COMPOUND AND METHOD FOR TREATING MYOTONIC DYSTROPHY

Info

Publication number: 20110269665
Type: Application
Filed: May 5, 2011
Publication Date: Nov 3, 2011
Applicant: AVI BIOPHARMA, INC. (Corvallis, OR)
Inventor: Ryszard Kole (Chapel Hill, NC)
Application Number: 13/101,942

Abstract

Provided are 9-base morpholino antisense compounds targeted to polyCUG repeats in the 3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA, and related methods for treating myotonic dystrophy DM1.

Description

Description

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No. 12/493,140, filed Jun. 26, 2009, now pending, which is incorporated by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 120178_—492_SEQUENCE_LISTING_txt. The text file is about 13 KB, was created on May 5, 2011, and is being submitted electronically via EFS-Web.

FIELD OF THE INVENTION

The present invention relates to antisense oligonucleotides targeted to polyCUG repeats in the 3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA, and methods for treating myotonic dystrophy DM1.

BACKGROUND OF THE INVENTION

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are associated with long polyCUG and polyCCUG repeats in the 3′-UTR and intron 1 regions of the transcript dystrophia myotonica protein kinase (DMPK) and zinc finger protein 9 (ZNF9), respectively (Wheeler and Thornton 2007). While normal individuals have as many as 30 CTG repeats, DM1 patients carry a larger number of repeats ranging from 50 to thousands. The severity of the disease and the age of onset correlates with the number of repeats. Patients with adult onsets show milder symptoms and have less than 100 repeats, juvenile onset DM1 patients carry as many as 500 repeats and congenital cases usually have around a thousand CTG repeats. The expanded transcripts containing CUG repeats form a secondary structure, accumulate in the nucleus in the form of nuclear foci and sequester RNA-binding proteins (RNA-BP).

Several RNA-BP have been implicated in the disease, including muscleblind-like (MBNL) proteins and CUG-binding protein (CUGBP). MBNL proteins are homologous to Drosophila muscleblind (Mbl) proteins necessary for photoreceptor and muscle differentiation. MBNL and CUGBP have been identified as antagonistic splicing regulators of transcripts affected in DM1 such as cardiac troponin T (cTNT), insulin receptor (IR) and muscle-specific chloride channel (ClC-1).

Myotonic dystrophy type 2 (DM2) is associated with repeats in the first intron of the ZNF9 gene on chromosome 3. CNBP (ZNF9) is the only gene known to be associated with myotonic dystrophy type 2. CNBP intron 1 contains a complex repeat motif, (TG)n(TCTG)n(CCTG)n, and expansion of the CCTG repeat causes DM2. The number of CCTG repeats in expanded alleles can range from approximately 75 to more than 11,000, with a mean of approximately 5000 repeats.

DM1 and DM2 are associated with a variety of serious pathologies including muscle abnormalities and weakness, and in the heart, conduction abnormalities.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected antisense activity of 9-base phosphorodiamidate morpholino oligomers (PMOs), relative, for example, to longer PMOs, for reducing or ameliorating one or more symptoms of myotonic dystrophy type 1 (DM1) or type 2 (DM2). These 9-base antisense oligomers described herein can employ a variety of PMO-based chemistries, including PMO, PMO+, PPMO, and PPMO+ chemistries, as described herein.

Embodiments of the present invention therefore include antisense compounds for treating myotonic dystrophy type 1 (DM1), comprising a morpholino antisense oligonucleotide of 9 bases, where the 9 bases are complementary to polyCUG repeats in the 3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA. In certain embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligonucleotide (PMO). In some embodiments, at least one and up to about 1 per every 2 intersubunit linkage(s) of the oligonucleotide contains a pendant cationic group. In specific embodiments, the cationic group comprises an optionally substituted piperazino group (PMO+). In certain embodiments, the oligonucleotide is conjugated to a cell-penetrating peptide, such as an arginine-rich peptide (PPMO or PPMO+).

Also included are methods of treating myotonic dystrophy DM1 in a mammalian subject, comprising administering to the subject, a morpholino antisense compound of 9 bases, where the 9 bases are complementary to polyCUG repeats in the 3′UTR region of dystrophia myotonica protein kinase (DMPK) mRNA, and optionally repeating said administering at least once every one week to 3 months. In some embodiments, at least one and up to about 1 per every 2 intersubunit linkage(s) of the oligonucleotide contains a pendant cationic group. In specific embodiments, the cationic group comprises an optionally substituted piperazino group. In certain embodiments, the oligonucleotide is conjugated to an arginine-rich peptide.

The compounds may be administered by intravenous or subcutaneous injection to the subject, at a dose between 1-5 or 1-20 mg/kg body weight antisense compound, and the administering step may be continued at regular intervals of every two weeks to three months. The subject may be monitored during the treatment for improvement in muscle performance, heart conduction properties, and/or for a reduction in serum creatine kinase.

Certain embodiments include antisense compounds for treating myotonic dystrophy type 2 (DM2), comprising a morpholino antisense oligonucleotide of 9 bases, where the 9 bases are complementary to polyCCUG repeats in the first intron region of zinc finger protein 9 (ZNF9) pre-mRNA. Also included are methods of treating myotonic dystrophy DM1 in a mammalian subject, comprising administering to the subject, a morpholino antisense oligonucleotide of 9 bases, where the 9 bases are complementary to polyCCUG repeats in the first intron region of zinc finger protein 9 (ZNF9) pre-mRNA, and optionally repeating said administering at least once every one week to 3 months.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show exemplary structures of a phosphorodiamidate-linked morpholino oligomer (PMO), a peptide-conjugated PMO (PPMO), and a peptide-conjugated PMO having cationic intersubunit linkages (PPMO+), respectively. Though multiple cationic linkage types are illustrated in FIG. 1C, a PMO+ or PPMO+oligomer will typically include just one type of cationic linkage.

DETAILED DESCRIPTION Definitions

The terms below, as used herein, have the following meanings, unless indicated otherwise:

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.

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.

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” 3′UTR 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.

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.

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.

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 FIG. 1A, which shows a preferred phosphorodiamidate linkage type. Variations can be made to this linkage as long as they do not interfere with binding or activity. For example, the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate). The 5′ oxygen may be substituted with amino or lower alkyl substituted amino. The pendant nitrogen attached to phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl. See also the discussion of cationic linkages below. The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, and PCT Pubn. No. WO 2008036127 (cationic linkages), all of which are incorporated herein by reference.

An “amino acid subunit” or “amino acid residue” can refer to an α-amino acid residue (—CO—CHR—NH—) or a β- or other amino acid residue (e.g. —CO—(CH₂)_nCHR—NH—), where R is a side chain (which may include hydrogen) and n is 1 to 6, preferably 1 to 4.

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 (β-Ala), 6-aminohexanoic acid (Ahx) and 6-aminopentanoic acid.

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.

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.

“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.

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)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.

“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.

A systemically administered antisense oligonucleotide can be targeted, for example, to heart muscle tissue by conjugation to the CPP(RXRRBR)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

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 β-alanine, and each X is independently —C(O)—(CHR¹)_n—NH—, where n is 4-6 and each R¹is independently H or methyl, such that at most two R¹'s are methyl. Preferably, each R¹is hydrogen. These peptides have the generic formula (RXRRR(B/X)R)₂XB (SEQ ID NO:55), where R is arginine; B is β-alanine; and each X is —C(O)—(CH₂)_n—NH—, where n is 4-6, preferably 6, and include both (RXRRBR)₂(SEQ ID NO:42) with an XB linkage, and (RXRRXR)₂(SEQ ID NO:40) with an XB linkage, and where R is arginine; B is β-alanine; and each X is —C(O)—(CH₂)_n—NH—, 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.

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)₂peptide (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)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 (RXRRXRR)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 1 Cell-Penetrating Peptides Name SEQ ID (Designation) Sequence NO.^a rTAT RRRQRRKKR 30 Tat RKKRRQRRR 31 R₉F₂ RRRRRRRRRFF 32 R₅F₂R₄ RRRRRFFRRRR 33 R₄ RRRR 34 R₅ RRRRR 35 R₆ RRRRRR 36 R₇ RRRRRRR 37 R₈ RRRRRRRR 38 R₉ RRRRRRRRR 39 (RAhxR)₄; RAhxRRAhxRRAhxRRAhxR 40 (P007) (RAhxR)₅; RAhxRRAhxRRAhxRRAhxRRAhxR 41 (CP04057) (RAhxRRBR)₂; RAhxRRBRRAhxRRBR 42 (CP06062) (RAR)₄F₂ RARRARRARRARFFC 43 (RGR)₄F₂ RGRRGRRGRRGRFFC 44 ^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

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.

Treatment of Myotonic Dystrophy. 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.

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).

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 3′UTR 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 (RXRRR(B/X)R)₂XB (SEQ ID NO:55), where R is arginine; B is β-alanine; and each X is —C(O)—(CH₂)_n—NH—, where n is 4-6.

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 3′UTR 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+).

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.

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

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).

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.

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 2 Examples of Muscle-specific Homing Peptides (HP) Peptide Sequence SEQ ID Target Tissue (NH₂ to COOH) NO. Skeletal Muscle-SMP1 ASSLNIA 45 SMP2 SLGSFP 46 SMP3 SGASAV 47 SMP4 GRSGAR 48 SMP5 TARGEHKEEELI 49 Cardiac Muscle-CMP1 WLSEAGPVVTVRALRGTGSW 50 CMP2 VTVRALRGTSW 51 CMP3 VVTVRALRGTGSW 52 CMP4 CRPPR 53 CMP5 SKTFNTHPQSTP 54 CRVASVLPC 56

Peptide-Antisense Oligomer Conjugate Compositions Conjugates for Specific Muscle Treatments

Therapeutic conjugates comprising selected transport peptide sequences are also provided by the invention. These include conjugates comprising a carrier peptide (RXRR(B/X)R)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.

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.

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 3′UTR 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)₂XB (SEQ ID NO:55), where R is arginine; B is β-alanine; and each X is —C(O)—(CH₂)_n—NH—, 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

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 WO120081036127, which is incorporated by reference.

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 FIGS. 1B-C.

The intersubunit linkages in these oligomers are preferably phosphorus-containing linkages, having the structure:

where
W is S or O, and is preferably O,

X=NR¹R²or OR⁶, Y=O or NR⁷,

and each said linkage in the oligomer is selected from:

(a) uncharged linkage (a), where each of R¹, R², R⁶and R⁷is independently selected from hydrogen and lower alkyl;

(b1) cationic linkage (b1), where X=NR¹R²and Y=O, and NR¹R²represents an optionally substituted piperazino group, such that R¹R²=—CHRCHRN(R³)(R⁴)CHRCHR—, where

each R is independently H or CH₃,

R⁴is H, CH₃, or an electron pair, and

R³is selected from H, lower alkyl, e.g. CH₃, C(═NH)NH₂, Z-L-NHC(═NH)NH₂, and {C(O)CHR′NH}_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;

(b2) cationic linkage (b2), where X=NR¹R²and Y=O, R¹=H or CH₃, and R²=LNR³R⁴R⁵, where L, R³, and R⁴are as defined above, and R⁵is H, lower alkyl, or lower (alkoxy)alkyl; and

(b3) cationic linkage (b3), where Y=NR⁷and X=OR⁶, and R⁷=LNR³R⁴R⁵, where L, R³, R⁴and R⁵are as defined above, and R⁶is H or lower alkyl;

and at least one said linkage is selected from cationic linkages (b1), (b2), and (b3).

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.

In one embodiment, at least one linkage is of type (b1), where, preferably, each R is H, R⁴is H, CH₃, or an electron pair, and R³is selected from H, lower alkyl, e.g. CH₃, C(═NH)NH₂, and C(O)-L-NHC(═NH)NH₂The latter two embodiments of R³provide 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 R³is preferably C(O) (carbonyl), as shown.

The linker group L, as noted above, contains bonds in its backbone selected from alkyl (e.g. —CH₂—CH₂—), alkoxy (—C—O—), and alkylamino (e.g. —CH₂—NH—), 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. —CH₂—CHCH₃—) are possible, the linker is preferably unbranched. In one embodiment, the linker is a hydrocarbon linker. Such a linker may have the structure —(CH₂)_n—, where n is 1-12, preferably 2-8, and more preferably 2-6.

The use of embodiments of linkage types (b1), (b2) and (b3) above to link morpholino subunits may be illustrated graphically as follows:

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.

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:

In the structures above, W is S or O, and is preferably O; each of R¹and R²is 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.

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″).

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, R³is H or CH₃, and R⁴is H, CH₃, or an electron pair.

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.

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.

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%.

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.

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.

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.

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%400% of these charged linkages localized in the 10 centermost linkages.

Other Oligomer Types

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).

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.

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

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

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 A PMO, PMO+, PPMO,and PPMO+ agents targeted to polyCUG repeats in the 3′UTR region of dystrophia myotonica protein kinase (DMPK). Sample Name Sequence 5′End 3′End CAG 9 mer CAG CAG CAG (SEQ ID NO: 1) EG3 H CAG 9 mer−B CAG CAG CAG (SEQ ID NO: 1) EG3 CP06062 CAG 9 mer−R9F2 CAG CAG CAG (SEQ ID NO: 1) EG3 R9F2 CAG 9 mer−rTat CAG CAG CAG (SEQ ID NO: 1) EG3 rTat CAG 12 mer CAG CAG CAG CAG (SEQ ID NO: 2) EG3 H CAG 12 mer−B CAG CAG CAG CAG (SEQ ID NO: 2) EG3 CP06062 CAG 15 mer CAG CAG CAG CAG CAG (SEQ ID NO: 3) EG3 H CAG 15 mer−B CAG CAG CAG CAG CAG (SEQ ID NO: 3) EG3 CP06062 CAG 18 mer CAG CAG CAG CAG CAG CAG (SEQ ID NO: 4) EG3 H CAG 18 mer−B CAG CAG CAG CAG CAG CAG (SEQ ID NO: 4) EG3 CP06062 AGC 9 mer AGC AGC AGC (SEQ ID NO: 5) EG3 H AGC 9 mer−B AGC AGC AGC (SEQ ID NO: 5) EG3 CP06062 AGC 12 mer AGC AGC AGC AGC (SEQ ID NO: 6) EG3 H AGC 12 mer-B AGC AGC AGC AGC (SEQ ID NO: 6) EG3 CP06062 AGC 15 mer AGC AGC AGC AGC AGC (SEQ ID NO: 7) EG3 H AGC 15 mer−B AGC AGC AGC AGC AGC (SEQ ID NO: 7) EG3 CP06062 AGC 18 mer AGC AGC AGC AGC AGC AGC (SEQ ID NO: 8) EG3 H AGC 18 mer−B AGC AGC AGC AGC AGC AGC (SEQ ID NO: 8) EG3 CP06062 GCA 9 mer GCA GCA GCA (SEQ ID NO: 9) EG3 H GCA 9 mer-B GCA GCA GCA (SEQ ID NO: 9) EG3 CP06062 GCA 12 mer GCA GCA GCA GCA (SEQ ID NO: 10) EG3 H GCA 12 mer−B GCA GCA GCA GCA (SEQ ID NO: 10) EG3 CP06062 GCA 15 mer GCA GCA GCA GCA GCA (SEQ ID NO: 11) EG3 H GCA 15 mer-B GCA GCA GCA GCA GCA (SEQ ID NO: 11) EG3 CP06062 GCA 18 mer GCA GCA GCA GCA GCA GCA (SEQ ID NO: 12) EG3 H GCA 18 mer−B GCA GCA GCA GCA GCA GCA (SEQ ID NO: 12) EG3 CP06062 AGC 25 mer AGC AGC AGC AGC AGC AGC AGC AGC A EG3 H (SEQ ID NO: 13) AGC 25 mer−B AGC AGC AGC AGC AGC AGC AGC AGC A EG3 CP06062 (SEQ ID NO: 13) CAG 25 mer CAG CAG CAG CAG CAG CAG CAG CAG C EG3 H (SEQ ID NO: 14) CAG 25 mer−B CAG CAG CAG CAG CAG CAG CAG CAG C EG3 CP06062 (SEQ ID NO: 14) CAG 25 mer−R9F2 CAG CAG CAG CAG CAG CAG CAG CAG C EG3 R9F2 (SEQ ID NO: 14) CAG 25 mer−rTat CAG CAG CAG CAG CAG CAG CAG CAG C EG3 rTat (SEQ ID NO: 14) CAG 9 mer+ C+AG C+AG C+AG (SEQ ID NO: 15) EG3 H CAG 9 mer+B C+AG C+AG C+AG (SEQ ID NO: 15) EG3 CP06062 CAG 9 mer+ C+AG CAG CAG (SEQ ID NO: 16) EG3 H CAG 9 mer+ CAG CAG C+AG (SEQ ID NO: 17) EG3 H CAG 9 mer+ CAG C+AG CAG (SEQ ID NO: 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.

HSA^LRtransgenic 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 an 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.

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, ClC-1, Serca-1, m-Titin, Tnnt3 and Zasp genes (Mulders, van den Broek et al. 2009). HSA^LRtransgenic 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.

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^LRmouse model. Myotonia can be measured through determination of delayed muscle relaxation and repetitive action potentials and are expected to improve in HSA^LRmice 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

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 B PMO, PMO+, PPMO, and PPMO+ agents targeted to polyCCUG repeats in the first intron of zinc finger protein 9 (ZNF9) pre-mRNA. Sample Name Sequence 5′End 3′End CAGG 9 mer CAG GCA GGC (SEQ ID NO: 19) EG3 H CAGG 9 mer−B CAG GCA GGC (SEQ ID NO: 19) EG3 CP06062 CAGG 9 mer−R9F2 CAG GCA GGC (SEQ ID NO: 19) EG3 R9F2 CCAG 9 mer−rTat CAG GCA GGC (SEQ ID NO: 19) EG3 rTat CCAG 12 mer CAG GCA GGC AGG (SEQ ID NO: 20) EG3 H CCAG 12 mer−B CAG GCA GGC AGG (SEQ ID NO: 20) EG3 CP06062 AGCC 9 mer AGG CAG GCA (SEQ ID NO: 21) EG3 H AGCC 9 mer−B AGG CAG GCA (SEQ ID NO: 21) EG3 CP06062 AGCC 12 mer AGG CAG GCA GGC (SEQ ID NO: 22) EG3 H AGCC 12 mer−B AGG CAG GCA GGC (SEQ ID NO: 22) EG3 CP06062 GCCA 9 mer GGC AGG CAG (SEQ ID NO: 23) EG3 H GCCA 9 mer−B GGC AGG CAG (SEQ ID NO: 23) EG3 CP06062 GCCA 12 mer GGC AGG CAG GCA (SEQ ID NO: 24) EG3 H GCCA 12 mer−B GGC AGG CAG GCA (SEQ ID NO: 24) EG3 CP06062 CAGG 24 mer CAG GCA GGC AGG CAG GCA GGC AGG EG3 H (SEQ ID NO: 25) CAGG 24 mer−B CAG GCA GGC AGG CAG GCA GGC AGG EG3 CP06062 (SEQ ID NO: 25) CAGG 24 mer−R9F2 CAG GCA GGC AGG CAG GCA GGC AGG EG3 R9F2 (SEQ ID NO: 25) CAGG 24 mer−rTat CAG GCA GGC AGG CAG GCA GGC AGG EG3 rTat (SEQ ID NO: 25) CAGG 9 mer+ C+AG GC+A GGC (SEQ ID NO: 26) EG3 H CAGG 9 mer+B C+AG GC+A GGC (SEQ ID NO: 27) EG3 CP06062 CAGG 9 mer+ C+AG GCA GGC (SEQ ID NO: 28) EG3 H CAGG 9 mer+ CAG GC+A GGC (SEQ ID NO: 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.

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.

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Sequence Listing Table Sample Name Sequence SEQ ID NO: CAG 9 mer CAG CAG CAG 1 CAG 12 mer CAG CAG CAG CAG 2 CAG 15 mer CAG CAG CAG CAG CAG 3 CAG 18 mer CAG CAG CAG CAG CAG CAG 4 AGC 9 mer AGC AGC AGC 5 AGC 12 mer AGC AGC AGC AGC 6 AGC 15 mer AGC AGC AGC AGC AGC 7 AGC 18 mer AGC AGC AGC AGC AGC AGC 8 GCA 9 mer GCA GCA GCA 9 GCA 12 mer GCA GCA GCA GCA 10 GCA 15 mer GCA GCA GCA GCA GCA 11 GCA 18 mer GCA GCA GCA GCA GCA GCA 12 AGC 25 mer AGC AGC AGC AGC AGC AGC AGC AGC A 13 CAG 25 mer CAG CAG CAG CAG CAG CAG CAG CAG C 14 CAG 9 mer+ C+AG C+AG C+AG 15 CAG 9 mer+ C+AG CAG CAG 16 CAG 9 mer+ CAG CAG C+AG 17 CAG 9 mer+ CAG C+AG CAG 18 CAGG 9 mer CAG GCA GGC 19 CAGG 12 mer CAG GCA GGC AGG 20 AGGC 9 mer AGG CAG GCA 21 AGGC 12 mer AGG CAG GCA GGC 22 GGCA 9 mer GGC AGG CAG 23 GGCA 12 mer GGC AGG CAG GCA 24 CAGG 24 mer CAG GCA GGC AGG CAG GCA GGC AGG 25 CAGG 9 mer+ C+AG GC+A GGC 26 CAGG 9 mer+B C+AG GC+A GGC 27 CAGG 9 mer+ C+AG GCA GGC 28 CAGG 9 mer+ CAG GC+A GGC 29 rTAT RRRQRRKKR 30 Tat RKKRRQRRR 31 R₉F₂ RRRRRRRRRFF 32 R₅F₂R₄ RRRRRFFRRRR 33 R₄ RRRR 34 R₅ RRRRR 35 R₆ RRRRRR 36 R₇ RRRRRRR 37 R₈ RRRRRRRR 38 R₉ RRRRRRRRR 39 (RAhxR)₄; (P007) RAhxRRAhxRRAhxRRAhxR 40 (RAhxR)₅; RAhxRRAhxRRAhxRRAhxRRAhxR 41 (CP04057) (RAhxRRBR)₂; RAhxRRBRRAhxRRBR 42 (CP06062) (RAR)₄F₂ RARRARRARRARFFC 43 (RGR)₄F₂ 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)₂XB 55 CRVASVLPC 56 *In SEQ ID NOS: 15-18 and 26-29, “+” refers to a cationic linkage, such as 1-piperazinyl.

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 3′UTR 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 3′UTR 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.