Patent Description:
This invention was made with government support under R01 NS043264 awarded by the National Institutes of Neurologic Diseases and Stroke. The government has certain rights in the invention.

The present invention relates to the rAAv delivery of oligomers for use in treating patients with a <NUM>' mutation in their DMD gene other than a DMD exon <NUM> duplication for the treatment of Duchenne Muscular Dystrophy or Becker Muscular Dystrophy.

Muscular dystrophies (MDs) are a group of genetic diseases. The group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. The disorders differ in terms of the distribution and extent of muscle weakness (some forms of MD also affect cardiac muscle), the age of onset, the rate of progression, and the pattern of inheritance.

One form of MD is Duchenne Muscular Dystrophy (DMD). It is the most common severe childhood form of muscular dystrophy affecting <NUM> in <NUM> newborn males. DMD is caused by mutations in the DMD gene leading to absence of dystrophin protein (<NUM> KDa) in skeletal and cardiac muscles, as well as GI tract and retina. Dystrophin not only protects the sarcolemma from eccentric contractions, but also anchors a number of signaling proteins in close proximity to sarcolemma. Many clinical cases of DMD are linked to deletion mutations in the DMD gene. Despite many lines of research following the identification of the DMD gene, treatment options are limited. Corticosteroids are clearly beneficial but even with added years of ambulation the benefits are offset by long-term side effects. The original controlled, randomized, double-blind study reported more than <NUM> years ago showed benefits using prednisone [<NPL>)]. Subsequent reports showed equal efficacy using deflazacort, a sodium-sparing steroid [<NPL>)]. Recent studies also demonstrate efficacy by exon skipping, prolonging walking distance on the 6MWT. Thus far, published clinical studies have reported benefit for only mutations where the reading frame is restored by skipping exon <NUM> [<NPL>) and <NPL>)]. In the only report of a double blind, randomized treatment trial promising results were demonstrated with eteplirsen, a phosphorodiamidate morpholino oligomer (PMO) [<NPL>)]. In all of these exon-skipping trials, the common denominator of findings has been a plateau in walking ability after an initial modest improvement. Another exon-skipping article is <NPL>).

In contrast to the deletion mutations, DMD exon duplications account for around <NUM>% of disease-causing mutations in unbiased samples of dystrophinopathy patients [<NPL>)], although in some catalogues of mutations the number of duplications is higher [including that published by the United Dystrophinopathy Project in <NPL>), in which it was <NUM> %].

Mutations in the DMD gene result in either the more severe DMD or the milder Becker muscular dystrophy (BMD). The phenotype generally depends upon whether the mutation results in the complete absence of the protein product dystrophin (in DMD) or preserves a reading frame that allows translation of a partially functional dystrophin protein (in BMD) [<NPL>)]. We previously identified a particular BMD founder allele (c. Trp3X) that did not follow this reading frame rule [<NPL>) and Flanigan et al. , Human Mutation, <NUM>: <NUM>-<NUM> (<NUM>)]. Although this nonsense mutation is predicted to result in no protein translation, muscle biopsy revealed significant amounts (∼<NUM>%) of dystrophin expression of minimally decreased size and the clinical phenotype is one of a very mild dystrophinopathy [<NPL>)]. In cellulo and in vitro translation studies demonstrated that in p. Trp3X patients translation is initiated from AUGs in exon <NUM>, suggesting alternate translation initiation as a mechanism of phenotypic amelioration [<NPL>)]. Noting that most truncating mutations reported in <NUM>' exons were in fact associated with BMD rather than DMD, we proposed that altered translation initiation may be a general mechanism of phenotypic rescue for <NUM>' mutations in this gene, a prediction supported by subsequent reports [<NPL>) and <NPL>)]. The canonical actin-binding domain <NUM> (ABD1) was previously proposed to be essential for protein function [<NPL>).

Translation initiation is commonly understood to occur by cap-dependent initiation. Internal ribosome entry sites (IRESs) are RNA regulatory sequences that govern cap-independent translation initiation in eukaryotic cells, which is activated when cap-dependent translation is compromised (e.g., during cell stress). Ribosomes are recruited directly to these IRESs on the mRNA and can then continue scanning in a <NUM>' to <NUM>' direction for alternative initiation codons. They were first described in viruses, and among the earliest characterized was the encephalomyocarditis virus (EMCV) IRES. Almost <NUM> cellular IRESs have been described to date and are mainly located in <NUM>'UTR regions; for example, the <NUM>'UTR of utrophin A, an autosomal homologue of dystrophin, contains an IRES that is both particularly active in regenerating muscle and inducible by exposure to glucocorticoid (the mainstay of therapy for DMD) [<NPL>) and<NPL>)]. However, other eukaryotic IRESs have been described within coding sequences, and some have also been implicated in the modulation of pathology. These include an IRES in the APC gene linked to a mild version of familial adenomatous polyposis coli in which patients with certain <NUM>' mutations still produce a partially functional protein through the use of a downstream initiation codon.

Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about <NUM> kb in length including <NUM> nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-<NUM> is provided in GenBank Accession No. NC_002077; the complete genome of AAV-<NUM> is provided in GenBank Accession No. NC_001401 and <NPL>); the complete genome of AAV-<NUM> is provided in GenBank Accession No. NC_1829; the complete genome of AAV-<NUM> is provided in GenBank Accession No. NC_001829; the AAV-<NUM> genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-<NUM> is provided in GenBank Accession No. NC_00 <NUM>; at least portions of AAV-<NUM> and AAV-<NUM> genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also <CIT> and <CIT> relating to AAV-<NUM>); the AAV-<NUM> genome is provided in <NPL>); the AAV-<NUM> genome is provided in <NPL>); and the AAV-<NUM> genome is provided in <NPL>). The sequence of the AAV rh. <NUM> genome is provided herein. Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides <NUM> and <NUM>), result in the production of four rep proteins (rep <NUM>, rep <NUM>, rep <NUM>, and rep <NUM>) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position <NUM> of the AAV genome. The life cycle and genetics of AAV are reviewed in <NPL>).

AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately <NUM> kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56o to <NUM> for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

<NPL> appears to disclose an out-of-frame exon-skipping approach to generate a truncated reading frame upstream of the IRES in patient-derived cell lines and in a DMD mouse model, leading to synthesis of a functional N-truncated isoform.

There remains a need in the art for treatments for muscular dystrophies including DMD and BMD.

The present disclosure contemplates products for use in methods of preventing disease, delaying the progression of disease, and/or treating patients with one or more <NUM>' mutations of the DMD gene. The use in the methods are based on the identification of a glucocorticoid-inducible IRES in exon <NUM> of the DMD gene, the activation of which can generate a functional N-terminally truncated dystrophin isoform.

The disclosure contemplates use in methods of ameliorating Duchenne Muscular Dystrophy or Becker Muscular Dystrophy in a patient with a <NUM>' mutation in the DMD gene comprising the step of administering a DMD exon <NUM> IRES-activating oligomer construct to the patient, wherein the patient does not have a DMD exon <NUM> duplication.

The invention is defined in the claims. The invention provides a recombinant adeno-associated virus (rAAV) comprising a Duchenne Muscular Dystrophy (DMD) exon <NUM> internal ribosome entry site (IRES)-activating oligomer construct comprising:.

In some embodiments, the DMD exon <NUM> IRES-activating oligomer construct is a U7snRNA polynucleotide construct in the genome of the rAAV.

In some embodiments, the rAAV comprises the nucleotide sequence set forth in SEQ ID NO: <NUM>.

In some embodiments, treating further comprises administering a glucocorticoid to the patient.

In some embodiments, a) the progression of a dystrophic pathology is inhibited in the patient; b) muscle function is improved in the patient; c) muscle function is improved in the patient and the improvement in muscle function is an improvement in muscle strength; d) muscle function is improved in the patient and the improvement in muscle function is an improvement in stability in standing and walking; or e) a functional truncated isoform of dystrophin is expressed in muscle tissue of the patient.

In some embodiments, a) the genome of the rAAV lacks adeno-associated virus rep and cap DNA; b) the rAAV genome is self-complementary or is single-stranded; or c) the rAAV is rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, or rAAVrh74.

In some embodiments, the DMD exon <NUM> IRES-activating oligomer construct comprises a polynucleotide comprising two copies of (a) the nucleotide sequence set forth in SEQ ID NO: <NUM> and the nucleotide sequence set forth in SEQ ID NO: <NUM>; or (b) the nucleotide sequence that expresses an RNA transcript comprising the nucleotide sequence set forth in SEQ ID NO: <NUM> and the nucleotide sequence that expresses an RNA transcript comprising the nucleotide sequence set forth in SEQ ID NO: <NUM>.

In some embodiments, the AAV comprises the U7-C antisense polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: <NUM>.

In some embodiments, the polynucleotide that expresses the U7-C antisense oligomer comprises the nucleotide sequence set forth in SEQ ID NO: <NUM>.

As noted above, the present disclosure contemplates products for use in preventing, delaying the progression of, and/or treating patients with one or more <NUM>' mutations of the DMD gene that are based on the activation of a glucocorticoid-inducible IRES in exon <NUM> of the DMD gene. The activation of the inducible IRES in exon <NUM> of the DMD gene generates a functional N-terminally truncated dystrophin isoform.

As used herein, a "<NUM>' mutation of the DMD gene" is a mutation within or affecting exon <NUM>, <NUM>, <NUM> or <NUM> of the DMD gene. In the use in the methods of the invention, the patients treated do not have a DMD exon <NUM> duplication, but a "mutation affecting exon <NUM>, <NUM>, <NUM> or <NUM>" as contemplated herein can be a duplication other than a DMD exon <NUM> duplication.

The use in the methods involve using an "DMD exon <NUM> IRES-activating oligomer construct" as defined in the claims. As used herein, a DMD exon <NUM> IRES-activating oligomer construct targets exon <NUM> to induce altered splicing that results in the exclusion of exon <NUM> from the mature RNA causing a frameshift in the DMD gene reading frame and inducing utilization of the IRES in exon <NUM> for translational initiation.

The DMD exon <NUM> IRES-activating oligomer used in the invention is defined in the claims. In some aspects of the disclosure, the DMD exon <NUM> IRES-activating oligomer construct targets one of the following portions (shown <NUM>' to <NUM>') of exon <NUM> of the DMD gene.

An rAAV is used to deliver a U7 small nuclear RNA polynucleotide construct that is targeted to DMD exon <NUM> by an antisense polynucleotide, as defined in the claims. In some embodiments, the U7 small nuclear RNA is a human U7 small nuclear RNA. In some embodiments, the polynucleotide construct is inserted in the genome of a rAAV9, the genome of a rAAV6 or the genome of a rAAVrh74. The DMD exon <NUM> IRES-activating oligomer construct for use according to the invention is defined in the claims. In some aspects of the disclosure, the U7 small nucleotide RNA construct comprises exemplary targeting antisense polynucleotides including, but not limited to the following where, for example, the "U7-AL antisense polynucleotide" is respectively complementary to and targets the "AL" exon <NUM> sequence in the preceding paragraph.

The DMD exon <NUM> IRES-activating oligomer construct as defined in the claims is an exon <NUM>-targeting antisense oligomer. In some embodiments, the antisense oligomers are contemplated to include modifications compared to the native phosphodiester oligodeoxynucleotide polymer to limit their nuclease sensitivity. Contemplated modifications include, but are not limited to, phosphorodiamidate morpholino oligomers (PPOs), cell penetrating peptide-conjugated PMOs (PPMOs), PMO internalizing peptides (PIP) [(<NPL>)], tricyclo-DNA (tcDNA) [<NPL>)] and <NUM>'O-methyl-phosphorothioate modifications. The DMD exon <NUM> IRES-activating oligomer construct of the invention is defined in the claims. Exemplary DMD exon <NUM> IRES-activating oligomer constructs of the disclosure that are exon <NUM>-targeting antisense oligomers include, but are not limited to, the following antisense oligomers (shown <NUM>' to <NUM>') where, for example, the "B antisense oligomer" respectively targets the "B" exon <NUM> target in paragraph [<NUM>].

In another aspect, use in a method of ameliorating a muscular dystrophy (such as DMD or BMD) in a patient with a <NUM>' mutation of the DMD gene is provided. The use in the method comprises the step of administering a rAAV to the patient, wherein the genome of the rAAV comprises a DMD exon <NUM> IRES-activating oligomer construct as defined in the claims. The use in the method comprises the step of administering a DMD exon <NUM> IRES-activating oligomer construct that is an exon <NUM>-targeting antisense oligomer as defined in the claims. In some embodiments, the patient is also treated with a glucocorticoid.

In yet another aspect, the invention provides use in a method of inhibiting the progression of dystrophic pathology associated with a muscular dystrophy (such as DMD or BMD). The use in the method comprises the step of administering a rAAV to a patient with a <NUM>' mutation of the DMD gene, wherein the genome of the rAAV comprises a DMD exon <NUM> IRES-activating oligomer construct as defined in the claims. The use in the method comprises the step of administering a DMD exon <NUM> IRES-activating oligomer construct that is an exon <NUM>-targeting antisense oligomer as defined in the claims. In some embodiments, the patient is also treated with a glucocorticoid.

In still another aspect, use in a method of improving muscle function in a patient with a <NUM>' mutation of the DMD gene is provided. the use in the method comprises the step of administering a rAAV to the patient, wherein the genome of the rAAV comprises a DMD exon <NUM> IRES-activating oligomer construct as defined in the claims. The method comprises the step of administering a DMD exon <NUM> IRES-activating oligomer construct that is an exon <NUM>-targeting antisense oligomer as defined in the claims. In some embodiments, the improvement in muscle function is an improvement in muscle strength. The improvement in muscle strength is determined by techniques known in the art such as the maximal voluntary isometric contraction testing (MVICT). In some instances, the improvement in muscle function is an improvement in stability in standing and walking. The improvement in stability strength is determined by techniques known in the art such as the <NUM>-minute walk test (6MWT) or timed stair climb. In some embodiments, the patient is also treated with a glucocorticoid.

In another aspect, the disclosure provides use in a method of delivering a DMD exon <NUM> IRES-activating oligomer construct to an animal (including, but not limited to, a human) with a <NUM>' mutation of the DMD gene. In some aspects of the disclosure, the use in the method comprises the step of a rAAV to the patient, wherein the genome of the rAAV comprises a DMD exon <NUM> IRES-activating oligomer construct. In some aspects of the disclosure, the use in the method comprises the step of administering a DMD exon <NUM> IRES-activating oligomer construct that is an exon <NUM>-targeting antisense oligomer. In some aspects of the disclosure, the animal is also treated with a glucocorticoid.

Cell transduction efficiencies of the use in the methods of the invention described herein may be at least about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM> or about <NUM> percent.

In some embodiments of the foregoing use in the methods of the invention, the virus genome is a self-complementary genome. In some embodiments of the use in the methods, the genome of the rAAV lacks AAV rep and cap DNA. In some embodiments of the use in the methods, the rAAV is a SC rAAV U7_ACCA comprising the exemplary genome set out in <FIG>. In some embodiments, the rAAV is a rAAV6. In some embodiments, the rAAV is a rAAV9. In some embodiments the rAAV is a rAAV rh74 (<FIG>).

In yet another aspect, the disclosure provides a rAAV comprising the AAV rAAV9 capsid and a genome comprising the exemplary DMD exon <NUM> IRES-activating U7 snRNA polynucleotide construct U7_ACCA. In some aspects of the disclosure, the genome of the rAAV lacks AAV rep and cap DNA. In some aspects of the disclosure, the rAAV comprises a self-complementary genome. In some aspects of the methods of the disclosure, the rAAV is a SC rAAV U7_ACCA comprising the exemplary genome is set out in <FIG>. In some aspects of the disclosure, the rAAV is a rAAV6. In some aspects of the disclosure, the rAAV is a rAAV9. In some aspects of the disclosure, the rAAV is a rAAV rh74 (<FIG>).

Recombinant AAV genomes for use according to the invention comprise one or more AAV ITRs flanking at least one DMD exon <NUM> IRES-activating U7 snRNA polynucleotide construct. Genomes with DMD exon <NUM> IRES-activating U7 snRNA polynucleotide constructs comprising each of the targeting antisense sequences set out in paragraph [<NUM>] are specifically contemplated, as well as genomes with DMD exon <NUM> IRES-activating U7 snRNA polynucleotide constructs comprising each possible combination of two or more of the targeting antisense sequences set out in paragraph [<NUM>]. In some embodiments, including the exemplified embodiments, the U7 snRNA polynucleotide includes its own promoter. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM> and AAV rh. As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. In some embodiments of the invention, the promoter DNAs are muscle-specific control elements, including, but not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family [See<NPL>)], the myocyte-specific enhancer binding factor MEF-<NUM> [<NPL>)], control elements derived from the human skeletal actin gene [<NPL>)], the cardiac actin gene, muscle creatine kinase sequence elements [<NPL>)] and the murine creatine kinase enhancer (MCK) element, desmin promoter, control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene: hypoxia-inducible nuclear factors [<NPL>)], steroidinducible elements and promoters including the glucocorticoid response element (GRE) [See <NPL>)], and other control elements.

DNA plasmids for use according to the invention comprise rAAV genomes as defined in the claims.

The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM>, AAV-<NUM> and AAV rh74. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, <CIT>.

A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing [<NPL>)], addition of synthetic linkers containing restriction endonuclease cleavage sites [<NPL>)] or by direct, blunt-end ligation [<NPL>)]. The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example, <NPL>); and<NPL>). Various approaches are described in <NPL>);<NPL>); <NPL>); <NPL>); and <NPL>). <NPL>); <CIT>; <CIT> and corresponding <CIT> ; <CIT>; <CIT>; <CIT>; <CIT> (<CIT>); <CIT> (<CIT>); <CIT> (<CIT>); <CIT> (<CIT>); <CIT>;<NPL>); <NPL>); <NPL>); <CIT>; <CIT>; and <CIT>.

The disclosure thus provides packaging cells that produce infectious rAAV. In one aspect of the disclosure, packaging cells may be stably transformed cancer cells such as HeLa cells, <NUM> cells and PerC. <NUM> cells (a cognate <NUM> line). In another aspect of the disclosure, packaging cells are cells that are not transformed cancer cells, such as low passage <NUM> cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-<NUM> cells (human fetal fibroblasts), WI-<NUM> cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-<NUM> cells (rhesus fetal lung cells).

The rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example,<NPL>); <NPL>); <CIT> and <CIT>.

In another aspect, the disclosure contemplates compositions comprising a DMD exon <NUM> IRES-activating oligomer construct of the present disclosure in a viral delivery vector or other delivery vehicle. Compositions of the disclosure comprise a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients such as diluents. Acceptable carriers and diluents are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formig counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

Titers of rAAV to be administered in use in the methods of the invention will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about <NUM>×<NUM><NUM>, about <NUM>×<NUM><NUM>, about <NUM>×<NUM><NUM>, about <NUM>×<NUM><NUM>, about <NUM>×<NUM><NUM>, about <NUM>×<NUM><NUM>, about <NUM>×<NUM><NUM>, about <NUM>×<NUM><NUM> to about <NUM>×<NUM><NUM> or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) (i.e., <NUM>×<NUM><NUM> vg, <NUM>×<NUM><NUM> vg, <NUM>×<NUM><NUM> vg, <NUM>×<NUM><NUM> vg, <NUM>×<NUM><NUM> vg, <NUM>×<NUM><NUM> vg, <NUM>×<NUM><NUM> vg, <NUM>×<NUM><NUM> vg, respectively).

Use of a rAAV as defined in the claims in methods of transducing a target cell (e.g., a skeletal muscle) of a patient with a <NUM>' mutation of the DMD gene , in vivo or in vitro, are contemplated herein as part of the disclosure. The use in the methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV as defined in the claims to an animal (including a human being) with a <NUM>' mutation of the DMD gene. If the dose is administered prior to development of DMD, the administration is prophylactic. If the dose is administered after the development of DMD, the administration is therapeutic. In embodiments of the invention, an "effective dose" is a dose that alleviates (eliminates or reduces) at least one symptom associated with DMD being treated, that slows or prevents progression to DMD, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival.

Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration and serotype(s) of AAV components of rAAV (in particular, the AAV ITRs and capsid protein) as defined in the claims may be chosen and/or matched by those skilled in the art taking into account the infection and/or disease state being treated and the target cells/tissue(s). In some embodiments, the route of administration is intramuscular. In some embodiments, the route of administration is intravenous.

Combination therapies are also contemplated by the invention. Combination therapy as used herein includes simultaneous treatment or sequential treatments. Combinations of methods of the invention with standard medical treatments (e.g., corticosteroids and/or immunosuppressive drugs) are specifically contemplated, as are combinations with other therapies such as those mentioned in the Background section above. In some embodiments, the corticosteroid is a glucocorticoid such as prednisone, deflazacort or Medrol (<NUM>-methyl-prednisolone; PDN).

Aspects and embodiments of the invention are illustrated by the following examples. Any examples falling outside the scope of the claims are provided for comparative purposes.

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to DMD. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon <NUM> that leads to expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an IRES within exon <NUM> that is glucocorticoid-inducible. IRES activity was confirmed in patient muscle by both peptide sequencing and ribosome profiling as described below. Generation of a truncated reading frame upstream of the IRES by exon skipping led to synthesis of a functional N-truncated isoform in both patient-derived cell lines and in a DMD mouse model, where expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. These results support a novel therapeutic approach for patients with mutations within the <NUM>' exons of the DMD gene. See also, <NPL>).

We previously published that nonsense and frameshifting mutations leading to a stop codon within at least the first two DMD exons should result in the mild BMD phenotype via exon <NUM> translation initiation [<NPL>)]. However, duplication of exon <NUM> - which is the most common single exon duplication and results in a premature stop codon within the duplicated exon <NUM> sequence - would seem to be an exception to this prediction, as it is usually associated with DMD [<NPL>)]. However, a deletion of exon <NUM>, which also results in a premature stop codon, has not been described, either in our large cohort [<NPL>)] or in other large publicly available catalogues (www. We interpreted this lack of reported cases to mean that the clinical features in patients with exon <NUM> deletions are either asymptomatic or exceedingly mild due to expression of the N-truncated isoform.

This interpretation was confirmed by the detection of a deletion of exon <NUM> (DEL2) in an Italian boy who first presented at age <NUM> years for evaluation of an incidentally detected elevation of serum creatine kinase (<NUM> iu/l; normal value < <NUM> iu/l). Normal early motor milestones were reported and no muscle dystrophy was ever reported in the family. His neurological examination was entirely normal at <NUM> years of age. Muscle biopsy showed slight fiber size variability (<FIG>), and in some sections an increased number of central nuclei along with some densely stained hypercontracted fibers. Immunofluorescent analysis using a C-terminal antibody showed the presence of dystrophin at the membrane (<FIG>). Interestingly, western blot revealed that the detected dystrophin had a smaller molecular weight (∼410kDa) (<FIG>) , and mutational analysis revealed a deletion of exon <NUM> (<FIG>). Subsequent peptide sequencing using tandem mass spectrometry (LC-MS/MS)<NUM> confirmed the absence of any residues encoded by exons <NUM> through <NUM> among the <NUM> unique peptides detected and matched to dystrophin, consistent with translation initiation within exon <NUM> (<FIG> and Table <NUM>).

In a complementary approach, we examined DMD translation efficiency, promoter usage, and alternate splicing using muscle RNA isolated from a mild BMD patient with an exon <NUM> frameshift mutation (c. 40_41del [p. Glu14ArgfsX17], referred to as FS) whose western blot also revealed expression of the same smaller molecular weight dystrophin (~410kDa) which lacked the N-terminal epitope (<FIG>; <FIG>). To confirm our western blot results, muscle homogenate from the same FS patient was used to construct RNA-Seq libraries for ribosome-protected fragments (i.e., ribosome footprints isolated after RNase digestion) and for total RNA. We compared the mRNA translation efficiency in normal versus patient muscle using the ratio of reads from ribosome-protected fragments (RPFs) to reads from RNA-Seq. Among the top <NUM> most abundant muscle mRNAs, DMD displayed the greatest change in translation efficiency (<FIG>), indicating a -<NUM>-fold reduction in the amount of ribosomes translating the DMD muscle transcript in the frameshifted patient FS. This decreased amount of translation is consistent with both the expected reduction in dystrophin level given the patient's mild BMD phenotype, and with the amount of dystrophin seen in p. Trp3X patients<NUM> and other <NUM>' mutation alleles (<FIG>).

The saw-tooth RNA-Seq pattern observed in DMD introns <NUM> through <NUM> (<FIG>) confirmed that the major transcription start was located at the dystrophin muscle-specific promoter (Dp427m) and that DMD exons <NUM> through <NUM> underwent efficient co-transcriptional splicing [<NPL>)] in both the control and FS patient samples. Two alternate <NUM> kD isoforms of dystrophin (Dp427p and Dp427c) are expressed primarily in the central nervous system, and differ from Dp427m only in the use of alternate exon <NUM> sequences. The lack of a strong nascent RNA signal from either the Dp427p or Dp427c promoters confirmed that up-regulation of alternate promoters does not contribute to alternate AUG usage in exon <NUM> (<FIG>). In both samples, RNA-Seq reads spanning exonexon junctions mapped exclusively to the known junctions between Dp427m exon <NUM> and exon <NUM>, indicating that splicing of novel <NUM>' UTRs from alternate promoters did not contribute to exon <NUM> AUG usage. The distribution of ribosome footprints mapped on Dp427m exons <NUM> through <NUM> revealed normal levels of exon <NUM> AUG initiation, followed by premature termination in exon <NUM> and resumption of translation following the exon <NUM> in-frame AUG codons (<FIG>) that continued into the body of the DMD transcript (<FIG>), consistent with efficient alternate translation initiation.

Having demonstrated new evidence for efficient alternate translation initiation using both ribosome profiling and protein analysis directly in patient muscle, we sought to characterize the elements contributing to the high translation efficiency. To determine whether exons <NUM> through <NUM> of DMD contain an IRES, we cloned the <NUM>' portion of the cDNA encompassing exons <NUM> through part of exon <NUM>, beginning at the +<NUM> position to exclude the native AUG initiation codon (c. <NUM>, referred as exon <NUM> to <NUM>), into the dicistronic dual luciferase reporter vector pRDEF. This vector contains an upstream cap-dependent renilla luciferase (RLuc) open reading frame (ORF) under control of an SV40 promoter and a downstream cap-independent firefly luciferase (FLuc) ORF under the control of the sequences of interest, with the two ORFs separated by a secondary structure element (dEMCV) that prevents ribosomal scanning (<FIG>). We used the EMCV IRES sequence as a positive control, and normalized all values to the empty vector. In each case we included <NUM> nucleotides from exon <NUM> that placed the exon <NUM> AUGs in-frame with the downstream FLuc reporter. This sequence corresponds to the first <NUM> nt, inclusive of the two in-frame AUGs (M124 and M128), and <NUM> additional nucleotides used for cloning purposes. T7 mediated RNA were generated from the different constructs and were used to perform rabbit reticulocyte lysate (RRL) translation assays (<FIG>, left panel). Size and integrity of the corresponding RNAs were checked using a formaldehyde agarose gel (<FIG>). Cap-independent translation activity (represented as the ratio of downstream FLuc to the RLuc luminescence) of the exons <NUM>-<NUM> of DMD results in a <NUM>-<NUM> fold increase in FLuc signal, less than the <NUM>-<NUM> increase seen with the control EMCV IRES but consistent with IRES activity (<FIG>, left panel).

RRL-based translation may underestimate IRES activity of either viral or eukaryotic cellular IRESs, possibly due to the limiting amounts of RNA binding proteins in this specialized extract or due to the lack of tissue-specific IRES trans-acting factors (ITAFs). Therefore, the assay was performed in C2C12 myoblasts which express dystrophin, and we observed that the presence of the exon <NUM> to <NUM> construct leads to ~<NUM> fold higher FLuc expression relative to exon <NUM> alone vector (<FIG>, right panel). This represents -<NUM>% of the activity of the control EMCV IRES, suggesting the presence of a relatively strong IRES within exons <NUM>-<NUM>. To map the position of the IRES, deletion constructs consisting of the <NUM>' portion of the DMD gene (exons <NUM>-<NUM>) or appropriate controls were cloned into pRDEF (<FIG>). Deletion of the first <NUM> nucleotides (nt) of this sequence did not significantly change the FLuc expression, whereas removal or inversion of the last <NUM> nt (representing nearly all of exon <NUM>) completely abrogates expression of the FLuc reporter, and further deletions within exon <NUM> result in greatly reduced FLuc expression. To test the hypothesis that the putative IRES required muscle specific factors, we repeated the experiments in HEK293K cells, which do not endogenously express dystrophin, and in a commercial human myoblast cell line (hSKMM). Unlike the ECMV IRES, the putative DMD IRES did not stimulate FLuc expression in <NUM> cells whereas the level of stimulation in hSKMM cells replicated the C2C12 results (<FIG>), suggesting that the IRES is preferentially active in muscle.

Control experiments were performed to exclude the possibility of aberrant splicing events, cryptic promoter activities, or other potential artifacts leading to misinterpretation of the dicistronic assay. We removed the upstream SV40 promoter to generate a promoterless version of the pRDEF vector containing the exon <NUM> to <NUM> (c. <NUM>) DMD sequence. Transfection of this construct into C2C12 myoblasts showed only minimal background luminescence from both RLuc and FLuc, strongly arguing against any cryptic promoter activity in the DMD coding sequence (data not shown). No aberrant splicing was detected by RT-PCR (<FIG> and <FIG>), and RNA integrity was confirmed by a northern blot (<FIG> and <FIG>).

Notably, although either duplication or deletion of exon <NUM> results in an interrupted reading frame, the disparate associated clinical phenotypes led to the hypothesis that IRES activity may be diminished in the presence of an exon <NUM> duplication. We tested this hypothesis in C2C12 cells and showed that IRES activation was equivalent between the full length (exons <NUM>-<NUM>) and deletion <NUM> cDNAs, but was markedly reduced in the presence of an exon <NUM> duplication (<FIG>) confirming that duplication but not deletion of exon <NUM> ablates IRES activity.

In considering skipping of exons prior to the exon <NUM> IRES, only the removal of exon <NUM> will disrupt the reading frame and result in a premature stop codon (<FIG>). We contemplated that deletion of this exon could be used therapeutically to increase activation of the IRES, whether by use of antisense oligonucleotides (AONs) [<NPL>); <NPL>) and <NPL>)] or by use of AAV-U7 mediated antisense delivery [<NPL>) and <NPL>)]. We selected four different sequences (respectively labeled "B", "AL", "AS" and "C" in <FIG>) for U7snRNA targeting and cloned each into AAV1 to assess exon-skipping efficiency in myoblasts generated from either a wild type or an exon <NUM> duplication fibroblast cell lines that expresses a doxycycline-inducible MyoD (referred as FibroMyoD) [<NPL>)]. All constructs were able to skip either one or two copies of exon <NUM> (<FIG>). Subsequently, in order to increase skipping efficiency, two copies of each of the U7-C and U7-AL targeting antisense sequences were cloned into the single self-complementary (sc) AAV1 vector (and designated AAV1. U7-ACCA), the genome of which is shown in <FIG> in the <NUM>' to <NUM>' orientation. U7-C and U7-AL were used to avoid any possible overlap in the antisense sequence between AL and B. A known antisense sequence (AON H2A) was used as a positive control of skipping [<NPL>)]. Infection of FibroMyoD cells resulted in <NUM>% of the DMD transcript with complete skipping of exon <NUM> leading to the production of N-terminally truncated dystrophin (<FIG> and <FIG>).

We tested the ability of the U7-ACCA vector to skip exon <NUM> in vivo in a mouse model carrying a duplication of exon <NUM> on a C57BL/<NUM> background (the Dup2 mouse; described in Example <NUM> below). The resulting DMD mRNA contains two copies of exon <NUM>, disrupting the reading frame and resulting in nearly complete absence of dystrophin expression. U7-ACCA (1e11vg) was injected directly into the tibialis anterior muscle in six to eight week-old Dup2 mice (n=<NUM>) or BI6 control mice. Four weeks later, RT-PCR analysis from injected muscles demonstrates nearly complete exon-skipping of exon <NUM> in Dup2 or BI6 (<FIG>). Consistent with the RT-PCR results, the saw-tooth RNA-Seq pattern observed in Dmd introns <NUM> and <NUM> confirmed the suppression of co-transcriptional splicing of the duplicated exon <NUM> as well as the high-efficiency of co-transcriptional splicing of exon <NUM> to exon <NUM> in the treated mice (<FIG>). Western blot and immunostaining demonstrate expression of the N-truncated protein. Sarcolemmal staining is restored for β-dystroglycan and nNOS (<FIG>, <FIG>), suggesting the presence of a functional dystroglycan complex.

We also performed a dose escalation study using intramuscular injection (IM) into the tibialis anterior (TA) of Dup2 mice in order to assess the degree of dose response for exon skipping and protein expression. IM escalating doses are set out in <FIG>. As seen in <FIG>, the degree of skipped transcript shows an expected dose response. <FIG> shows a similar expected dose response in protein expression, maximal at <NUM>. 1E11 vg per injection, with significant correction of physiologic force defects (<FIG>).

We examined the effect of glucocorticoid exposure on IRES activity as a muscle-specific IRES found in the <NUM>' UTR of utrophin, an analog of dystrophin, was found to be glucocorticoid-activated [<NPL>)]. Furthermore, treatment with the glucocorticoids prednisone and deflazacort are standard treatment for DMD. We assayed exon <NUM> IRES activity using the exon <NUM> to <NUM> construct in C2C12 cells in the presence of increasing concentrations of <NUM>-methyl-prednisolone (PDN) and found that downstream FLuc activity increased in a dose-dependent fashion from around <NUM> fold change in the absence of PDN to over <NUM> fold at <NUM> PDN (<FIG>). This glucocorticoid activation was not seen after transfection of the exon <NUM> alone or the inverted exon <NUM> control constructs or in <NUM> (<FIG> and S5a). An increase in dystrophin expression was seen in Dup2 FibroMyoD cells treated with <NUM> PDN (<FIG>) and co-treatment of Dup2 mice (n=<NUM>) with both U7-ACCA and PDN resulted in an increase in dystrophin expression over U7-ACCA alone (<FIG>), consistent with glucocorticoid inducibility. An increase to less than <NUM>% compared to untreated Dup2 was seen with PDN alone in rare samples (represented in <FIG>), suggesting some leakiness of the IRES in the Dup2 model. In all cases, this increase of dystrophin expression was not due to a difference in the AAV vector genome copy number (data not shown). Because utrophin translation may be regulated by corticosteroids and overexpression can compensate for absent dystrophin, we assessed utrophin levels in the same injected muscles (<FIG>). In untreated Dup2 animals, utrophin levels were increased in comparison to BI6, similar to what has been reported in mdx, the standard dystrophinopathy mouse model. Comparison of the four groups reveals no statistically significant difference in utrophin levels between PDN treated and untreated animals (<FIG>), excluding utrophin upregulation as a cause of functional rescue following PDN treatment.

We examined whether expression of the IRES-driven isoform improved muscle integrity and physiology in the Dup2 mouse. Similar to the case in mdx mice, dystrophic changes in Dup2 mice are quantifiable at <NUM> weeks of age as widespread muscle regeneration characterized by centralized nuclei (Vulin et al. , manuscript in press). One month after intramuscular injection of AAV1. U7-ACCA into the tibialis anterior muscle of <NUM>-week old Dup2 mice, expression of the IRES driven isoform results in a significant reduction of centralized nuclei (<FIG>). To demonstrate that this isoform restores membrane integrity, treated and untreated Dup2 mice were subjected to a downhill running protocol and injected with Evans blue dye (EBD), which enters skeletal muscle fibers that have been permeabilized by membrane damage. Following intraperitoneal injection of EBD, uptake is found only in fibers without dystrophin staining, suggesting the N-truncated protein stabilizes the sarcolemma and provides further evidence for the functionality of this protein in vivo (<FIG>). Quantification of the number of EBD positive fiber confirms that expression of the IRES driven isoform results in protection of muscle fibers in these mice (<FIG>). Importantly, this membrane protection is associated with restoration of hindlimb grip strength (<FIG>) and muscle specific force (<FIG>) to the levels seen in BI6 control mice. Dup2 muscles injected with U7-ACCA with or without prednisone were significantly more resistant to contraction-induced injury than untreated Dup2 muscle, and the combination of both treatments showed no significant difference from BI6 controls (<FIG>), Despite the minimal (<<NUM>%) expression of dystrophin seen in some Dup2 muscles by PDN (<FIG>), treatment of the Dup2 muscles by PDN alone does not result in a significant amelioration of the muscle physiology (<FIG>).

Examples of models of the DMD exon <NUM> duplication include in vivo and in vitro models as follows.

Mice carrying a duplication of exon <NUM> within the DMD locus were developed. The exon <NUM> duplication mutation is the most common human duplication mutation and results in relatively severe DMD.

A map of the insertion vector is shown in Figure D. In the map, the numbers indicate the relative positions of cloning sites and exons and restriction sites. The neo cassette is in the same direction of the gene and the insertion point is precisely at <NUM>/<NUM> bp in the intron2. At least 150bp extra intronic sequences are kept on each side of inserted exon <NUM>, E2 region is <NUM>-2195bp. Sizes of exon <NUM> and intron <NUM> are 62bp and 209572bp respectively.

Male C57BL/<NUM> ES cells were transfected with the vector (Figure D) carrying an exon2 construct and then insertion was checked by PCR. One good clone was found, amplified and injected in dozens of albino BL/<NUM> blastocysts. Injected blastocysts were implanted into recipient mice. The dystrophin gene from chimeric males was checked by PCR and then by RT-PCR. The colony was expanded and includes some female mice bred to homozygosity. Dystrophin expression in muscles from a <NUM> week old hemizygous mdxdup2 mouse was essentially absent.

Expression of the MyoD gene in mammalian fibroblasts results in transdifferentiation of cells into the myogenic lineage. Such cells can be further differentiated into myotubes, and they express muscle genes, including the DMD gene.

Immortalized cell lines that conditionally express MyoD under the control of a tetracycline-inducible promoter were generated. This is achieved by stable transfection of the primary fibroblast lines of a lentivirus the tet- inducible MyoD and containing the human telomerase gene (TER). The resultant stable line allows MyoD expression to be initiated by treatment with doxycycline. Such cell lines were generated from patients with DMD who carry a duplication of exon <NUM>.

Using the line, duplication skipping using <NUM>'-O- methyl antisense oligomers (AONs) provided by Dr. Steve Wilton (Perth, Australia) was demonstrated. Multiple cell lines were tested.

Proof-of-principle experiments using primary fibroblast lines transiently transfected with adenovirus-MyoD were conducted. The adenovirus constructs were not integrated in the cell genomes, yet MyoD was transiently expressed. The resulting DMD expression was sufficient to perform exon skipping experiments (although reproducibility favors the stably transfected lines.

We tested the ability of an AAV9- U7-ACCA genome to skip exon <NUM> in vivo in Dup2 mice upon intravenous injection. The U7-ACCA genome was cloned into a rAAV9 vector (designated AAV9-U7_ACCA herein) for administration to the mice. AAV9-U7_ACCA was injected into the tail vein (<NUM>. 3E12 vg/kg) of five Dup2 mice. One month after injection, treated animals were examined.

Results of the experiment are shown in <FIG>.

We also carried out dose escalation studies of intravenous dosing (<FIG>). As seen in <FIG>, the degree of skipped transcript shows an expected dose response, as was seen in the IM studies. At the highest level, the majority of transcript consists of either wild-type transcript, which is translated into full-length dystrophin, or exon <NUM>-deleted transcript, which is translated into the N-truncated isoform; importantly, either isoform provides a functional benefit to the mouse (as to humans). <FIG> shows a similar expected dose response in protein expression. Quite importantly, in terms of clinical utility, at the higher doses there is unquestionable and abundant expression of dystrophin in the diaphragm and heart muscles. Quantification of protein expression on immunoblot (<FIG>) confirms the dose escalation response.

Newborn screening (NBS) for DMD in human newborns is now feasible, therefore we tested the benefits of early expression of the N-trucated isoform by delivery of AAV9. U7-ACCA vector (<NUM>×<NUM><NUM> vg) results at postnatal day <NUM> (P1) in Dup2 mice. This single injection results in widespread expression of the N-truncated isoform in all muscles, with sustained protection of muscle fibers through one and six months post treatment (<FIG>).

PPMOs having following sequences (shown <NUM>' to <NUM>') are administered to Dup2 mice.

We transfected the AL-PPMO into wild type C2C12 mouse myoblasts (<FIG>). Three days following transfection, an RT-PCR was performed and demonstrated an efficient exon <NUM> skipping (<FIG>). A similar experiment was performed in the Dup2 mouse model. Intramuscular injection of the AL-PPMO into the tibialis anterior (TA) of Dup2 mice was performed in order to assess the degree of exon <NUM> skipping and protein expression. As seen in <FIG>, exon <NUM> skipping was achieved efficiently. <FIG> was obtained using the same treated TA muscles. Immunostaining of dystrophin was carried out and of dystophrin he results demonstrated efficient production and localization to the plasma membrane protein.

In another experiment, systemic injections are given in the tail vein of another cohort of mice of three doses weekly at <NUM>/kg. We will evaluate skipping and dystrophin restoration at <NUM> weeks after the first injection.

Patients harboring a nonsense mutation within exon <NUM> or <NUM> still express the highly functional N-terminally truncated dystrophin isoform. This is due to the presence of IRES in exon <NUM> that allow re-entry of the ribosome and translation from exon <NUM>. Therefore we hypothesize that creation of a nonsense mutation should force activation of the IRES in human patient cell lines carrying either missense mutation or in frame deletion duplication, within exon <NUM> to <NUM>. Only removal of exon <NUM> generates a stop codon in exon <NUM>. Therefore complete skipping of exon <NUM> in patient carrying the above mentioned mutation, would induce a stop codon in exon <NUM>, and thereby production of the IRES-mediated N-terminally truncated isoform.

We collected cells from human patients carrying mutation in these exons. The cells were then infected with a lentivirus expressing an inducible MyoD that forces conversion of fibroblasts to myoblasts which can then be further differentiated into myotubes, the cell type that expresses dystrophin (referred to hereafter as "myofibroblasts"). Despite aiming to collect cells from patients harboring missense mutation or in frame deletion or duplication within exon <NUM> to <NUM>, only cells from patient carrying a nonsense mutation were available. These cells were derived from BMD patients, and as they carry a nonsense mutation they already naturally expressed the N-terminally truncated dystrophin isoform. However, treatment with AAV1. U7-ACCA at differentiation resulted in higher expression of the IRES-initiated isoform by day <NUM> (<FIG>).

We have demonstrated the presence of a glucocorticoid-responsive IRES within DMD exon <NUM> that can drive the expression of an N-truncated but functional dystrophin. Ribosome profiling from a BMD patient with an exon <NUM> frameshifting mutation demonstrated a mild reduction in dystrophin translation efficiency and a ribosome footprint pattern consistent with ribosome loading beginning in exons <NUM> and <NUM>. The relevance of this IRES-induced isoform to the amelioration of disease severity, which we first described in patients with exon <NUM> nonsense mutations [<NPL>)], is also confirmed by the mass spectrometric data from the first ever reported case of an exon <NUM> deletion, found in an entirely asymptomatic subject. Finally, in a novel therapeutic approach, we have induced out-of-frame exon-skipping to generate a premature stop codon and consequently force activation of the IRES in both patient-derived cell lines and in a novel DMD mouse model, in which we restored components of the dystrophin complex and corrected the pathologic and physiologic features of muscle injury.

Most eukaryotic mRNAs are monocistronic and possess a specialized cap structure at their <NUM>' terminus, which is required for translation initiation as this is where scanning by the <NUM> ribosomal subunit begins. Despite clear evidence for the cap-dependent <NUM>' →<NUM>' scanning model of initiation, bioinformatic analysis has suggested that -<NUM>% of human transcripts contain <NUM>'UTR short upstream open reading frames (uORFs) that may mediate transcript-specific translation efficiency and control. uORFs may function by modulating either leaky scanning or termination-dependent reinitiation, although uORFs can also dynamically regulate access to IRES elements as shown for the mammalian cationic amino acid transporter <NUM> gene, CAT1/SLC7A1. Recognizing the cautions raised regarding IRES identification via reporter assays, all control experiments performed in this study - including assessment of RNA integrity by RT-PCR and Northern blot, use of a promoterless plasmid, and use of an appropriate positive IRES control - were consistent with cap-independent initiation due to IRES activity. We mapped a minimal region harboring a DMD IRES activity to <NUM> nt, of a small length compared to EMCV (<NUM> nt) but similar in size to that identified in the c-myc <NUM>'UTR (<NUM> nt). This is an important feature as such small IRESs can be used in dicistronic vectors, where space is limited when packaged into viral vectors such as AAV.

Although the precise molecular mechanism by which cellular IRESs modulate translation has not been defined in the literature, the requirement of ITAFs has been strongly suggested. These cellular proteins act in trans to augment IRES activity. Almost all ITAFs have been shown to harbor RNA binding domains and have been hypothesized to act as RNA chaperones, helping the IRES primary sequence attain appropriate conformational state intrinsic to its activity. This is likely relevant to the loss of dystrophin IRES activity in the presence of an exon <NUM> duplication, which may ablate IRES function by formation of a complex secondary structure or cause the formation of an inhibitory uORF that interferes with ITAF access to the exon <NUM> IRES.

Our results provide a molecular explanation for the rescue of <NUM>' truncating mutations via a heretofore undescribed mechanism of post-transcriptional regulation of dystrophin expression. The identification of this new cellular IRES and the resultant dystrophin isoform has significant implications for understanding the basic biology of muscle and dystrophin. We note that exon <NUM> of DMD is highly conserved, with <NUM>% identity to human found in the dog, mouse, horse, and chicken DMD genes, and <NUM>% among <NUM> species including D. rerio and X. tropicalis. The presence of an IRES within such a highly conserved region strongly suggests selective pressure favoring a programmed role for alternate translation initiation. The role of the IRES under normal conditions is unclear, but ongoing efforts to understand the relevant cell lineage-specific and/or conditional activation signals will shed light on underlying mechanisms of IRES control and elucidate potentially novel functions of dystrophin.

An intriguing question is how the N-truncated isoform remains functional. A key cellular role for dystrophin is presumed to be transmitting the force of contraction across the sarcolemma to extracellular structures by serving as an important architectural bridge role between the F-actin cytoskeleton and the muscle plasma membrane. Two regions within dystrophin are responsible for F-actin binding: ABD1 (actin binding domain, spanning residues <NUM>-<NUM>) and ABD2 (spanning residues <NUM>-<NUM>). A number of studies have shown a lack of stability of dystrophin in the setting of deletions within the ABD1 domain. However, we note that most of these studies were performed with microdystrophin constructs lacking the ABD2 domain, which has been shown to enhance the interaction between ABD1 and actin. Such miniproteins bind actin and modify actin dynamics in a different manner compared to the full length version. Although results with such constructs show that absence of ABD2 does not completely abrogate binding of dystrophin to actin, it is unlikely that absence of ABD1 completely disrupts the interaction between dystrophin and actin. Expression of transgenes deleted for ABD1 lessens the mdx phenotype and restores the costameric pattern of the M band and Z lines, suggesting that the link between dystrophin and the subsarcolemmal cytoskeleton involves more than an interaction with ABD1. In agreement with this, other members of the cytoskeleton have been shown to interact with the dystrophin spectrin-repeat.

Claim 1:
A recombinant adeno-associated virus (rAAV) comprising a Duchenne Muscular Dystrophy (DMD) exon <NUM> internal ribosome entry site (IRES)-activating oligomer construct comprising:
(a) a U7-C antisense polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: <NUM>, or
(b) a polynucleotide that encodes the U7-C antisense oligomer comprising the nucleotide sequence set forth in SEQ ID NO: <NUM>
for use in treating DMD or Becker Muscular Dystrophy in a patient with a <NUM>' mutation in the DMD gene, wherein the patient does not have a DMD exon <NUM> duplication.