPMO+ agentstargetedtopolyCCUGrepeats inthefirstintronofzincfingerprotein9(ZNF9)pre-mRNA. SampleName Sequence 5End 3End CAGG9mer CAGGCAGGC(SEQIDNO:19) EG3 H CAGG9mer-B CAGGCAGGC(SEQIDNO:19) EG3 CP06062 CAGG9mer-R9F2 CAGGCAGGC(SEQIDNO:19) EG3 R9F2 CCAG9mer-rTat CAGGCAGGC(SEQIDNO:19) EG3 rTat CCAG12mer CAGGCAGGCAGG(SEQIDNO:20) EG3 H CCAG12mer-B CAGGCAGGCAGG(SEQIDNO:20) EG3 CP06062 AGCC9mer AGGCAGGCA(SEQIDNO:21) EG3 H AGCC9mer-B AGGCAGGCA(SEQIDNO:21) EG3 CP06062 AGCC12mer AGGCAGGCAGGC(SEQIDNO:22) EG3 H AGCC12mer-B AGGCAGGCAGGC(SEQIDNO:22) EG3 CP06062 GCCA9mer GGCAGGCAG(SEQIDNO:23) EG3 H GCCA9mer-B GGCAGGCAG(SEQIDNO:23) EG3 CP06062 GCCA12mer GGCAGGCAGGCA(SEQIDNO:24) EG3 H GCCA12mer-B GGCAGGCAGGCA(SEQIDNO:24) EG3 CP06062 CAGG24mer CAGGCAGGCAGGCAGGCAGGCAGG EG3 H (SEQIDNO:25) CAGG24mer-B CAGGCAGGCAGGCAGGCAGGCAGG EG3 CP06062 (SEQIDNO:25) CAGG24mer-R9F2 CAGGCAGGCAGGCAGGCAGGCAGG EG3 R9F2 (SEQIDNO:25) CAGG24mer-rTat CAGGCAGGCAGGCAGGCAGGCAGG EG3 rTat (SEQIDNO:25) CAGG9mer+ C+AGGC+AGGC(SEQIDNO:26) EG3 H CAGG9mer+B C+AGGC+AGGC(SEQIDNO:27) EG3 CP06062 CAGG9mer+ C+AGGCAGGC(SEQIDNO:28) EG3 H CAGG9mer+ CAGGC+AGGC(SEQIDNO:29) EG3 H * The linkage(s) between the oligonucleotide and the cell-penetrating peptide can included a variety of linkages, but preferred linkages are C, AhxB, G, and B.
[0085] Although the invention has been described with respect to certain embodiments and examples, it will be appreciated that various changes, modifications, and additions may be made without departing from the claimed invention.
REFERENCES
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TABLE-US-00005 SEQUENCELISTINGTABLE SEQ ID SampleName Sequence NO: CAG9mer CAGCAGCAG 1 CAG12mer CAGCAGCAGCAG 2 CAG15mer CAGCAGCAGCAGCAG 3 CAG18mer CAGCAGCAGCAGCAGCAG 4 AGC9mer AGCAGCAGC 5 AGC12mer AGCAGCAGCAGC 6 AGC15mer AGCAGCAGCAGCAGC 7 AGC18mer AGCAGCAGCAGCAGCAGC 8 GCA9mer GCAGCAGCA 9 GCA12mer GCAGCAGCAGCA 10 GCA15mer GCAGCAGCAGCAGCA 11 GCA18mer GCAGCAGCAGCAGCAGCA 12 AGC25mer AGCAGCAGCAGCAGCAGCAGCAGCA 13 CAG25mer CAGCAGCAGCAGCAGCAGCAGCAGC 14 CAG9mer+ C+AGC+AGC+AG 15 CAG9mer+ C+AGCAGCAG 16 CAG9mer+ CAGCAGC+AG 17 CAG9mer+ CAGC+AGCAG 18 CAGG9mer CAGGCAGGC 19 CAGG12mer CAGGCAGGCAGG 20 AGGC9mer AGGCAGGCA 21 AGGC12mer AGGCAGGCAGGC 22 GGCA9mer GGCAGGCAG 23 GGCA12mer GGCAGGCAGGCA 24 CAGG24mer CAGGCAGGCAGGCAGGCAGGCAGG 25 CAGG9mer+ C+AGGC+AGGC 26 CAGG9mer+B C+AGGC+AGGC 27 CAGG9mer+ C+AGGCAGGC 28 CAGG9mer+ CAGGC+AGGC 29 rTAT RRRQRRKKR 30 Tat RKKRRQRRR 31 R.sub.9F.sub.2 RRRRRRRRRFF 32 R.sub.5F.sub.2R.sub.4 RRRRRFFRRRR 33 R.sub.4 RRRR 34 R.sub.5 RRRRR 35 R.sub.6 RRRRRR 36 R.sub.7 RRRRRRR 37 R.sub.8 RRRRRRRR 38 R.sub.9 RRRRRRRRR 39 (RAhxR).sub.4; RAhxRRAhxRRAhxRRAhxR 40 (P007) (RAhxR).sub.5; RAhxRRAhxRRAhxRRAhxRRAhxR 41 (CP04057) (RAhxRRBR).sub.2; RAhxRRBRRAhxRRBR 42 (CP06062) (RAR).sub.4F.sub.2 RARRARRARRARFFC 43 (RGR).sub.4F.sub.2 RGRRGRRGRRGRFFC 44 SMP1 ASSLNIA 45 SMP2 SLGSFP 46 SMP3 SGASAV 47 SMP4 GRSGAR 48 SMP5 TARGEHKEEELI 49 CMP1 WLSEAGPVVTVRALRGTGSW 50 CMP2 VTVRALRGTSW 51 CMP3 VVTVRALRGTGSW 52 CMP4 CRPPR 53 CMP5 SKTFNTHPQSTP 54 (RXRR(X/B)R).sub.2XB 55 CRVASVLPC 56 *In SEQ ID NOS: 15-18 and 26-29, + refers to a cationic linkage, such as 1-piperazinyl.