EA045951B1 - GENE THERAPY AGAINST PELOVIC-BRACHALERAL MUSCULAR DYSTROPHY TYPE 2C - Google Patents

Description

Cross-reference to related patent application

This application claims priority under 35 U.S.C. § 119(e) by original U.S. Patent Application No. 62/624,616, filed January 31, 2018, the contents of which are incorporated herein by reference in their entirety.

List of sequences

The currently pending application contains a sequence listing that is filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on January 25, 2019, is named 1068877141_SL.txt and is 18760 bytes in size.

Field of technology to which the invention relates

The invention relates to gene therapy. More specifically, the disclosure provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, for the treatment of muscular dystrophy, such as limb-girdle muscular dystrophy type 2C (LGMD2C). type 2C).

Prior Art

Muscular dystrophy (MD - from the English muscular dystrophy) is a group of genetic diseases. This group is characterized by progressive weakening 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 the distribution and extent of muscle weakness—some forms of MD also affect the heart muscle—age of onset, rate of progression, and pattern of inheritance.

One group of MD is the pelvic-brachial group (LGMD) of MD. LGMDs are rare conditions that present differently among individuals with respect to age of onset, areas of muscle weakness, involvement of the heart and respiratory system, rate of progression, and severity. LGMD can begin in childhood, adolescence, early adulthood, or even later. Both genders are affected equally. LGMDs cause weakening in the shoulder girdle and pelvic girdle, and nearby muscles in the upper legs and arms sometimes also weaken over time. Leg weakness often occurs before arm weakness occurs. The facial muscles are usually not affected. As the condition progresses, affected individuals may develop problems with walking, and over time the use of a wheelchair may become necessary. Affection of the shoulder and arm muscles may lead to difficulty raising the arms above the head and lifting objects. Some types of LGMD may involve the heart and respiratory muscles.

LGMD2C (sarcoglycan dystrophy type 2C) is caused by a deficiency of gamma O-sarcoglycan (SGCG - from the English gamma(Y)-sarcoglycan). Like other sarcoglycanopathies, it manifests as a progressive muscular dystrophy, beginning in the girdle muscles before spreading to the muscles of the lower and eventually the upper extremities. Onset usually occurs in mid to late adolescence. When attempting to treat LGMD2C, no form of drug therapy, even including corticosteroids, changed the course of the disease.

Functional improvement in patients with LGMD2C and other muscular dystrophies requires both gene restoration and fibrosis reduction. There is a need in the art for compositions and methods for treating LGMD2C and other muscular dystrophies.

Summary of the invention

Disclosed herein are gene therapy vectors, eg, recombinant adeno-associated virus (AAV) vectors encoding γ-sarcoglycan, and methods for delivering such γ-sarcoglycan encoding vectors to muscle to reduce or prevent fibrosis; to maintain or improve muscle function; to increase muscle strength; to increase muscle endurance; or for the treatment of γ-sarcoglycanopathy in a mammalian subject suffering from muscular dystrophy.

In addition, the disclosure provides therapies and approaches using gene therapy vectors to deliver γ-sarcoglycan to address the gene defect observed in LGMD2C (pelvic brachial dystrophy type 2C). In one aspect, provided herein is a method of one or more of the following: treating γ-sarcoglycanopathy; increasing muscle strength, muscle endurance and/or muscle mass; reduction of fibrosis; reduction of contraction-induced damage; reduction of fatty infiltration; and/or reducing central nucleation in a subject in need thereof and/or treating muscular dystrophy by reducing fiber degeneration or necrotic fibers; reduction of inflammation; increased levels of creatine kinase; treating muscle fiber atrophy and hypertrophy, and/or reducing dystrophic calcification in a subject suffering from muscular dystrophy, the methods comprising or essentially consisting of or further consisting of administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (AAV) vector, wherein the rAAV vector contains or essentially consists of or further consists of a gene expression cassette that contains or essentially consists of

- 1 045951 consists of or additionally consists of a polynucleotide sequence encoding a γsarcoglycan under the transcriptional control of a promoter, said cassette being flanked by one or more AAV inverted terminal repeats.

In one aspect, described herein is a recombinant AAV vector (rAAV) comprising either essentially consisting of or further consisting of a polynucleotide sequence encoding a γ-sarcoglycan under the transcriptional control of a promoter. In some embodiments, the polynucleotide sequence encoding a γ-sarcoglycan contains or essentially consists of, or further consists of, e.g., at least 65%, at least 70%, at least 75%, at least 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical nucleotide sequence, set forth in SEQ ID NO: 1, and encodes a protein that retains γ-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding a γ-sarcoglycan comprises or essentially consists of, or further consists of, a nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, a polynucleotide sequence encoding a γ-sarcoglycan consists of a nucleotide sequence set forth in in SEQ ID NO: 1, or the sequence, for example, at least 65%, at least 70%, at least 75%, at least 80, 81, 82, 83, 84, 85, 86 , 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the nucleotide sequence set forth in SEQ ID NO: 1, and encodes a protein that retains activity of a γ-sarcoglycan, which in one aspect retains the nucleotide substitutions of SEQ ID NO: 1, compared to the corresponding nucleotides in a wild-type human polynucleotide encoding a γ-sarcoglycan.

In another aspect, the rAAV vector described herein contains or essentially consists of, or further consists of, a polynucleotide sequence encoding a γsarcoglycan that has at least 65%, at least 70%, at least 75 %, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically at least 90%, 91%, 92%, 93% or 94%, and even more typically at least 95%, 96% 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and the protein retains γ-sarcoglycan activity.

γ-sarcoglycan activity is critical for muscle function. γ-Sarcoglycan is one of several sarcolemmal transmembrane glycoproteins that interact with dystrophin and form a dystrophin-glycoprotein complex that spans the sarcolemma and consists of dystrophin, syntrophin, α-dystroglycans and β-dystroglycans and sarcoglycans, including γsarcoglycan. The dystrophin-glycoprotein complex provides a structural link between the subsarcolemmal cytoskeleton and the extracellular matrix of muscle cells. Non-limiting examples of muscle cells include muscle cells of the heart, diaphragm, leg, pelvic girdle, shoulder and arm. Additionally, non-limiting examples of γ-sarcoglycan activity and the consequences of γ-sarcoglycanopathy are described in Blake et al. (2002) Physiol Rev.; 82(2):291-329 and Tarakci et al. (2016) Front Biosci (Landmark Ed); 21:744-56.

In another aspect, the rAAV vectors described herein can be operably linked to a muscle-specific promoter and/or regulatory element to restrict expression to muscle. For example, a muscle-specific regulatory element is a human skeletal actin gene element (GenBank accession no. NG_006672.1), a cardiac actin gene element (GenBank accession no. NG_007553.1), myocyte-specific enhancer binding factor MEF (MEF accession no. GenBank accession NG_016443.2), muscle creatine kinase (MCK) (GenBank accession number AF188002.1), tMCK (truncated MCK), myosin heavy chain (MHC) , MHCK7 (hybrid version of MHC and MCK), C5-12 (synthetic promoter), mouse creatine kinase enhancer element, fast-twitch skeletal muscle troponin C gene element, slow-twitch cardiac muscle troponin C gene element, slow-twitch muscle troponin I gene element, nuclear factors , induced by hypoxia, steroid-inducible element or glucocorticoid response element (GRE - from English glucocorticoid response element).

In some embodiments, the muscle-specific promoter is MHCK7 (SEQ ID NO: 4) or its equivalent. An exemplary rAAV vector described herein is pAAV.MHCK7.hSCGC, which contains or essentially consists of or further consists of the nucleotide sequence of SEQ ID NO: 3 or its equivalent; where the MHCK7 promoter spans nucleotides 136-927, the CMV intron spans nucleotides 937-1084, the γ-sarcoglycan sequence spans nucleotides 1094-1968, and polyA spans nucleotides 1976-2028. In some cases, pAAV.MHCK7.hSCGC is packaged within the aAv rh74 capsid.

AAV can be any serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13 or AAV rh74. In some incarnations

- 2 045951 The rAAV vector contains AAV2 inverted terminal repeat (ITR) sequences.

The production of pseudotyped rAAV is disclosed, for example, in WO 01/83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also being considered. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900–1909 (2014).

Compositions containing or essentially consisting of any of the rAAV vectors described herein are also contemplated.

Also provided are methods for producing a recombinant AAV vector particle, comprising culturing a cell that has been transfected with any recombinant AAV vector described herein and isolating the recombinant AAV particles from the supernatant of the transfected cells. Viral particles containing or essentially consisting of any of the recombinant AAV vectors described herein are also contemplated.

Methods for reducing fibrosis in a mammalian subject in need thereof are also provided. In this regard, the method includes or essentially consists of or further consists of administering to a mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition containing or essentially consisting of an AAV vector described herein) . In some embodiments, the subject, which is a mammal, suffers from muscular dystrophy. In some embodiments, administration of an AAV vector described herein (or a composition containing or essentially consisting of an AAV vector described herein) reduces fibrosis in skeletal muscle or cardiac muscle of a subject.

In another aspect, described herein is a method of increasing muscle strength or muscle mass or muscle endurance in a mammalian subject, comprising or essentially consisting of or further consisting of administering to the mammalian subject a therapeutically effective amount of an AAV vector described herein (or a composition containing or essentially consisting of an AAV vector described herein).

In any of the methods of the disclosure, the subject may suffer from a muscular dystrophy, such as pelvic-brachial muscular dystrophy or any other dystrophin-associated muscular dystrophy.

Also provided is a method of treating muscular dystrophy in a mammalian subject, comprising or consisting essentially of or further consisting of administering a therapeutically effective amount of an AAV vector described herein (or a composition containing or essentially consisting of an AAV vector described herein). document) to a subject that is a mammal. In some embodiments, the muscular dystrophy is pelvic-brachial muscular dystrophy.

In any of the methods of the disclosure, rAAV is administered by any appropriate route of administration, for example, by intramuscular injection or intravenous injection. Additionally, in any method of the disclosure, rAAV is administered systemically, such as parenterally by injection, infusion, or implantation.

The disclosure compositions are prepared for intramuscular injection or intravenous injection. In addition, the compositions of the disclosure are prepared for systemic administration, such as parenteral administration by injection, infusion or implantation.

In addition, any of the compositions prepared for administration to a subject suffering from muscular dystrophy (such as pelvic-brachial muscular dystrophy or any other dystrophin-associated muscular dystrophy). Also described herein are combination therapies comprising or essentially consisting of one or more of the compositions disclosed herein and a corticosteroid. Provided herein are host cells containing the rAAV vector of this disclosure. Also provided herein are kits containing any one or more embodiments disclosed herein and instructions for use. Kits may contain or consist essentially of one or more of the compositions disclosed herein and a corticosteroid or one or more combination therapies provided herein. In any of the uses of the disclosure, the medicament is prepared for administration, for example, by intramuscular injection or intravenous injection. Moreover, in any of the uses of the disclosure, the drug is formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation. In addition, any of the drugs can be prepared for administration to a subject suffering from muscular dystrophy (such as hip-brachial muscular dystrophy or any other dystrophin-associated muscular dystrophy).

The above paragraphs are not intended to define every aspect of the invention, and additional aspects are described in other sections such as the Detailed Description. The entire document is intended to be bound as a single disclosure, and it is understood that all combinations of features described herein are considered, even if the combination of features is not found together in the same sentence or paragraph or section of this document. Invent

- 3 045951 this includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined in the specific paragraphs above. For example, where certain aspects of an invention are described as a genus, it is understood that each member of the genus, individually, constitutes an aspect of the invention.

Brief description of graphic materials

In fig. 1 depicts an AAV vector (scAAVrh74.MHCK7.hSGCG) containing full-length codon-optimized human γ-sarcoglycan (hSCGB) cDNA (SEQ ID NO: 1). The construct is flanked by two AAV inverted terminal repeats (ITRs), approximately 100 bp in size, includes codon-optimized human γ-sarcoglycan (hSGCG) cDNA, a chimeric intron (Intron), a synthetic polyadenylation signal (pA), and is driven by the MHCK7 promoter. specific for skeletal and cardiac muscle.

In fig. Figure 2 shows hematoxylin and eosin (H&E) staining of the tibialis anterior (TA) muscle from BL6 WT mice at 8 weeks of age and γ-sarcoglycan knockout (γ-SG KO) mice demonstrating a dystrophic phenotype in diseased mice. .

In fig. 3A-3C depict the in vivo activity of the vector. scAAVrh74.MHCK7. hSGCG was injected into the tibialis (TA) muscle of γ-SG KO mice at a total dose of 3e10 vg. In fig. Figure 3A shows immunofluorescence staining of TA muscle in γ-SG KO mice. Almost 100% of γ-sarcoglycan protein expression in the sarcolemma occurred as a result of vector delivery. In fig. 3B shows a Western blot of γ-sarcoglycan expression in TA-injected muscles from treated mice #794, 795. FIG. Figure 3C shows immunofluorescent staining of TA muscle from wild-type control mice (BL6 WT TA) or uninjected control γ-SG KO mice (GSG KO TA), as well as an unstained sample (GSG KO No Primary).

In fig. 4A-4B depict in vivo vector activity and toxicity in wild-type (WT) BL6 mice. In fig. 4A shows immunofluorescence staining indicating γsarcoglycan overexpression through membrane staining and intracellular staining. In fig. 4B shows a Western blot showing γ-sarcoglycan overexpression in LTA-injected muscle.

In fig. 5 depicts in vivo vector activity and toxicity in wild-type (WT) BL6 mice. H&E staining of uninjected and TA-injected BL6 WT muscles demonstrates no toxicity in the complete absence of any central nuclei, necrotic fibers, inflammatory infiltration, or fibrous tissue.

In fig. 6 depicts immunofluorescent staining of γ-sarcoglycan on TA, gastrocnemius (GAS), quadriceps (QUAD), gluteus (GLUT), psoas (PSOAS), triceps (TRICEP), diaphragm and cardiac muscle, demonstrating extensive γ-sarcoglycan expression.

In fig. Figure 7 shows immunofluorescent staining of tissues when determining the activity of the vector during i.v. administered. IF staining of γ-sarcoglycan in various skeletal muscles, diaphragm, and heart shows strong expression with sparse negative fibers 6 weeks after systemic delivery of scAAVrh.74.MHCK7.hSGCG.

In fig. 8A-8B show SGCG expression in animals treated with i.v. Immunofluorescence imaging of skeletal muscle, diaphragm, and heart from SGCG −/− mice intravenously injected with a total dose of 1e13 vg scAAVrh.74.MHCK7.hSGCG is shown in representative 20X images (Fig. 8A). Western blot analysis demonstrates hSGCG expression in all skeletal muscle and heart from mice that received intravenous delivery of scAAVrh.74.MHCK7.hSGCG (Fig. 8B).

In fig. 9A-9B show histological evaluation of tissues after systemic treatment. Hematoxylin and eosin staining of skeletal muscle TRI and DIA in BL6 WT, untreated SGCG −/− and AAV.MHCK7.hSGCG-treated SGCG −/− mice demonstrates reversal of pathology-dystrophy after treatment (Fig. 9A). Quantification of the percentage of centrally nucleated fibers demonstrates a decrease in treated muscles. BL6 WT (n is 5), untreated SGCG-/- (n is 6), AAV.MHCK7.hSGCG-treated (n is 5) (Fig. 9I). *** means p less than 0.001, **** means p less than 0.0001.

In fig. 10A-10F show a quantification of fiber diameter. Fiber diameters were quantified in GAS (Fig. 10A), PSOAS (Fig. 10B), and TRI (Fig. 10C) BL6 WT (n = 5), untreated SGCG-/- (n = 6), and AAV.MHCK7.hSGCG -treated SGCG-/- (n=5) mice, and normalization of fiber diameter distribution after treatment. Average fiber diameter is reduced in GAS (Fig. 10D), PSOAS (Fig. 10E), and TRI (Fig. 10F) muscle of untreated SGCG −/− mice and increased to WT levels in each muscle after treatment with AAV.MHCK7.hSGCG in SGCG mice -/-. **** represents p less than 0.0001.

In fig. Figures 11A-11C show the physiology of the TA and diaphragm. TA and DIA muscles of BL6 WT (n = 5), untreated SGCG−/− (n = 6), and AAV.MHCK7.hSGCG-treated (n = 5) mice subjected to normalized specific force production measurements. TA muscle subjected to an eccentric contraction-induced injury protocol (Fig. 11A). We observed an improvement in specific

- 4 045951 TA output strength and resistance to contraction-induced damage in SGCG-/treated mice (Fig. 11B). DIA output specific force was restored to WT levels in SGCG-/--treated mice (Fig. 11C). * means p less than 0.05, **** means p less than 0.0001.

In fig. Figure 12 shows laser monitoring of activity in an open field chamber. The net ability to move in the x- and y-planes is reduced in SGCG-/- mice and improved in AAV.MCHK7.hSGCG-treated mice. BL6 WT (n is 6), untreated SGCG-/- (n is 6), and AAV.MHCK7.hSGCG-treated (n is 5).

In fig. Figure 13 shows the biodistribution of vector genomes. Vector genome distribution of average vg copy numbers per microgram of genomic DNA (gDNA) was measured in different tissues from two SGCG−/mice 3 months after i.v. delivery of a total dose of 1e13 vg scAAVrh.74.MHCK7.hSGCG.

In fig. 14A-14B show a comparison of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in serum. Serum from BL6 WT mice (n = 6), untreated SGCG −/− mice (n = 6), and AAV.MHCK7.hSGCG − treated i.v. SGCG-/- mice (n = 5) (total dose 1e13 vg) were analyzed for levels of biochemical components. Levels of the liver enzymes aspartate aminotransferase (ALT, Fig. 14A) and aspartate aminotransferase (AST, Fig. 14B) were elevated in sick SGCG-/- mice and were restored to almost WT levels after treatment. * means p less than 0.05. The dotted lines represent the lower and upper limits of the normal interval.

Detailed description

The present disclosure relates to the administration of a recombinant adeno-associated virus (rAAV) vector containing a γ-sarcoglycan expressing polynucleotide to reduce or reverse muscle fibrosis in an individual suffering from pelvis-brachial muscular dystrophy. As demonstrated in the Examples section, administration of the rAAV vector described herein resulted in restoration of γ-sarcoglycan expression in knockout mice. Administration of the rAAV vector described herein will result in reversal of signs of dystrophy, including fewer degenerating fibers, reduced levels of inflammation, and improved recovery of function, resulting from protection from eccentric contraction with increased force generation. The disclosure covers the treatment of pelvic brachial muscular dystrophy in a subject (eg, a human subject) by administering the rAAV vector described herein.

As used herein, any concentration range, percentage range, fractions range, or whole number range is intended to include the value of any whole number within the range listed, and, as appropriate, fractions thereof (such as one tenth and one hundredth whole number) unless otherwise noted. It should be understood that the singular terms as used herein refer to one or more of the listed components unless otherwise indicated. It should be understood that the use of an alternative (eg, or) implies one, both, or any combination of these alternatives. As used in this document, the terms includes and contains are used interchangeably. As used herein, the term multiple may refer to one or more components (eg, one or more miRNA (miRNA) target sequences). In this application, the use of either means and/or, unless otherwise indicated.

As used in this application, the terms approximately and approximately are used as equivalents. Any numerals used in this application, with or without the term approximately/approximately, are intended to cover any normal variations as judged by one of ordinary skill in the art. In some embodiments, the term approximately or approximately refers to a range of values that fall within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1% or less in either direction (more than or less than) the stated reference value, unless otherwise stated or otherwise obvious from the context (unless such number would exceed 100% of the possible value) .

The term reduces or reduces refers to reducing or reducing a specific value by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%, compared to the reference value. A decrease or decrease in a particular value can also be represented as a multiple change in value compared to a reference value, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more reduction compared to the reference value.

The term increases refers to increasing a specific value by at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100, 200, 300, 400, 500% or more compared to the reference value. An increase in a particular value can also be represented as a multiple change in value compared to a reference value, for example, at least 1-fold, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 or more times the reference level.

- 5 045951

Complementarity refers to the ability to form a pair due to base stacking and specific hydrogen bond formation between two sequences containing naturally occurring or non-naturally occurring (eg, modified as described above) bases (nucleotides) or their analogues. For example, if a base at one position of a nucleic acid is capable of forming hydrogen bonds with a base at the corresponding target position, then the bases are considered to be complementary to each other at that position. Nucleic acids may contain universal bases or inert spacers with the nitrogenous base removed, which provide neither a positive nor a negative contribution to hydrogen bonding. Base pairing can include both classical Watson-Crick base pairing and non-Watson-Crick base pairing (eg, ambiguous base pairing and Hoogsten base pairing). For complementary base pairing, it is believed that bases like adenosine (A) are complementary to bases like thymidine (T) or bases like uracil (U), that bases like cytosine (C) are complementary to bases like guanosine (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole, can hybridize with and are considered complementary to any A, C, U or T. Nichols et al., Nature, 1994;369:492-493 and Loakes et al., Nucleic Acids Res., 1994; 22:4039–4043. It is also believed in the art that inosine (I) is a universal base and is considered complementary to any A, C, U or T. See Watkins and Santa Lucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.

The term subject includes animals, such as, for example, mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subjects are farm animals such as cattle, sheep, goats, cows, pigs, and the like; or domesticated animals such as dogs and cats. In some embodiments (eg, particularly in the context of research), the subjects are rodents (eg, mice, rats, hamsters), rabbits, primates, or pigs, such as inbred pigs and the like. The terms subject and patient are used interchangeably throughout this document.

Administration herein refers to the introduction of an agent or composition to a subject.

The term Treatment, as used herein, refers to the delivery of an agent or composition to a subject to affect a physiological outcome. In some embodiments, treatment refers to treating a disease in a subject, such as a human, comprising (a) inhibiting the disease, such as stopping the development of the disease or preventing the progression of the disease; (b) alleviating the disease, for example by causing regression of the disease state; (c) cure the disease; and (d) preventing the onset of a disease, for example, stopping the development of a disease in an asymptomatic subject identified as a carrier of a genetic defect. In one aspect, treatment excludes prophylaxis or prevention.

When the disease is muscular dystrophy, the following clinical endpoints are non-limiting examples of treatment: decrease in specific force, increase in resistance to damage, increase in muscle strength, increase in muscle endurance, increase in muscle mass, decrease in contraction-induced damage, decrease in fatty infiltration, decrease in central nucleation , decreased fiber degeneration or necrotic fibers, decreased inflammation, increased creatine kinase levels, decreased muscle fiber atrophy and hypertrophy, and/or decreased dystrophic calcification.

When the disease is fibrosis, the following clinical endpoints are non-limiting examples of treatment: reduction in fibrous tissue, reduction in inflammation, reduction in fibroblastic lesions, reduction in activated fibroblast proliferation, reduction in myofibroblast formation, reduction in the rate of decline in forced vital capacity (FVC). Vital Capacity, where FVC represents the total amount of air exhaled during pulmonary function testing, absolute and relative increases from baseline in FVC, absolute increase from baseline in FVC (% predicted), progression-free survival increase, decrease from baseline total score level on the St George's Respiratory Questionnaire (SGRQ), where the SGRQ is a questionnaire assessing health-related quality of life, divided into 3 components: symptoms, activity and impact, and a total score (total weight) can range from 0 to 100, with a lower score indicating better health and a relative decrease from baseline in the Quantitative Pulmonary Fibrosis (QLF) score. quantitative lung fibrosis) based on high-resolution computerized tomography (HRCT), where the QLF score ranges from 0 to 100%, and higher values represent more lung fibrosis and are considered worse health status. Non-limiting examples of clinical endpoints for fibrosis treatment and testing that can be performed to evaluate these clinical endpoints are described in the following clinical trials: NCT03733444 (clinicaltrials.gov/ct2/show/NCT03733444) (last accessed January 9, 2019), NCT00287729 (clinical tri

- 6 045951 als.gov/ct2/show/NCT00287729) (last accessed January 9, 2019), NCT00287716 (clinicaltrials.gov/ct2/show/NCT00287716) (last accessed January 9, 2019

NCT02503657(clinicaltrials.gov/ct2/show/NCT02503657) (last accessed January 9, 2019

NCT00047645

NCT02802345

NCT01979952

NCT00650091

NCT01335464

NCT01335477 (clinicaltrials.gov/ct2/show/NCT00047645) (clinicaltrials.gov/ct2/show/NCT02802345) (clinicaltrials.gov/ct2/show/NCT01979952) (clinicaltrials.gov/ct2/show/NCT00650091) (clinicaltrials.gov/ ct2/show/NCT01335464) (clinicaltrials.gov/ct2/show/NCT01335477) year), year), year), year), year), year), year), year), (last accessed January 9, 2019 (last accessed 9 January2019 (last accessed January 9, 2019 (last accessed January 9, 2019 (last accessed January 9, 2019 (last accessed January 9, 2019)

NCT01366209 (clinicaltrials.gov/ct2/show/NCT01366209) (last accessed January 9, 2019). Additional non-limiting examples of clinical endpoints for the treatment of fibrosis and testing that can be performed to measure these clinical endpoints are described in King et al, (2014) N Engl J Med. May 29; 70(22):2083-92 and Richeldi et al., (2014) N Engl J Med. May 29;

370(22):2071-82.

The term effective amount or therapeutically effective amount refers to the minimum amount of an agent or composition required to result in a particular physiological effect (eg, the amount required to enhance, activate or enhance a particular physiological effect). The effective amount or therapeutically effective amount of a particular agent can be represented in a variety of ways depending on the nature of the agent, such as weight/volume ratio, cell number/volume, particle number/volume, (weight of agent)/(weight of subject), number of cells/ (subject's mass) or number of particles/(subject's mass). An effective amount or therapeutically effective amount of a particular agent may also be expressed as the half-maximal effective concentration (EC ₅₀ ), which refers to the concentration of the agent that results in a magnitude of a particular physiological response that is midway between the control level and the maximum response level.

Cell population refers to any number of cells greater than 1, but preferably is at least 1x 10 ³ cells, at least 1x 10 ⁴ cells, at least 1x 10 ⁵ cells, at least 1x 10 6 cells, at least 1x 10 ⁷ cells, at least 1x 10 8 cells, at least 1x 10 9 cells, at least 1x 10 ¹⁰ cells or more cells. The cell population may refer to an in vitro population (eg, a population of cells in culture) or an in vivo population (eg, a population of cells found in a particular tissue).

The phrase pharmaceutically acceptable is used herein to refer to those compounds, substances, compositions and/or dosage forms that are within the scope of informed medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problem or complication corresponding to a reasonable benefit/risk ratio.

As used herein, the term pharmaceutically acceptable carrier, diluent or excipient includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, color/color, flavor enhancer and odorless agent, surfactant, humectant, dispersant, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans and/or pets.

As used herein, the term vector refers to a nucleic acid molecule capable of carrying or transporting a nucleic acid molecule into a cell along with, in a viral vector, one or more viral proteins, such as in the case of encapsulated viruses , the capsid of the virus. The transferred nucleic acid is typically linked to, for example inserted into, a vector nucleic acid molecule. The vector may include sequences that direct autonomous replication or reverse transcription in the cell or may include sequences sufficient to promote integration into the DNA of the host cell. The term vectors includes gene therapy vectors. As used herein, the term gene therapy vector refers to a vector capable of being used in gene therapy, eg, delivering a polynucleotide sequence encoding a therapeutic polypeptide to a subject. Gene therapy vectors may contain a polynucleotide (transgene) encoding a protein, such as γ-sarcoglycan.

As used herein, the term expression cassette refers to a segment of DNA that is capable, when appropriately positioned, of directing the expression of a polynucleotide (e.g., transgene) encoding a protein (e.g., γ-sarcoglycan) that is included in said expression cassette . When introduced into a host cell, the expression cassette is inter alia capable of directing the cell's machinery to transcribe the transgene into RNA, which is then usually complemented

- 7 045951 is extensively processed and ultimately translated into a therapeutically active polypeptide. The gene therapy vector may contain or essentially consist of an expression cassette. The term expression cassette excludes polynucleotide sequences from the 5' to 5' ITR and from the 3' to 3' ITR. Provided herein are host cells containing or essentially consisting of, or further consisting of, the rAAV vector of this disclosure. The cells may be of any suitable species, for example mammalian cells.

As used herein, the phrases operably linked to or under transcriptional control of a polynucleotide refer interchangeably to the configuration of a promoter or muscle-specific regulatory element and a polynucleotide that allows the polynucleotide to be transcribed by a polymerase capable of binding to the promoter . In one aspect, the muscle-specific regulatory element must restrict expression to the muscle. Non-limiting examples of muscle-specific regulatory elements include the human skeletal actin gene element (GenBank accession number NG_006672.1), the cardiac actin gene element (GenBank accession number NG_007553.1), the myocyte-specific enhancer binding factor MEF (accession number GenBank accession NG_016443.2), muscle creatine kinase (MCK) (GenBank accession no. AF188002.1), tMCK (truncated MCK), myosin heavy chain (MHC), MHCK7 (a hybrid version of MHC and MCK), C5-12 ( synthetic promoter), mouse creatine kinase enhancer element, fast-twitch skeletal muscle troponin C gene element, slow-twitch cardiac troponin C gene element, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factors, steroid-inducible element or glucocorticoid response element (GRE) .

Common techniques in molecular and cellular biochemistry can be found in the following standard manuals on molecular cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

As used herein, the term AAV is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are achieved through co-infection with a helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). There can be no doubt that the same principles described in these reviews will apply to additional AAV serotypes characterized since the publication dates of the reviews, since it is well known that the different serotypes are quite closely related, both structurally and functionally, even at genetic level (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further indicated by heteroduplex analysis, which reveals large-scale cross-hybridization among serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends that correspond to inverted terminal repeat (ITR) sequences. Similar patterns of infectivity also suggest that replication functions in each serotype are under similar regulatory control.

The term AAV vector, as used herein, refers to a vector containing one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat (ITR) sequences. Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products.

The term AAV virion or AAV viral particle or AAV vector particle refers to a viral particle consisting of at least one AAV capsid protein and an AAV polynucleotide vector capsid. If the particle contains a heterologous polynucleotide (namely, a polynucleotide different from the wild-type AAV genome, such as a transgene to be delivered into a mammalian cell), it is commonly referred to as an AAV vector particle or simply an AAV vector. Thus, the production of an AAV vector particle necessarily includes the production of an AAV vector, and therefore the vector is contained within the AAV vector particle.

Adeno-associated virus (AAV) is a replication-defective parvovirus with a single-stranded DNA genome of approximately 4.7 kb. in length, including two inverted terminal repeats (ITRs) of 145 nucleotides. There are many serotypes of AAV.

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The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank, accession number NC_002077; The complete AAV-2 genome is provided to GenBank, accession number NC_001401 and Srivastava et al., J. Virol., 45: 555-564 {1983); The complete AAV-3 genome has been submitted to GenBank, accession number NC_1829; The complete AAV-4 genome has been submitted to GenBank, accession number NC_001829; The AAV-5 genome has been submitted to GenBank, accession number AF085716; The complete AAV-6 genome has been submitted to GenBank, accession number NC_00 1862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank, accession numbers AX753246 and AX753249, respectively; the AAV-9 genome is provided by Gao et al., J. Virol., 78: 6381-6388 (2004); The AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome provided in Virology, 330(2): 375-383 (2004). The genome sequence of AAV rh.74 is provided in US Pat. No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences that direct viral DNA replication (rep), encapsidation/packaging, and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (called p5, pI9, and p40 for their respective positions on the map) regulate the expression of two AAV internal open reading frames encoding the rep and cap genes. These two rep promoters (p5 and pi9), along with differential splicing of the single AAV intron (at nucleotides 2107 and 2227), lead to the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. rep proteins have multiple enzymatic properties that are ultimately responsible for viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins VP1, VP2 and VP3. Alternative splicing and non-consensus translation initiation sites drive the production of three related capsid proteins. One consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

AAV has unique features that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. Infection of AAV cells in culture is not cytopathic, and natural infection in humans and other animals is silent and asymptomatic. In addition, AAV infects many mammalian cells, allowing targeting to many different tissues in vivo. In addition, AAV transduces slowly dividing and non-dividing cells, and can essentially continue to exist throughout the life of those cells as a transcriptionally active nuclear episome (an extrachromosomal element). The AAV provirus genome is inserted as cloned DNA into plasmids, making the construction of recombinant genomes possible. In addition, because the signals directing AAV replication and genome encapsidation are contained in the ITR of the AAV genome, some or all of the approximately 4.3 kb genome (encoding replication proteins and structural capsid proteins, repcap) may be replaced by foreign DNA. To create AAV vectors, the rep and cap proteins can be provided while being trans-active. Another significant characteristic of AAV is that it is an extremely stable and strong virus. It easily withstands the conditions used to inactivate adenovirus (56°-65°C for several hours), making cold preservation of AAV less critical. AAV can even be lyophilized. Ultimately, AAV-infected cells are not resistant to superinfection.

Multiple studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 8098-8108 (1996). Also see Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). In addition, since muscle is highly vascularized, transduction with recombinant AAV resulted in the appearance of transgene products in the systemic circulation after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al. al., Proc Natl Acad Sci USA, 94: 13921–13926 (1997). In addition, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal muscle fiber possesses the necessary cellular factors for proper glycosylation, folding and secretion of antibodies, indicating that the muscle is capable of stable expression of secreted protein therapeutics. Recombinant AAV (rAAV) genomes are disclosed to contain or consist essentially of, or further consist of, a nucleic acid molecule encoding a γ-sarcoglycan (eg, SEQ ID NO: 1) and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in rAAV genomes can come from any AAV serotype for which a recombinant virus can be produced, including but not limited to the following AAV serotypes: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01/83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also considered. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900–1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art. AAV1, AAV5, AAV6, AAV8 or AAV9 can be used to stimulate skeletal muscle-specific expression. Thus, in one aspect, this document describes a recombinant AAV vector containing or essentially consisting of a polynucleotide sequence

- 9 045951 sti, encoding γ-sarcoglycan under the transcriptional control of a promoter and/or muscle-specific regulatory element. In some embodiments, the polynucleotide sequence encoding a γ-sarcoglycan contains or essentially consists of, or further consists of, e.g., at least 65%, at least 70%, at least 75%, at least 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical nucleotide sequence to human γ-sarcoglycan with optimized codons, which is set forth in SEQ ID NO: 1 (see Table 1) and encodes a protein that retains γ-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding the γ-sarcoglycan contains the nucleotide sequence set forth in SEQ ID NO: 1, or a sequence, for example, at least 65%, at least 70%, at least 75%, according to at least 80, 81, 82, 83, 84, 85, 86, 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO . 1, which encodes a protein that maintains γ-sarcoglycan activity.

The term sequence identity refers to the percentage of bases or amino acids of two polynucleotide or polypeptide sequences that are the same and in the same relative position. In this regard, one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. In the case of sequence comparisons, typically one sequence serves as a reference sequence to which the analyzed sequences are compared. The term reference sequence refers to the molecule to which the analyzed sequence is compared. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a specified percentage (e.g., 60, 65, 70, 75, 80, 85, 90, 95, 98, or 99%) of sequence identity with a reference sequence means that when aligned the percentage of bases (or amino acids) at each position in the analyzed sequence is identical to the base (or amino acid) at the same position in the reference sequence. This alignment and the percentage homology or sequence identity can be determined using software known in the art, for example, the software described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, the default settings are used for alignment. One alignment program is BLAST, which uses default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: genetic code - standard; filter - absence; chain - both; threshold value - 60; waiting - 10; matrix - BLOSUM62; Descriptions - 50 sequences; sort by - high indicator; Databases - non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/blast/Blast.cgi. A polypeptide or protein equivalent is a peptide or protein that has a defined sequence identity to a reference polypeptide or protein sequence (e.g., 60%, 65%, 70%, 75%, 80%, 85% -, 90%, 95%, 98%, or 99% identity with the reference) and retains similar activity or function when compared with the reference polypeptide or protein.

The term comprising or comprises is intended to mean that the compositions, eg media, and methods include the listed elements, but not to the exclusion of others. The phrase essentially consisting of when used to define compositions and methods will mean the exclusion of other elements of any significant significance to the combination for the stated purpose. Thus, a composition substantially consisting of the elements as defined herein will not exclude other substances or steps that do not materially affect the basic and novel feature(s) of the claimed invention. The term consisting of will imply the exclusion of elements in greater than trace amounts, other ingredients and essential process steps. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

In one aspect, the rAAV vector gene expression cassette of this disclosure is flanked by one or more AAV inverted terminal repeats. In another aspect, the polynucleotide sequence encoding the γ-sarcoglycan of the rAAV vector contains or essentially consists of or further consists of a nucleotide sequence at least 95% identical to SEQ ID NO: 1 and/or a nucleotide sequence set forth in SEQ ID NO: 1, and encodes a protein that retains γ-sarcoglycan activity. In yet another aspect, the polynucleotide sequence encoding a rAAV vector γ-sarcoglycan encodes an amino acid sequence that is at least 95% identical, at least 99% identical, or 100% identical to SEQ ID NO: 2, and encodes a protein that retains γ-sarcoglycan activity.

In some embodiments, the rAAV vector disclosed herein belongs to the serotype

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AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV rh74. In other embodiments, the rAAV vector genome contains or consists essentially of a muscle-specific regulatory element, and wherein the muscle-specific regulatory element is operably linked to a polynucleotide sequence. Non-limiting examples of muscle-specific regulatory elements include human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), MHCK7, C512, mouse creatine kinase enhancer element, fast-twitch skeletal muscle troponin gene element, slow-twitch cardiac troponin gene element, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factors, steroid-inducible element and glucocorticoid response element (gre) . In one aspect, the muscle-specific regulatory element of the rAAV vector is a truncated MCK (tMCK). In another aspect, the promoter and/or muscle-specific regulatory element of the rAAV vector is the MHCK7 promoter. In yet another aspect, the MHCK promoter comprises or consists essentially of, or further consists of, the nucleotide sequence set forth in SEQ ID NO: 3, or an equivalent thereof, and provides promoter function. In one embodiment, the genome of the rAAV vector disclosed herein contains or essentially consists of, or further consists of, an intron containing the nucleotide sequence set forth in SEQ ID NO: 5.

In some embodiments, the polynucleotide sequence encoding γ-sarcoglycan consists of a nucleotide sequence set forth in SEQ ID NO: 1, or a polynucleotide sequence encoding γ-sarcoglycan that has at least 65%, at least 70% , at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88% or 89%, more typically at least 90%, 91%, 92%, 93% or 94%, and even more typically at least 95% , 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, and retains γ-sarcoglycan activity.

In another aspect, the recombinant AAV vector described herein contains or essentially consists of a polynucleotide sequence encoding a γ-sarcoglycan that has at least 65%, at least 70%, at least 75% at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, more typically at least 90%, 91%, 92%, 93% or 94% and even more typically at least 95%, 96% , 97%, 98% or 99% sequence identity with the amino acid sequence of human γ-sarcoglycan, which is set forth in SEQ ID NO: 2 (see Table 1), and the protein retains γ-sarcoglycan activity.

Table 1

Non-limiting Examples of Protein and Nucleotide Sequences

Sequence Description Subsequence SEQ ID NO Nucleotide sequence of human γsarcoglycan (with codon optimization) ATGGTGAGGGAGGCAGTACACCACAGCAACCGAGG GAATCTGCATCGAGAGGCCAGAGAACCAGTACGTG TATAAGATCGGCATCTACGGCTGGCGGAAGAGATG TCTGTATCTGTTCGTGCTGCTGCTGCTGATCATCCT GGTGGTGAATCTGGCCCTGACCATCTGGATCCTGA AAGTGATGTGGTTTTCCCCAGCAGGAATGGGACAC CTGTGCGTGACAAAGGACGGACTGCGG CTGGAGG GAGAGTCTGAGTTCCTGTTTCCCCTGTATGCCAAGG AGATCCACAGCAGAGTGGATAGCTCCCTGCTGCTG CAGTCCACCCAGAACGTGACAGTGAACGCAAGGAA TAGCGAGGGAGAGGTGACCGGCAGACTGAAGGTC GGCCCCAAGATGGTGGAGGTGCAGAATCAGCAGTT CCAGATCAACTCCAATGACGGCAAGCCTCTGTTTAC AGTGGATGAGAAGGAGGTG GTGGTGGGCACCGAC AAGCTGAGGGTGACAGGACCTGAGGGCGCCCTGTT CGAGCACTCTGTGGAGACCCCACTGGTGCGCGCA 1

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GACCCTTTTCAGGATCTGAGGCTGGAGAGCCCAAC ACGCAGCCTGTCCATGGACGCACCCAGAGGCGTG CACATCCAGGCACACGCAGGCAAGATCGAGGCCCT GAGCCAGATGGATATCCTGTTCCACTCTAGCGACG GCATGCTGGTGCTGGATGCCGAGACCGTGTGCCTG CCTAAGCTGGTGCAGGGCACATGGGGCCCATCTGG CTCCTCTCAGAGCCTGTACGAGATCTGCGTGTGCC CAGATGGCAAGCTGTATCTGTCCGTGGCCGGCGTG TCTACCACATGCCAGGAGCACAACCACATCTGTCTG TGA Amino acid sequence of human γsarcoglycan MVREQYTTATEGICIERPENQYVYKIGIYGWRKRCLYL FVLLLLIILVVNLALTIWILKVMWFSPAGMGHLCVTKDG LRLEGESEFLFPLYAKEIHSRVDSSLLLQSTQNVTVNA RNSEGEVTGRLKVGPKMVEVQNQQFQINSNDGKPLF TVDEKEWVGTDKLRVTGPEGALFEHSVETPLVRADP FQDLRLESPTRS LSDAPRGVHIQAHAGKIEALSQMDI LFHSSDGMLVLDAETVCLPKLVQGTWGPSGSSQSLY EICVCPDGKLYLSVAGVSTTCQEHNHICL 2 5’ITR-MHCK7 Chimeric intron-hSGCGPolyA-3’ITR (full-length sequence between ITRs) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG CAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCC GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA GTGGGGTTAACCAATTGGCGCGGCCGCAAGCTTGC ATGTCTAAGCTAGACCCTTCAGATTAAAAATAACTG AGGTAAGGGCCTGGGTAGGGGAGGTGGTGTGAGA CGCTCCTGTCTCTCCTTACTGCCCAT CGGCCCTT TGGGGAGGAGGAATGTGCCCAAGGACTAAAAAAAG GCCATGGAGCCAGAGGGGCGAGGGCAACAGACCT TTCATGGGCAAACCTTGGGGCCCTGCTGTCTAGCA TGCCCCACTACGGGTCTAGGCTGCCCATGTAAGGA GGCAAGGCCTGGGGACACCCGAGATGCCTGGTTAT AATTAACCCAGACATGTGGCTGCCCCCCCCCCCCC AACACCTGCTGCCTCTAAAAATAAC CCTGTCCCTGG TGGATCCCCTGCATGCGAAGATCTTCGAACAAGGC TGTGGGGGACTGAGGGCAGGCTGTAACAGGCTTG GGGGCCAGGGCTTATACGTGCCTGGGACTCCCAAA GTATTACTGTTCCATGTTCCCGGCGAAGGGCCAGC 3

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TGTCCCCCGCCAGCTAGACTCAGCACTTAGTTTAG GAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCCA TACAAGGCCATGGGGCTGGGCAAGCTGCACGCCT GGGTCCGGGGTGGGCACGGTGCCCGGGCAACGAG CTGAAAGCTCATCTGCTCTCAGGGGCCCCTCCCTG GGGACAGCCCCTCCTGGCTAGTCACACCCTGTAGG CTCCTCTATATAACCCAGGGCACAGG GGCTGCCC TCATTCTACCACCACCTCCACAGCACAGACAGACAC TCAGGAGCAGCCAGCGGCGCGCCCAGGTAAGTTTA GTC1 1 1 1 1GTCTTTTATTTCAGGTCCCGGATCCGGT GGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGAT GTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTAC TTCTGCTCTAAAAGCTGCGGAATTGTACCCGGTACC ACCAT TGAGGGAGCAGTACACCACAGCAACCGA GGGAATCTGCATCGAGAGGCCAGAGAACCAGTACG TGTATAAGATCGGCATCTACGGCTGGCGGAAGAGA TGTCTGTATCTGTTCGTGCTGCTGCTGCTGATCATC CTGGTGGTGAATCTGGCCCTGACCATCTGGATCCT GAAAGTGATGTGGTTTTCCCCAGCAGGAATGGGAC ACCTGTGCGTGACAAAGGACGGACTGCGGCTGGA GGGAGAGTCTGAGTTCCTGTTTCCCCTGTATGCCAA GGAGATCCACAGCAGAGTGGATAGCTCCCTGCTGC TGCAGTCCACCCAGAACGTGACAGTGAACGCAAGG AATAGCGAGGGAGAGGTGACCGGCAGACTGAAGG TCGGCCCCAAGATGGTGGAGGTGCAGAATCAGCAG TTCCAGATCAACTCCAATGACGGCAAGCCTCTGTTT ACAGTGGATGAGAAGGAGGTGGTGGTG GGCACCG ACAAGCTGAGGGTGACAGGACCTGAGGGCGCCCT GTTCGAGCACTCTGTGGAGACCCCACTGGTGCGCG CAGACCCTTTTCAGGATCTGAGGCTGGAGAGCCCA ACACGCAGCCTGTCCATGGACGCACCCAGAGGCGT GCACATCCAGGCACACGCAGGCAAGATCGAGGCC CTGAGCCAGATGGATATCCTGTTCCACTCTAGCGAC GGCATGCTGGTGCTGGATGCCGAGACCGTG TGCCT GCCTAAGCTGGTGCAGGGCACATGGGGCCCATCTG GCTCCTCTCAGAGCCTGTACGAGATCTGCGTGTGC

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CCAGATGGCAAGCTGTATCTGTCCGTGGCCGGCGT GTCTACCACATGCCAGGAGCACAACCACATCTGTCT GTGACTCGAGGGCCGCAATAAAAGATCTTTATTTTC ATTAGATCTGTGTGTTGG 1 1 1 1 1 1 GTGTGTCCTGCA GGGGCGCGCCTAATCTAGAGCATGGCTACGTAGAT AAGTAGCATGGCGGGTTAATCATTAACTACAAGGAA CCCCTAG TGATGGAGTTGGCCACTCCCTCTCTGCG CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAG GTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGC Promoter MNSK7 AAGCTTGCATGTCTAAGCTAGACCCTTCAGATTAAA AATAACTGAGGTAAGGGCCTGGGTAGGGGAGGTG GTGTGAGACGCTCCTGTCTCTCCTCTATCTGCCCAT CGGCCCTTTGGGGAGGAGGAATGTGCCCAAGGACT AAAAAAAGGCCATGGAGCCAGAGGGGCGAGGGCA ACAGACCTTTCATGGGCAAACCTTGGGGCCCTGCT GTCTAGCATGCCCCACTACGGGTCTAGGCTGCCCA TGTAAGGAGGCAAGGCCTGGGGACACCCGAGATG CCTGGTTATAATTAACCCAGACATGTGGCTGCCCCC CCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCT GTCCCTGGTGGATCCCCTGCATGCGAAGATCTTCG AACAAGGCTGTGGGGGACTGAGGGCAGGCTGTAA CAGGCTTGGGGGCCAGGCTTATACGTGCCTGGG ACTCCCAAAGTATTACTGTTCCATGTTCCCGGC GAA GGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACT TAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGG GCAGCCCATACAAGGCCATGGGGCTGGGCAAGCT GCACGCCTGGGTCCGGGGTGGGCACGGTGCCCGG GCAACGAGCTGAAAGCTCATCTGCTCTCAGGGGCC CCTCCCTGGGGACAGCCCCTCCTGGCTAGTCACAC CCTGTAGGCTCTCTATATAACCCAGG CACAGG GGCTGCCCTCATTCTACCACCACCTCCACAGCACA GACAGACACTCAGGAGCAGCCAGC 4 Chimeric intron AGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCC CGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTC CTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACG 5

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GAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGT ACCC PolyA GGCCGCAATAAAAGATCTTTATTTTCATTAGATCTGT GTGTTGGTTTTTTGTG 6 5' ITR CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGG CAAAGCCCGGGCGTCGGGCGACCTTTTGGTCGCCC GGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA GTGGGGTT 7 3' ITR CCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT TTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCG C 8 Nucleotide sequence of human γsarcoglycan (wild type), GenBank U34976.1 ATGGTGCGTGAGCAGTACACTACAGCCACAGAAGG CATCTGCATAGAGAGGCCAGAGAATCAGTATGTCTA CAAAATTGGCATTTATGGCTGGAGAAAGCGCTGTCT CTACTTGTTTGTTCTTCTTTTACTCATCATCCTCGTT GTGAATTTAGCTCTTACAATTTGGATTCTTAAAGTGA TGTGGTTTTCTCCAGCAGGAATGGGCCACTTGTGT GTAACAAAAGATGGACTGCGCTTGGAAGGGG AATC AGAA I I I I IATTCCCATTGTATGCCAAAGAAATACAC TCCAGAGTGGACTCATCTCTGCTGCTACAATCAACC CAGAATGTGACTGTAAATGCGCGCAACTCAGAAGG GGAGGTCACAGGCAGGTTAAAAGTCGGTCCCAAAA TGGTAGAAGTCCAGAATCAACAGTTTCAGATCAACT CCAACGACGGCAAGCCACTATTTACTGTAGATGAGA AGGAAGTTGTGGTTGGTA CAGATAAACTTCGAGTAA CTGGGCCTGAAGGGGCTCI I I I IGAACATTCAGTG GAGACACCCCTTGTCAGAGCCGACCCGTTTCAAGA CCTTAGATTAGAATCCCCCACTCGGAGTCTAAGCAT GGATGCCCCAAGGGGTGTGCATATTCAAGCTCACG CTGGGAAAATTGAGGCGCTTTCTCAAATGGATATTC I I I I I CATAGTAGTGATGGAATGCTTGTGCTTGATG C TGAAACTGTGTGCTTACCCAAGCTGGTGCAGGGG ACGTGGGGTCCCTCTGGCAGCTCACAGAGCCTCTA CGAAATCTGTGTGTGTCCAGATGGGAAGCTGTACC 9

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TGTCTGTGGCCGGTGTGAGCACCACGTGCCAGGAG CACAGCCACATCTGCCTCTGA Amino acid sequence of the AAV rh.74 capsid MAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLG DRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNT YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGF RPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTD SEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNN GSQAVGRSSF YCLEYFPSQMLRTGNNFEFSYNFEDV PFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTA GTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVST TLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATH KDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSE EEIKTTNPVATEQYGWADNLQQ QNAAPIVGAVNSQG ALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMG GFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYS TGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVD FAVNTEGTYSEPRPIGTRYLTRNL 10 Polynucleotide sequence of the rAAV vector ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGA GGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGG ACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAAC CAGCAAAAGCAGGACAACGGCCGGGGTCTGGTGC TTCCTGGCTACAAGTACCTCGGACCCTTCAACGGA CTCGACAAGGGGGAGCCCGTCAACGCGGCGGACG CAGCGGCCCTCGAGCACGACAAGGCCTACGAC CA GCAGCTCCAAGCGGGTGACAATCCGTACCTGCGGT ATAATCACGCCGACGCCGAGTTTCAGGAGCGTCTG CAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCG CGCAGTCTTCCAGGCCAAAAAGCGGGTTCTCGAAC CTCTGGGCCTGGTTGAATCGCCGGTTAAGACGGCT CCTGGAAAGAAGAGACCGGTAGAGCCATCACCCCA GCGCTCTCCAGACTCCTCTACGGGCAT CGGCAAGA AAGGCCAGCAGCCCGCAAAAAAGAGACTCAATTTT GGGCAGACTGGCGACTCAGAGTCAGTCCCCGACC CTCAACCAATCGGAGAACCACCAGCAGGCCCCTCT GGTCTGGGATCTGGTACAATGGCTGCAGGCGGTGG CGCTCCAATGGCAGACAATAACGAAGGCGCCGACG eleven

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GAGTGGGTAGTTCCTCAGGAAATTGGCATTGCGATT CCACATGGCTGGGCGACAGAGTCATCACCACCAGC ACCCGCACCTGGGCCCTGCCCACCTACAACAACCA CCTCTACAAGCAAATCTCCAACGGGACCTCGGGAG GAAGCACCAACGACAACACCTACTTCGGCTACAGC ACCCCCTGGGGGTATTTTGACTTCAACAGATTCCAC TGCCACTTTTCACCACGTGACTGGCAGCGACT CATC AACAACAACTGGGGATTCCGGCCCAAGAGGCTCAA CTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCAC GCAGAATGAAGGCACCAAGACCATCGCCAATAACC TTACCAGCACGATTCAGGTCTTTACGACTCGGAAT ACCAGCTCCCGTACGTGCTCGGCTCGGCGCACCAG GGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCAT GATTCCTCAGTACGGGTACCTGACTCT GAACAATGG CAGTCAGGCTGTGGGCCGGTCGTCCTTCTACTGCC TGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCA ACAACTTTGAATTCAGCTACAACTTCGAGGACGTGC CCTTCCACAGCAGCTACGCGCACAGCCAGAGCCTG GACCGGCTGATGAACCCTCTCATCGACCAGTACTT GTACTACCTGTCCCGGACTCAAAGCACGGGCGGTA CTGCAGGAACTCAGCAG TTGCTATTTTCTCAGGCCG GGCCTAACAACATGTCGGCTCAGGCCAAGAACTGG CTACCCGGTCCCTGCTACCGGCAGCAACGCGTCTC CACGACACTGTCGCAGAACAACAACAGCAACTTTG CCTGGACGGGTGCCACCAAGTATCATCTGAATGGC AGAGACTCTCTGGTGAATCCTGGCGTTGCCATGGC TACCCACAAGGACGACGAAGAGCGATTTTTTCCATC CAGCGGAGTC TTAATGTTTGGGAAACAGGGAGCTG GAAAAGACAACGTGGACTATAGCAGCGTGATGCTA ACCAGCGAGGAAGAAATAAAGACCACCAACCCAGT GGCCACAGAACAGTACGGCGTGGTGGCCGATAACC TGCAACAGCAAAACGCCGCTCCTATTGTAGGGGCC GTCAATAGTCAAGGAGCCTTACCTGGCATGGTGTG GCAGAACGGGACGTGTACCTGCAGGGTCCCATCT GGGCCAAGAT TCCTCATACGGACGGCAACTTTCAT CCCTCGCCGCTGATGGGAGGCTTTGGACTGAAGCA TCCGCCTCCTCAGATCCTGATTAAAAACACACCTGT TCCCGCGGATCCTCCGACCACCTTCAATCAGGCCA AGCTGGCTTCTTTCATCACGCAGTACAGTACCGGC CAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGA AGGAGAACAGCAAACGCTGGAACCCAGAGATTCAG TACACTTCCAACTACTACAAATCTACAAATGTGGACT TTGCTGTCAATACTGAGGGTACTTATT CCGAGCCTC GCCCCATTGGCACCCGTTACCTCACCCGTAATCTGT AA

In another aspect, described herein is a recombinant AAV vector comprising a polynucleotide sequence encoding a functional γ-sarcoglycan that contains or essentially consists of or further consists of a nucleotide sequence that hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 1 or its complement. Functional γ-sarcoglycan involves a γ-sarcoglycan polypeptide that retains γ-sarcoglycan activity. γ-Sarcoglycan activity is critical to muscle function; γ-Sarcoglycan is one of several sarcolemmal transmembrane glycoproteins that interact with dystrophin to form the dystrophin-glycoprotein complex, which

- 17 045951 ry covers the sarcolemma and consists of dystrophin, syntrophin, α-dystroglycans and β-dystroglycans, and sarcoglycans, including γ-sarcoglycan. The dystrophin-glycoprotein complex provides a structural link between the subsarcolemmal cytoskeleton and the intracellular matrix of muscle cells. Non-limiting examples of muscle cells include muscle cells of the heart, diaphragm, leg, pelvic girdle, shoulder and arm. Additionally, non-limiting examples of γ-sarcoglycan activity and the consequences of γ-sarcoglycanopathy are described in Blake et al. (2002) Physiol Rev.;82(2):291329 and Tarakci et al. (2016) Front Biosci (Landmark Ed);21:744-56.

The term stringent is used to refer to conditions that are commonly understood in the art to mean stringent conditions. The stringency of hybridization is fundamentally determined by temperature, ionic strength and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher levels of formamide or other denaturing agent) can also be used, but the degree of hybridization will be affected. In examples where hybridization of deoxyoligonucleotides is contemplated, additional illustrative stringent hybridization conditions include a wash in 6x SSC 0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for oligonucleotides of 20 bases) and 60°C (for oligonucleotides of 23 bases).

Other agents may be included in hybridization and wash buffers to reduce nonspecific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodS04, (SDS), Ficoll, Denhardt's solution, salmon sperm DNA, fragmented sonication (or other non-complementary DNA) and dextran sulfate, although other suitable agents may also be used. The concentration and types of these additives can be varied without significantly affecting the severity of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4, however, under normal ionic strength conditions the degree of hybridization is almost independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art to ensure these variables and ensure the formation of DNA hybrids with different sequence relatedness.

In another aspect, the recombinant AAV vectors described herein may contain or essentially consist of, or further consist of, a polynucleotide sequence encoding a γ-sarcoglycan that is operably linked to a promoter and/or a muscle-specific regulatory element. For example, the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, myocyte-specific enhancer binding factor MEF, muscle creatine kinase (MCK), tMCK (truncated MCK), myosin heavy chain (MHC), MHCK7 (hybrid version of MHC and MCK), C5-12 (synthetic promoter), mouse creatine kinase enhancer element, fast-twitch skeletal muscle troponin C gene element, cardiac slow-twitch troponin C gene element, slow-twitch muscle troponin I gene element, nuclear factors, hypoxia-inducible, steroid-inducible element or glucocorticoid response element (GRE). In one embodiment, the rAAV vector contains the MHCK7 promoter (SEQ ID NO: 4).

An exemplary rAAV vector described herein is pAAV.MHCK7.hSCGC, which contains the nucleotide sequence SEQ ID NO: 3; wherein the MCHK7 promoter spans nucleotides 136-927 (SEQ ID NO: 4), the intron spans nucleotides 937-1084 (SEQ ID NO: 5), the γ-sarcoglycan sequence spans nucleotides 1094-1969 (SEQ ID NO: 1), and polyA spans nucleotides 1976-2028 (SEQ ID NO: 6). See fig. 1. In some cases, the only viral sequences included in the rAAV vector are inverted terminal repeats, which are required for viral DNA replication and packaging. In some cases, the intron (SEQ ID NO: 5), spanning nucleotides 7-116, and the 5' UTR (5' untranslated region) (SEQ ID NO: 7), spanning nucleotides 2128-2231, are derived from pCMVe plasmids (Clontech). In some cases, the 3' UTR contains the sequence set forth in SEQ ID NO: 8. In some cases, pAAV.MHCK7.hSCGC is packaged within the AAV rh.74 capsid.

The DNA plasmids are disclosed to contain rAAV genomes. Plasmid DNA is transferred into cells permissive to infection by an AAV helper virus (eg, adenovirus, EI-deleted adenovirus, or herpesvirus) to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV viral particles into which the AAV genome, rep and cap genes, and helper virus functions proposed in a cell are to be packaged are standard in the art. Production of rAAV requires that the following components be present in a single cell (denoted

- 18 045951 mine in this document as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (namely, not included in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype from which a recombinant virus can be derived, and may be derived from an AAV serotype other than the ITR of the rAAV genome, including but not limited to the following AAV serotypes: AAV-1, AAV-2 , AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. In some embodiments, the rAAV vector contains AAV2 inverted terminal repeat (ITR) sequences. The production of pseudotyped rAAV is disclosed, for example, in WO 01/83692, which is incorporated herein by reference in its entirety. In some aspects, the rAAV vector contains inverted AAV2 ITR sequences and is enclosed within an AAV rh.74 capsid. In some cases, the rAAV vector genome contains the polynucleotide sequence set forth in SEQ ID NO: 11. In some cases, the AAV rh.74 capsid contains the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the rAAV vector contains a polynucleotide that contains or essentially consists of or further consists of a sequence, for example, at least 65%, at least 70%, at least 75%, at least 80, 81, 82, 83, 84, 85, 86 , 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the nucleotide sequence set forth in SEQ ID NO: 11, and encodes VP1 capsid proteins, VP2 and VP3 rAAV. In some embodiments, the rAAV vector contains a polypeptide that contains or essentially consists of, or further consists of, e.g., at least 65%, at least 70%, at least 75%, at least 80 , 81, 82, 83, 84, 85, 86, 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of AAV rh. 74 VP3, which is set forth in SEQ ID NO: 10.

The method for producing a packaging cell line is to create a cell line that stably expresses all the necessary components to produce AAV particles. For example, a plasmid(s) containing the rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene are integrated into the genome of the cell. AAV genomes have been introduced into bacterial plasmids by methods such as the formation of GC tails (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al. al., 1983, Gene, 23:65-73), or direct blunt end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:46614666). The packaging cell line is then infected with a helper virus, such as an adenovirus. The advantages of this method are that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells. In some cases, the rAAV vector genome contains the polynucleotide sequence set forth in SEQ ID NO: 11. In some cases, the AAV rh.74 capsid contains the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the rAAV vector contains a polynucleotide that contains or essentially consists of or further consists of a sequence, for example, at least 65%, at least 70%, at least 75%, at least 80, 81, 82, 83, 84, 85, 86 , 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the nucleotide sequence set forth in SEQ ID NO: 11, and encodes VP1 capsid proteins, VP2 and VP3 rAAV. In some embodiments, the rAAV vector contains a polypeptide that contains or essentially consists of, or further consists of, e.g., at least 65%, at least 70%, at least 75%, at least 80 , 81, 82, 83, 84, 85, 86, 87, 88 or 89%, more typically 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of AAV rh. 74 VP3, which is set forth in SEQ ID NO: 10.

Conventional principles for the preparation of rAAV are discussed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62: 1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); US Patent No. 5173414; WO 95/13365 and related US Patent No. 5658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13: 1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3: 11241132; US Patent No. 5786211; US Patent No. 5871982; and US Pat. No. 6,258,595. The above documents are incorporated herein by reference in their entirety, with particular emphasis on those portions of the documents related to the production of rAAV.

Thus, the disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (related

- 19 045951 line 293). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as transient 293 cells (human embryonic kidney cells transformed with adenovirus EI), MRC-5 cells (human embryonic fibroblasts), WI-38 cells ( human embryonic fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus macaque embryonic lung cells).

Recombinant AAV (namely, infectious, encapsided rAAV particles) are disclosed to contain the rAAV genome. Embodiments include, but are not limited to, rAAV named pAAV.MHCK7.hSCGC, which contains the polynucleotide sequence set forth in SEQ ID NO: 3.

rAAV can be purified by methods standard in the art, such as column chromatography or cesium chloride density gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include those disclosed, for example, in Clark et al., Hum. Gene Then., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); US patent No. 6566118 and WO 98/09657.

In another embodiment, the disclosure covers compositions containing or essentially consisting of rAAV of the present disclosure. The compositions described herein contain or consist essentially of rAAV in a pharmaceutically acceptable carrier. In one specific embodiment, the composition of this disclosure contains or consists essentially of a Lactated Ringer's solution (LRS). The compositions may also contain other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are non-toxic to recipients and are preferably inert in the dosages and concentrations used, 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 (ethylenediaminetetraacetic acid); sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or non-ionic surfactants such as Tween, plurons or polyethylene glycol (PEG). The disclosed compositions can be used for one or more of: the treatment of γ-sarcoglycanopathy; increasing muscle strength, muscle endurance and/or muscle mass; reducing fibrosis; reducing contraction-induced damage; reducing fatty infiltration; and/or reducing central nucleation in a subject in need thereof and/or treating muscular dystrophy by reducing fiber degeneration or necrotic fibers; reducing inflammation; increased levels of creatine kinase; treating muscle fiber atrophy and hypertrophy, and/or reducing dystrophic calcification in a subject suffering from muscular dystrophy.

Titers of rAAV to be administered in the methods of the disclosure will vary, for example, depending on the specific rAAV, route of administration, purpose of treatment, individual and cell type(s) to be targeted, and can be determined by methods standard in the art. rAAV titers can range from about 1x106, about ^1x107 , about 1x108, about 1x109, about ^1x1010 , about 1x1011, about ^1x1012 , about ^1x1013 to about ^1x1014 or more DNAse-resistant particles (DRP). DNase resistant particle) per ml. Dosages can be expressed in units of viral genomes (vg - from the English viral genome).

Methods for transducing a target cell with rAAV, in vivo or in vitro, are covered by this disclosure. The term transduction is used to refer to the introduction/delivery of a polynucleotide of interest (e.g., a polynucleotide sequence encoding a γ-sarcoglycan) into a recipient cell, either in vivo or in vitro, by a described replication-defective rAAV, resulting in expression of the γ-sarcoglycan by the recipient cell .

In one aspect, provided herein are methods of one or more of: treating γsarcoglycanopathy; increasing muscle strength, muscle endurance and/or muscle mass; reducing fibrosis; reducing contraction-induced damage; reducing fatty infiltration; and/or reducing central nucleation in a subject in need thereof and/or treating muscular dystrophy by reducing fiber degeneration or necrotic fibers; reducing inflammation; increased levels of creatine kinase; treating muscle fiber atrophy and hypertrophy, and/or reducing dystrophic calcification in a subject suffering from muscular dystrophy, comprising or consisting essentially of or further consisting of administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (AAV) vector, wherein the rAAV vector comprises or essentially consists of or further consists of a gene expression cassette containing or essentially consisting of or further consisting of a polynucleotide sequence encoding a γ-sarcoglycan under the transcriptional control of a promoter, said cassette being flanked by one or more inverted

- 20 045951 terminal AAV repeats. In some embodiments, said promoter is a muscle-specific regulatory element. In one embodiment, the methods disclosed herein increase muscle strength, muscle endurance, and/or muscle mass of one or more muscles of a subject. Non-limiting examples of muscles include the heart, diaphragm, thighs, calves, pelvic girdle, shoulder and arm. In one specific embodiment, muscle strength, muscle endurance, and/or muscle mass are increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 50%, or at least about 80%, compared to an untreated control subject.

In one particular aspect, the subject suffers from pelvic-brachial muscular dystrophy. In yet another aspect, the subject suffers from pelvic-brachial muscular dystrophy, which is pelvic-brachial muscular dystrophy type 2C.

The terms administration or administration, with respect to delivery of polynucleotides to a subject, include any route of administration or delivery to a subject of polynucleotides to perform the intended function. Administration can be accomplished by any suitable route, including orally, intranasally, parenterally (intravenous, intramuscular, intraperitoneal or subcutaneous), intracranial or local. Additional routes of administration include intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intrapulmonary, intraspinal, intrathoracic, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Administration includes self-administration and administration by someone else.

In one aspect, the methods disclosed herein include or essentially consist of or further consist of administering a composition comprising or essentially consisting of or further consisting of an rAAV vector and a pharmaceutically acceptable carrier. In yet another aspect, the methods disclosed herein include or essentially consist of or further consist of administering an rAAV vector or a composition comprising or essentially consisting of or further consisting of an rAAV vector and a pharmaceutically acceptable carrier by intramuscular injection or intravenous injection. In yet another aspect, the methods disclosed herein include or essentially consist of or further consist of systemically administering an rAAV vector or a composition comprising or essentially consisting of or further consisting of an rAAV vector and a pharmaceutically acceptable carrier. In one specific aspect, the methods disclosed herein include or essentially consist of or further consist of administering a rAAV vector or a composition comprising or essentially consisting of or further consisting of an rAAV vector and a pharmaceutically acceptable carrier, parenterally by injection, infusion or implantation.

In one aspect, the polynucleotide sequence encoding a γ-sarcoglycan of an rAAV vector for use in the methods described herein comprises or essentially consists of, or further consists of, the nucleotide sequence set forth in SEQ ID NO: 1. In another aspect, the polynucleotide sequence , encoding the γ-sarcoglycan of the rAAV vector, encodes the amino acid sequence of SEQ ID NO: 2. In yet another aspect, the rAAV vector used in the methods disclosed herein includes or essentially consists of, or further consists of, a self-complementary AAV vector genome. In one specific embodiment, the rAAV vector contains or essentially consists of, or further consists of, a genome lacking AAV rep and cap DNA. In another embodiment, the rAAV vector belongs to the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV rh74. In yet another aspect, the rAAV vector belongs to the AAV rh74 serotype, and the rAAV vector contains or essentially consists of, or further consists of, an AAV rh.74 capsid. In yet another aspect, the AAV rh.74 capsid of the rAAV vector contains or essentially consists of, or further consists of, the amino acid sequence set forth in SEQ ID NO: 10, or its equivalent.

The rAAV vector used in the methods disclosed herein may further comprise or essentially consist of a promoter and/or a muscle-specific regulatory element, and wherein the muscle-specific regulatory element is operably linked to a polynucleotide encoding a γ-sarcoglycan. Non-limiting examples of some muscle-specific regulatory elements are human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer binding factor mef, muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC) , MHCK7, C5-12, mouse creatine kinase enhancer element, fast-twitch skeletal muscle troponin gene element, slow-twitch cardiac troponin gene element, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factors, steroid-inducible element, and glucocorticoid-responsive element (gre). In one aspect, the muscle-specific regulatory element of the rAAV vector is a truncated MCK (tMCK). IN

- 21 045951 In another aspect, the muscle-specific promoter and/or regulatory element of the rAAV vector is the MHCK7 promoter. In yet another aspect, the MHCK promoter comprises or essentially consists of, or further consists of, the nucleotide sequence set forth in SEQ ID NO: 3, or an equivalent thereof.

In one embodiment, the genome of the rAAV vector disclosed herein contains an intron containing the nucleotide sequence set forth in SEQ ID NO: 5.

The in vivo methods include or essentially consist of or further consist of the step of administering an effective dose or effective multiple doses of a composition containing or essentially consisting of or further consisting of rAAV as disclosed herein, to an animal (including a human) in need thereof. If the dose is administered before the onset of the disorder/disease, the administration is prophylactic. If the dose is administered after the disorder/disease has developed, the administration is therapeutic. In embodiments of the disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease being treated, which slows or prevents the progression of the disorder/disease condition, which slows or prevents the progression of the disorder/disease condition , which reduces the severity of the disease, which leads to remission (partial or complete) of the disease, and/or which prolongs survival. An example of a disease contemplated for prevention or treatment by the methods of the disclosure is muscular dystrophy, such as pelvic-brachial muscular dystrophy. In some embodiments, the disease contemplated for prevention or treatment by the methods of the disclosure is pelvis-brachial muscular dystrophy type 2C (LGMD2C).

The term muscular dystrophy, as used herein, refers to a disorder in which strength and muscle mass gradually decrease. Non-limiting examples of muscular dystrophies may include Becker muscular dystrophy, tibial muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreyfus muscular dystrophy, Landouzy-Dejerine muscular dystrophy, sarcoglycanopathies, congenital muscular dystrophy, such as congenital muscular dystrophy due to partial deficiency LAMA2, merosin deficiency congenital muscular dystrophy, congenital muscular dystrophy type ID, congenital muscular dystrophy Fukuyama type, pelvic-brachial muscular dystrophy type 1A, pelvic-brachial muscular dystrophy type 2A, pelvic-brachial muscular dystrophy type 2B, pelvic-brachial muscular dystrophy type 2C , pelvic-brachial muscular dystrophy type 2D, pelvic-brachial muscular dystrophy type 2E, pelvic-brachial muscular dystrophy type 2F, pelvic-brachial muscular dystrophy type 2G, pelvic-brachial muscular dystrophy type 2H, pelvic-brachial muscular dystrophy type 21, pelvic - brachial muscular dystrophy type 21, pelvic-brachial muscular dystrophy type 2J, pelvic-brachial muscular dystrophy type 2K, pelvic-brachial muscular dystrophy type IC, muscular dystrophy with spinal rigidity, with epidermolysis bullosa simplex, oculopharyngeal muscular dystrophy, Ullrich congenital muscular dystrophy and Ullrich's scleroatonic muscular dystrophy. In some embodiments, the subject suffers from pelvic-brachial muscular dystrophy. In some embodiments, the subject suffers from pelvis-brachial muscular dystrophy type 2C (LGMD2C).

There are at least nineteen forms of LGMD and these forms are classified according to their associated genetic defects.

Type Inheritance type Gene or chromosome LGMD1A autosomal dominant Myotilin gene LGMD1B autosomal dominant Gene Lamina A/S LGMD1C autosomal dominant Caveolin gene LGMD1D autosomal dominant Chromosome 7 LGMD1E autosomal dominant Chromosome 6

- 22 045951

LGMD1F autosomal dominant Chromosome 7 LGMD1G autosomal dominant Chromosome 4 LGMD2A autosomal recessive Calpain-3 gene LGMD2B autosomal recessive Dysferlin gene LGMD2C autosomal recessive Gamma sarcoglycan gene LGMD2D autosomal recessive Alpha sarcoglycan gene LGMD2E autosomal recessive Beta-sarcoglycan gene LGMD2F autosomal recessive Delta sarcoglycan gene LGMD2G autosomal recessive Teletonin gene LGMD2H autosomal recessive TRIM32 LGMD2I autosomal recessive FKRP gene LGMD2J autosomal recessive titin gene LGMD2K autosomal recessive POMT1 gene LGMD2L autosomal recessive ANO5 gene

In some aspects, the disclosure relates to a method of treating muscular dystrophy (e.g., LGMD2C) in a subject, comprising either essentially consisting of or further consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding a γsarcoglycan as described herein, or a composition comprising or essentially consisting of such an rAAV vector.

In some embodiments, the disclosure provides a method of increasing muscle strength, muscle endurance, and/or muscle mass in a subject suffering from muscular dystrophy (e.g., LGMD2C), comprising or essentially consisting of, or further consisting of administering to the subject a therapeutically effective amount of an rAAV vector encoding γ-sarcoglycan, as described herein, or a composition containing or essentially consisting of such an rAAV vector.

In some aspects, the disclosure covers a method of reducing contraction-induced damage in a subject suffering from muscular dystrophy (e.g., LGMD2C), comprising or essentially consisting of, or further consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding a γ-sarcoglycan, as described herein, or a composition containing or essentially consisting of such an rAAV vector.

In some aspects, the disclosure covers a method of treating a γ-sarcoglycanopathy in a subject, comprising or essentially consisting of, or further consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding a γ-sarcoglycan as described herein, or a composition comprising or essentially consisting of such a rAAV vector.

The disclosure also covers a method of reducing fibrosis in a subject suffering from muscular dystrophy (e.g., LGMD2C), comprising either essentially consisting of or further consisting of administering to the subject a therapeutically effective amount of a rAAV vector encoding a γsarcoglycan as described herein, or a composition comprising or essentially consisting of such an rAAV vector. The term fibrosis, as used herein, refers to the excessive or dysregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues of injury, including skeletal muscle, cardiac muscle, liver, lung, kidney and pancreas. gland. ECM components that are deposited include collagen, such as collagen 1, collagen 2 or collagen 3, and fibronectin.

In some embodiments, the subject being treated by the methods described herein may be a mammal. In some cases, the subject is a human, non-human primate, pig, horse, cow, dog, cat, rabbit, mouse or rat. The subject may be a female human or a male human. In some cases, the subject is a human subject aged 1-7, 7-15, 1625, 26-50, 50-70, or more than 70 years old. Other age ranges are considered and include, but are not limited to, ages 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 60-70 or greater than 70 years, as well as any interval contained in the above.

As used herein, the term patient or subject in need refers to a patient or subject at risk of or suffering from a disease, disorder or condition that is treatable or ameliorated by an rAAV containing a nucleic acid sequence, encoding a γ-sarcoglycan, or a composition containing or essentially consisting of such rAAV as provided herein. The patient or subject in need may, for example, be a patient or subject diagnosed with a disease associated with impaired γ-sarcoglycan function, such as LGMD2C. The subject may have a mutation or impaired function in the γ gene or protein

- 23 045951 sarcoglycan. The terms Subject and Patient are used interchangeably throughout this document.

Combination therapies including or essentially consisting of or further consisting of one or more of the compositions disclosed herein and a corticosteroid are also contemplated by this disclosure. Combination, as used herein, includes simultaneous treatment or sequential treatment. Combinations of the disclosed methods with standard medical treatments (eg, corticosteroids) are specifically addressed, as are combinations with new therapies. In some embodiments, the subject can be treated with a steroid (eg, prednisone, prednisone, deflazacort) to prevent or reduce the immune response to rAAV administration described herein. In some cases, a subject may receive apheresis or another immunomodulator if the subject expresses antibodies to the rAAV described herein.

A therapeutically effective amount of rAAV vector, in some embodiments, is a dose of rAAV in one or more administrations ranging from about 1e13 vg/kg to about 5e14 vg/kg, or from about 1e13 vg/kg to about 2e13 vg/kg, or from about 1e13 vg/kg to about 3e13 vg/kg, or from about 1e13 vg/kg to about 4e13 vg/kg, or from about 1e13 vg/kg to about 5e13 vg/kg, or from about 1e13 vg/kg to about 6e13 vg/kg, or from about 1e13 vg/kg to about 7e13 vg/kg, or from about 1e13 vg/kg to about 8e13 vg/kg, or from about 1e13 vg/kg to about 9e13 vg/kg, or from about 1e13 vg/kg to about 1e14 vg/kg, or from about 1e13 vg/kg to about 2e14 vg/kg, or from 1ei 3 vg/kg to about 3e14 vg/kg, or from about 1x13 to about 4e14 vg/kg, or from about 3e13 vg/kg to about 4e13 vg/kg, or from about 3e13 vg/kg to about 5e13 vg/kg, or from about 3e13 vg/kg to about 6e13 vg/kg, or from about 3e13 vg/kg to about 7e13 vg/kg, or from about 3e13 vg/kg to about 8e13 vg/kg, or from about 3e13 vg/kg to about 9e13 vg/kg, or from about 3e13 vg/kg to about 1e14 vg/kg, or from about 3e13 vg /kg to about 2e14 vg/kg, or from 3e13 vg/kg to about 3e14 vg/kg, or from about 3e13 to about 4e14 vg/kg, or from about 3e13 vg/kg to about 5e14 vg/kg, or from about 5e13 vg/kg to about 6e13 vg/kg, or from about 5e13 vg/kg to about 7e13 vg/kg, or from about 5e13 vg/kg to about 8e13 vg/kg, or from about 5e13 vg/kg to about 9e13 vg/ kg, or from about 5e13 vg/kg to about 1e14 vg/kg, or from about 5e13 vg/kg to about 2e14 vg/kg, or from 5e13 vg/kg to about 3e14 vg/kg, or from about 5e13 to about 4e14 vg/kg, or from about 5e13 vg/kg to about 5e14 vg/kg, or from about 1e14 vg/kg to about 2e14 vg/kg, or from 1e14 vg/kg to about 3e14 vg/kg, or from about 1e14 to approximately 4e14 vg/kg, or from approximately 1e14 vg/kg to approximately 5e14 vg/kg. The disclosure also includes or essentially consists of or further consists of rAAV vector interval data.

For example, a therapeutically effective amount of rAAV vector is a dose of about 1e13 vg/kg, about 2e13 vg/kg, about 3e13 vg/kg, about 4e13 vg/kg, about 5e13 vg/kg, about 6e13 vg/kg, about 7e13 vg/ kg, approximately 8e13 vg/kg, approximately 9e13 vg/kg, approximately 1e14 vg/kg, approximately 2e14 vg/kg, approximately 3e14 vg/kg, approximately 4e14 vg/kg and 5e14 vg/kg. The disclosure also includes or essentially consists of or furthermore consists of compositions containing or essentially consists of or furthermore consists of given doses of the rAAV vector.

A therapeutic effective amount of rAAV in some embodiments is a dose of rAAV ranging from about 1e14 vg/kg to about 1e15 vg/kg or from about 1e15 vg/kg to about 1e16 vg/kg. In some embodiments, the disclosure provides methods of administering to a subject the rAAV vector of the disclosure at a dose of about 1e14 vg/kg, about 1.5e14 vg/kg, about 2e14 vg/kg, about 2.5e14 vg/kg, about 3e14 vg/kg, about 3.5e14 vg/kg, approximately 4e14 vg/kg, approximately 4.5e14 vg/kg, approximately 5e14 vg/kg, approximately 5.5e14 vg/kg, approximately 6e14 vg/kg, approximately 6.5e14 vg/kg, approximately 7e14 vg/kg, approximately 7.5e14 vg/kg, approximately 8e14 vg/kg, approximately 8.5e14 vg/kg, approximately 9e14 vg/kg, approximately 9.5e14 vg/kg, approximately 1e15 vg/kg, approximately 1, 5e15 vg/kg, approximately 2e15 vg/kg, approximately 2.5e15 vg/kg, approximately 3e15 vg/kg, approximately 3.5e15 vg/kg, approximately 4e15 vg/kg, approximately 4.5e15 vg/kg, or approximately 5e15 vg/kg. In some embodiments, the disclosure provides methods of administering an rAAV vector as disclosed to a subject at a total dose of about 4.0e14 vg/kg, about 4.1e14 vg/kg, about 4.2e14 vg/kg, about 4.3e14 vg/kg, about 4 ,4e14 vg/kg, approximately 4.5e14 vg/kg, approximately 4.6e14 vg/kg, approximately 4.7e14 vg/kg, approximately 4.8e14 vg/kg, approximately 4.9e14 vg/kg, approximately 5.0e14 vg/kg, approximately 5.1e14 vg/kg, approximately 5.2e14 vg/kg, approximately 5.3e14 vg/kg, approximately 5.4e14 vg/kg, approximately 5.5e14 vg/kg, approximately 5.6e14 vg/ kg, approximately 5.7e14 vg/kg, approximately 5.8e14 vg/kg, approximately 5.9e14 vg/kg or approximately 6e14 vg/kg,

In various embodiments, the administration step may include administering the total dose in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more divided doses. For example, the total dose may be delivered by injection at multiple sites on or to a subject, several minutes, several hours, or several days apart.

Administration of an effective dose of the compositions may be administered by routes standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, inhalation, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of management and serotypes) of AAV rAAV components (in

- 24 045951 in particular, AAV ITR and capsid protein) according to the disclosure can be selected and/or combined by those skilled in the art, taking into account the infection and/or disease state to be treated and the target cells/tissue(s) that should express γ-sarcoglycan.

The disclosure provides local administration and systemic administration of an effective dose of rAAV and the compositions of the disclosure. For example, systemic administration is administration into the circulatory system in such a way that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration by injection, infusion or implantation.

In particular, the actual administration of the rAAV of the present disclosure may be accomplished by using any physical method that will transport the recombinant rAAV vector into the target tissue of the animal. Administration according to the disclosure includes, but is not limited to, injection into muscle, bloodstream, and/or directly into the liver. Demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known restrictions on vehicles or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual way with rAAV).

The rAAV capsid proteins can be modified so that the rAAV is targeted to the specific target tissue of interest, such as muscle. See, for example, WO 02/053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable preparations or as topical preparations to be delivered to the muscles via transdermal transport. Numerous preparations for both intramuscular injection and transdermal transport have previously been developed and can be used in practice upon disclosure. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

For intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol, as well as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered, if desired, and the liquid diluent is first made isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. It is also possible to obtain rAAV dispersions in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media used are readily available through standard techniques well known to those skilled in the art.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for immediate administration of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to such an extent that it can be easily administered by syringe. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, etc. In many cases it will be preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions may be due to the use of delayed absorption agents, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating rAAV in the required amount in an appropriate diluent with various other ingredients listed above, if necessary, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle that contains a base dispersion medium and the required other ingredients from the ingredients listed above. In the case of pregnancy powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and lyophilization techniques, which result in a powder of the active ingredient plus any additional desired ingredient from a previously sterilizing filtered solution thereof.

Transduction of rAAV can also be carried out in vitro. In one embodiment, desired target muscle cells are collected from a subject, transduced with rAAV, and reintroduced into the subject. As al

- 25 045951 alternatives, syngeneic or xenogeneic muscle cells can be used in cases where such cells will not generate an inappropriate immune response in the subject.

Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells, for example, in appropriate media, and screening for those cells carrying the DNA of interest using conventional techniques such as Southern blotting and/or PCR, or by use of selectable markers. The transduced cells can then be formulated into pharmaceutical compositions, and the composition administered to the subject through various techniques, such as intramuscular, intravenous, subcutaneous and intraperitoneal injection, or injection into smooth or cardiac muscle using, for example, a catheter.

Transduction of rAAV cells by opening leads to sustained expression of γ-sarcoglycan. The present invention thus provides methods for administering/delivering rAAVs that express γ-sarcoglycan to a mammalian, preferably human, subject. These methods involve transducing tissues (including but not limited to tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more rAAVs of the present disclosure. Transduction can be carried out using gene cassettes containing tissue-specific regulatory elements. For example, according to one embodiment of the disclosure, methods are provided for transducing muscle cells and muscle tissues that are targeted by muscle-specific regulatory elements, including, but not limited to, regulatory elements derived from the actin and myosin gene families, such as the myoD gene family [See . Weintraub et al, Science, 251: 761-766 (1991)], myocyte-specific enhancer-binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol 11: 4854-4862 (1991)], gene-derived regulatory elements human skeletal actin [Muscat et al, Mol Cell Biol, 7: 4089-4099 (1987)], cardiac actin gene, muscle creatine kinase sequence elements [See. Johnson et al, Mol Cell Biol, 9:3393-3399 (1989)] and the murine creatine kinase (mCK) enhancer element, regulatory elements derived from the fast-twitch skeletal muscle troponin C gene, the slow-twitch cardiac muscle troponin C gene, and the slow-twitch troponin I gene contractile muscle: hypoxia-inducible nuclear factors (Semenza et al., Proc Natl Acad Sci USA, 88: 5680-5684 (1991)), steroid-inducible elements and promoters, including glucocorticoid response element (GRE) (See Mader and White , Proc. Natl. Acad. Sci. USA 90: 5603-5607 (1993)), and other regulatory elements.

Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is readily accessible. The disclosure addresses sustained expression of a transgene (eg, γ-sarcoglycan) from transduced muscle fibers.

The term muscle cell or muscle tissue refers to a cell or group of cells derived from muscle of any kind (eg, skeletal muscle and smooth muscle, such as from the digestive tract, bladder, blood vessels or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts.

Thus, also described herein are methods of administering an effective dose (or doses administered substantially simultaneously or doses administered at intervals) of rAAV that encodes γsarcoglycan to a mammalian subject in need thereof.

Also provided herein are kits containing or essentially consisting of, or further consisting of, any one or more embodiments disclosed herein, and possible instructions for use. The kits may contain or essentially consist of or further consist of one or more of the compositions disclosed herein and a corticosteroid or one or more combination therapies provided herein and possible instructions for use.

It should be understood that the present disclosure is not limited to the specific aspects described, and therefore may, of course, vary. It should also be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

A number of embodiments of the disclosure are described. However, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, the following examples are intended to illustrate and not to limit the scope of the disclosure described in the claims.

It is to be assumed, without express specification and unless otherwise stated, that when the present technology relates to a polypeptide, protein, polynucleotide or antibody, the equivalent or biological equivalent thereof is presumed to be within the scope of the present technology.

- 26 045951

Reference to any patent, patent application, publication or any other document does not imply that any of the foregoing is prior art, nor does it constitute any representation as to the contents or date of such publications or documents.

Any of the features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless explicitly stated otherwise, the disclosed features (eg, antibodies) are an example of a class of equivalent or similar features.

As used herein, all numeric values or ranges of numeric values include integers within such ranges and fractions of values or integers within ranges unless the context clearly indicates otherwise. In addition, when a list of values is described herein (eg, about 50, 60, 70, 80, 85, or 86%), the list includes all intermediate and fractional values thereof (eg, 54, 85.4%). Thus, for illustration purposes, reference to 80% or more includes 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94%, etc., as well as 81. 1, 81.2, 81.3, 81.4, 81.5%, etc., 82.1, 82.2, 82.3, 82.4, 82.5%, etc., and so on.

A reference to an integer with greater than or less than includes any number greater than or less than the referenced number, respectively. Thus, for example, a reference to less than 100 includes 99, 98, 97, etc. up to the number one (1); and less than 10 includes 9, 8, 7, etc., up to the number one (1).

As used herein, all numerical values or ranges include fractions of values and whole numbers within such ranges unless the context clearly indicates otherwise. Thus, for illustration purposes, reference to a range of numerical values such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1. 3, 1.4, 1.5, etc. and so on. Reference to the interval 1-50 thus includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 etc., up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2, 3, 2.4, 2.5, etc., and so on.

A reference to a range of intervals includes intervals that combine the values of the boundaries of different intervals within a given series. Thus, for illustration purposes, reference is made to a range of intervals, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150 -200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-400 0 , 4000-4500, 4500-5000, 5500-6000, 6000-7000, 7000-8000 or 8000-9000 includes intervals 10-50, 50-100, 100-1000, 1000-3000, 2000-4000, etc.

Modifications to the above can be made without deviating from the basic aspects of the technology. Although the technology has been described in substantially detail with reference to one or more specific embodiments, those of ordinary skill in the art will appreciate that modifications can be made to the embodiments specifically disclosed herein, such modifications and improvements being within the scope and spirit of the technology.

The technology illustratively described herein may suitably be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each example herein, any of the terms containing, essentially consisting of, and consisting of may be replaced by either of the other two terms. The terms and expressions that have been used are used by description and not by limitation, and the use of such terms and expressions does not exclude any equivalent features shown and described, or segments thereof, and various modifications are possible within the scope of the claimed technology.

All publications and patents mentioned herein are incorporated herein by reference in their entirety as if each individual publication or patent were specifically and separately identified as being incorporated by reference. In the event of a conflict, this application, including any definitions herein, will control. However, there is no reference to any reference, article, publication, patent, patent publication and patent application cited herein and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the ordinary general knowledge in any country in the world.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the range listed and, where appropriate, fractions thereof (such as one tenth and one hundredth whole number) unless otherwise noted. The term approximately, immediately preceding a number or numeral, means that the number or numeral is within plus or minus 10%. It should be understood that the singular terms as used herein refer to one or more of the listed components unless otherwise indicated. It should be understood that the use of an alternative (eg or) means either one or both or any combination of alternatives. It should be understood that ter- 27 045951 min and/or means either one or both of the alternatives. As used herein, the terms includes or contains are used interchangeably.

The section headings used in this document are offered for organizational purposes only and should not be construed as limiting the subject matter described.

The disclosure is further described in the following Examples, which do not limit the scope of the disclosure described in the claims.

Examples

Example 1. Construction of scAAVrh74.tMCK.hSGCB and vector activity.

An SGCG AAV construct was constructed containing the codon optimized full-length human γsarcoglycan (SCGB) cDNA (SEQ ID NO: 1) as shown in FIG. 1. The SGCG AAV construct was configured packaged using a self-complementary AAV scaffold for more efficient transduction efficiency. The SGCG cDNA (969) was configured under the control of the MHCK7 promoter (792 bp). The intron and 5′ UTR were derived from plasmid pCMVe (Clontech). The SGCG AAV construct had a Kozak consensus sequence immediately upstream of the ATG start codon and a small synthetic polyadenylation signal of 53 bp. to terminate mRNA. The cDNA was codon optimized for human use and synthesized by GenScript (Piscataway, NJ). The only viral sequences included in this vector were the AAV2 inverted terminal repeats, which were required for both viral DNA replication and packaging.

The vector for this study was prepared using the triple transfection method of HEK293 cells under high purity conditions. Characterization of the vector after production included determination of titer by quantitative PCR with a supercoiled standard, determination of endotoxin level (EU/ml) and assessment of sterility. The resulting vector was analyzed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) to check the consistency of the banding pattern with the expected rAAV. Vector preparations were titrated using a standard, a linear plasmid, and re-titrated using a standard, a supercoiled plasmid. The vector was generated using a plasmid containing a full-length human γ-sarcoglycan cDNA (NC_000013.11), a muscle-specific MHCK7 promoter to drive expression, a Kozak consensus sequence (CCACC), a chimeric SV40 intron, a synthetic polyadenylation site (53 bp) (Fig. 1). The SGCG expression cassette is cloned between the AAV2 ITR and packaged into the self-complementary (sc) vector AAVrh.74 for enhanced transduction of cardiac tissue.

An overview of the study design is provided in Table. 2. Dose values are determined by qPCR-based estimation of the total number of vector genomes (vg). Inclusion of at least some partially complete AAV capsids during vector production may result in overestimation of dose by qPCR-based methods. Thus, determining efficacy at a given dose (eg, 5E+13) suggests that efficacy may be observed at a lower qPCR-measured vector dose when the vector is purified to remove partially complete AAV capsids. The total dose (vg) and dose expressed in vector genomes per kilogram of subject (vg/kg) are listed in Table. 2, and throughout the Examples section, do not include partially complete AAV capsids.

- 28 045951

table 2

Review of study design scAAVrh74.MHCK7.hSGCG

Study group Path to get to and Animal line Total dose (vg) Dose (vg/kg) # Mice Final processing point (months) Analysis Activity v.m. SGCG-/- WE+11 n.p. 2 1 IF Activity i.v. SGCG-/- 1E+13 5E+14 1 1.5 IF Efficiency i.v. SGCG-/- 1E+12 5E+13 6 3 IF, H&E, western blottin g, TA Phys, Dia Phys, Cell for activity assessment, Histopa t., Biodistribution qPCR, biochemical. serum analysis

- 29 045951

ki Efficiency i.v. SGCG-/- 4E+12 2E+14 6 3 IF, H&E, western blottin g, TA Phys, Dia Phys, Cell for activity assessment, Histopa t., Biodistribution qPCR, biochemical. serum test Efficiency i.v. SGCG-/- 1E+13 5E+14 5 3 IF, H&E, western blottin g, TA Phys, Dia Phys, Cell for activity assessment, Histopa t., Biodistribution qPCR, biochemical. serum analysis

- 30 045951

ki Efficiency i.v. SGCG-/- 6 IF, H&E, western blottin g, TA Phys, Dia Phys, Cell for assessing activity, Gistopa t., biochemical. serum test Efficiency i.v. C57BL/6 LRS 6 3 IF, H&E, western blottin g, TA Phys, Dia Phys, Cell for assessing activity, Gistopa t., biochemical. serum test

n.p.: not applicable.

IF: immunofluorescence; H&E: hematoxylin and eosin staining; TA Phys: specific force measurements and resistance to ECC damage in TA muscle; Dia Phys: specific force measurements in the diaphragm muscle; Histopath: formal examination of histopathology; --: uninjected.

All animals assessed for efficacy were treated at 4-8 weeks of age and necropsied 3 months after injection. Negative control SGCG −/− mice were necropsied at 4 months of age.

Determination of the activity of the test product scAAVrh.74.MHCK7.hSGCG was achieved by intramuscular and systemic injections of the SGCG-/- vector into mice. Wild-type mice injected with lactated Ringer's solution (LRS) serve as positive controls, and uninjected SGCG −/− mice serve as negative controls.

Tibialis anterior (TA) muscle from 8-week-old BL6 wild-type (WT) and γ-sarcoglycan knockout (γ-SG KO) mice was isolated, and tissue sections were stained with hematoxylin and eosin (H&E) to visualize the histology of each muscle. . Even at this very early age, γ-SG KO mice exhibited a disease phenotype in muscle with necrotic muscle fibers, inflammatory infiltrates, and the presence of fibrous tissue (Fig. 2).

The SGCG AAV construct was packaged into AAV serotype rh.74 to create a recombinant AAV (rAAV) named scAAVrh.74.MHCK7.hSGCG. A total of 3 mice were injected to determine the activity of scAAVrh.74.MHCK7.hSGCG. One LRS-injected C57BL/6 WT mouse and one uninjected SGCG −/− mouse served as positive and negative controls, respectively. The remaining 3 mice were SGCG-/- and received injection or i.m. (intramuscular) in LTA (n is 2), or i.v. (intravenously) into the tail vein (n is 1) scAAVrh.74.MHCK7.hSGCG to determine whether the vector batch is active. The study design is summarized in Table. 3.

- 31 045951

Table 3

Activity assay of scAAVrh.74.MHCK7.hSGCG

Number mice Line mice Injectable substance Dose (total, vg) Delivery way Volume (µl) 1 SGCG-/- n.p. Negative control n.p. n.p. 1 C57BL/6 LRS Positive control i.v. 200 2 SGCG-/- AAV. hSGCG 3x10 ¹¹ vg v.m. thirty 1 SGCG-/- AAV. hSGCG 1x10 ¹³ vg centuries 460 (230/230)

γ-SG KO mice were injected with scAAVrh.74.MHCK7.hSGCG via intramuscular (IM) injection into the TA muscle at 4 weeks of age at a total dose of 3e10 vg. Mice were sacrificed 4 weeks after injection (8 weeks of age), and TA muscle was isolated and fresh frozen in methylbutane cooled with liquid nitrogen. Immunofluorescence (IF) staining for γ-sarcoglycan showed absence of γ-sarcoglycan in injected right TA (RTA) muscle and showed almost complete restoration of γ-sarcoglycan membrane protein expression in injected left TA (LTA) muscle (Fig. 3A). Western blot analysis for γ-sarcoglycan (Fig. 3B) showed γ-sarcoglycan expression in two BL6 WT TA muscles; lack of protein in TA muscle γ-SG KO; and restoration of γsarcoglycan protein expression in TA muscle from injected mice #794 and #795.

Delivery of scAAVrh.74.MHCK7.hSGCG via i.m. administration of SGCG −/− mice at a fixed total dose of 3x1011 vg resulted in 93.03% hSGCG expression in LTA-injected muscles, which is similar to levels of a β-sarcoglycan vector previously studied by us (scAAVrh.74.MHCK7.hSGCB). Immunofluorescence imaging of vector-dosed mice (animal ID: 794, 795) confirmed expression of the hSGCG transgene (Fig. 3A). 20X images are included to visualize the amount of expression in the injected muscle. As expected, the C57BL/6 WT mouse showed 100% γ-sarcoglycan protein expression, and the SGCG −/− mouse completely lacked γ-sarcoglycan expression ( Fig. 3C ).

Systemic tail vein injection of one SGCG −/− mouse (#797) resulted in high levels of hSGCG transgene expression. Applicant was able to achieve 94.00% or higher transduction in all skeletal muscle of a given mouse treated with a total dose of 1x10 ¹³ vg (5x1014 vg/kg) scAAVrh.74.MHCK7.hSGCG for activity assays. The average percentage expression level of the AAV-delivered hSGCG transgene among all skeletal muscles analyzed was 95.98%. Applicant was also able to achieve very high levels of transduction in the heart when delivered systemically. Representative 20X immunofluorescence images of all skeletal muscle along with the diaphragm and heart illustrating the extensive expression of hSGCG are shown in FIG. 7.

Example 2. Activity and toxicity of the scAAVrh74.tMCK.hSGCB vector in BL6 WT mice.

BL6 WT mice were injected with scAAVrh.74.MHCK7.hSGCG via intramuscular (IM) injection into the TA muscle at 4 weeks of age at a total dose of 3e10 vg. Mice were sacrificed 4 weeks after injection (8 weeks of age), and TA muscle was isolated and fresh frozen in methylbutane cooled with liquid nitrogen. Immunofluorescence (IF) staining for γ-sarcoglycan demonstrated membrane staining for γ-sarcoglycan in uninjected right TA (RTA) muscle and demonstrated intracellular staining indicating overexpression of γ-sarcoglycan protein in injected left TA (LTA) muscle (Fig. 4A). Western blot analysis for γ-sarcoglycan (Fig. 4B) showed overexpression of γ-sarcoglycan protein in LTA-injected muscle. H&E staining of TA muscle demonstrated a lack of toxicity with a complete absence of any central nuclei, necrotic fibers, inflammatory infiltration, or fibrous tissue in both uninjected RTA and injected LTA (Fig. 5).

Example 3: Gene expression after systemic delivery of scAAVrh.74.tMCK.hSGCB.

4-5 week old γ-SG KO mice were injected intravenously into the tail vein with a total dose of 1e12 vg (5e13 vg/kg). Mice were sacrificed after 6 weeks of treatment. Immunofluorescent staining on TA, gastrocnemius (GAS), quadriceps (QUAD), gluteus (GLUT), psoas (PSOAS), triceps (TRICEP), diaphragm, and cardiac muscles demonstrated extensive γsarcoglycan expression (Fig. 6).

Determination of the effectiveness of the test product scAAVrh.74.MHCK7.hSGCG was achieved by performing systemic injections of SGCG-/- mice (genotype: sgcgC57) using a single dose (total dose 1x101 ³ vg, 5x1014 vg/kg). Systemic injection of scAAVrh.74.MHCK7.hSGCG at clinical dose (total dose 1x1012 vg (5x ¹⁰¹³ vg/kg)), intermediate dose (total dose 4x1012 vg (2x1014 vg/kg)) and

- 32 045951 high dose (total dose 1x 10 ¹³ vg (5x 10 ¹⁴ vg/kg)) into the tail vein of an SGCG-/- mouse when sacrificed 3 months after injection.

In accordance with the results of the activity analysis of scAAVrh.74.MHCK7.hSGCG by the authors of the invention, the applicant delivered the vector by injection through the tail vein to 5 SGCG-/mice at a total dose of the activity analysis by the authors of the invention 1 x 10 ¹³ vg (5 x 10 ¹⁴ vg/kg) for assessing transgene expression and the effectiveness of the inventors' vector when delivered systemically over an extended period of 3 months. Mice were injected at 4 weeks of age, and complete necropsy was performed 3 months after injection. All skeletal muscles discussed above in the activity analysis along with the diaphragm and heart were isolated for analysis. Organs including lungs, kidneys, liver, spleen and gonads were also removed for toxicology and biodistribution studies. Briefly, expression of the hSGCG transgene remained at high levels after 3 months of treatment, and again all muscles from treated mice were transduced at high levels. This was accompanied by improved muscle histopathology and improved function of the TA muscle and diaphragm. Systemic delivery of the scAAVrh.74.MHCK7.hSGCG vector did not cause any toxicity in muscles or organs.

Expression of γ-Sarcoglycan.

Immunofluorescent staining for human γ-sarcoglycan was used to determine hSGCG transgene expression in six skeletal muscles, both left and right, in addition to the diaphragm and heart of all SGCG −/− mice systemically injected with the scAAVrh.74.MHCK7.hSGCG vector. These muscles included TA, GAS, QUAD, GLUT, PSOAS, TRI. For the purpose of expression analysis and transduction efficiency, images of left and right muscles from 5 treated mice were used for quantification. Four 20X images were taken for each muscle, and the percentage of hSGCG-positive fibers (number of positive expressing fibers/total number of fibers) was determined for each image, providing an average percentage of transduction for each muscle from each mouse. In fig. 8A shows representative images from treated mice and demonstrates high expression levels averaging 92.26% across all muscles quantified, including the diaphragm. The Applicant again also observed high levels of transduction in cardiac muscle in all vector-treated mice. In fig. 8B shows a Western blot that confirms expression of the hSGCG transgene in all skeletal muscle and heart from mice treated with intravenous scAAVrh.74.MHCK7.hSGCG vector. In table Figure 4 lists the average percentage expression across four 20X images for each muscle from each mouse, along with the average for each muscle across all 5 mice.

Table 4

Average percentage expression of γ-Sarcoglycan transgene

Animal ID 5229 5230 5231 5232 5233 Average Muscle TA 97.79 97.79 99.54 99.13 96.59 98.17 G.A.S. 90.88 90.88 74.14 97.67 93.49 89.41 QUAD 88.37 88.37 96.85 95.01 76.15 88.95 GLUT 96.19 96.19 91.12 100 92.83 95.27 PSOAS 97.55 97.55 99.01 49.88 97.92 88.38 TRI 97.32 97.32 99.31 94.98 93.31 96.45 DIA 93.02 93.02 75.56 97.14 87.22 89.19

Histopathology of the treated muscle.

Muscle from SGCG −/− mice, both skeletal and cardiac, exhibit extensive myopathy, including severe atrophy and hypertrophy of muscle fibers with multiple foci of necrosis. There is also an increase in the number of inflammatory mononuclear cells (lymphocytes and macrophages, with scattered neutrophils) and increased dystrophic calcification, fatty infiltration, central nucleation and fibrosis. Hematoxylin and eosin staining in Fig. 9A illustrates this dystrophic phenotype in SGCG -/- mice when compared with normal WT mice and improvement in muscle pathology after treatment. Quantification of histological parameters shows a significant increase in the number of centrally nucleated fibers in the skeletal muscle of SGCG −/− mice, followed by a decrease in central nucleation in numerous different skeletal muscles as a result of γ-sarcoglycan gene transfer (Fig. 9B). A more in-depth analysis of the histopathology of the muscle

- 33 045951 shows normalization of fiber size distribution along with an increase in mean fiber diameter in vector-treated SGCG-/- mice in all three muscles tested (GAS, PSOAS and TRI) (FIGS. 10A-10F). The number of individual central nuclei and average fiber diameters for different muscles were analyzed from each mouse.

Example 4. Physiological deficiency in γ-SG KO mice.

Sirius red staining will be performed to quantify the amount of fibrous tissue. γ-SG KO mice and BL6 WT mice will be tested at 4 months of age to assess whether there are strength deficits in skeletal muscle. The tibialis anterior (TA) muscle will be tested for a significant reduction in specific force and resistance to damage compared to controls. The diaphragm muscle will also be tested in a similar manner to detect any significant decreases. This measurable reduction will provide a measure of functional disease outcome in order to establish the effectiveness of AAV.hSGCB-based therapy.

Example 5. Functional outcome of the disease after treatment with scAAVrh74.tMCK.hSGCB.

Cohorts of γ-SG KO mice will be injected on a chronic basis over three-month studies to quantify efficacy and toxicity (Table 5). Mice will undergo a cage assay before sacrifice to determine the overall activity of treated mice compared to γ-SG KO controls. The TA muscle and diaphragm muscle will be subjected to physiology analysis to determine specific strength and resistance to injury/fatigue. γ-SG KO muscles will be compared to BL6 WT controls to establish measures of functional disease outcome that can be used to determine treatment efficacy in treated mice. All skeletal muscle will be subjected to IF (immunofluorescence) staining for γ-sarcoglycan expression, H&E staining for histopathology. Quantitative polymerase chain reaction (qPCR) will be performed on muscles and organs from injected mice to determine the biodistribution of the vector genome.

Table 5

Line mice Product being tested Human dose Sample size End point C57/BL6 LR* n.p. 6 12 weeks SGCG KO LR n.p. 6 12 weeks SGCG KO scAAVrh74.MHCK7.SGCG 5x10 ¹³ vg/kg 6 12 weeks SGCG KO scAAVrh74.MHCK7.SGCG 1 x 10 ¹⁴ vg/kg 6 12 weeks SGCG KO scAAVrh74.MHCK7.SGCG 2 x 10 ¹⁴ vg/kg 6 12 weeks

Example 6: Functional assessment of system delivery.

To determine whether hSGCG gene transfer provides a functional benefit to diseased muscle, we assessed the functional properties of TA muscle and diaphragm muscle from SGCG-/- mice treated with scAAVrh.74.MHCK7.hSCGG. In accordance with Examples 1-5, the applicant first demonstrated histopathology in the skeletal muscle of the limb and diaphragms of mice in the absence of γ-sarcoglycan. In situ analysis of TA muscle from untreated SGCG-/- mice revealed a statistically significant reduction of 37.68% in normalized specific force generation compared to TA muscle from BL6 WT (BL6 WT: 291.65 mN/ ^mm2 vs SGCG -/-: 181.77 mN/mm ² ). Specific force results were significantly increased to normal WT levels compared to SGCG-/muscle after treatment (SGCG-/-: 181.77 mN/ ^mm2 , vs. treated: 266.02 mN/ ^mm2 ) (Fig. 11A and 11C). One additional measure of functional outcome to determine the functional benefit of hSGCG gene transfer is to assess resistance to contraction injury in the TA muscle after repeated eccentric contractions. The TA muscle of normal BL6 WT untreated SGCG-/- mice lost only 18% of force generation after a cycle of 10 eccentric contractions, compared to a 37% loss of force in untreated SGCG-/-TA muscle. Vector-treated SGCG-/- muscles had improvement to the above WT levels, where only a 10% loss of strength was observed following the eccentric contraction (ECC) protocol (Figure 11B).

To further analyze the possible functional benefit derived from systemic delivery of the hSGCG therapeutic transgene and ultimately improving the disease phenotype of SGCG −/− mice, laser activity monitoring was performed in an open field chamber in all groups of mice. In the graph in Fig. Figure 12 depicts a 23.64% decrease in total x- and y-plane locomotion in SGCG-/- mice compared to normal BL6 WT (BL6 WT: 7655.42 ray interruptions/h, vs. SGCG-/-: 5846 .00 beam interruptions/hour). scAAVrh.74.MHCK7.hSGCG-treated mice were generally more active compared to SGCG-/- mice by quantitative observation, and quantitative activity measurement in the open field chamber showed a 24.90% increase in overall ambulation ability (SGCG-/- -: 5846.00 beam interruptions/hour, compared to treated: 7301.80 beam interruptions/hour). The exact values for each parameter are available for individual

- 34 045951 mice were also measured.

Example 7. Toxicology and biodistribution of the vector.

The purpose of this study was to evaluate any potential toxicity or safety concerns of hSGCG gene therapy in SGCG −/− mice 3 months after delivery of scAAVrh.74.MHCK7.hSGCG test product using the same animals described above. The test product was administered to 5 SGCG-/- at a total dose of 1.0 x 10 ¹³ vg (5 x 10 ¹⁴ vg/kg) by intravenous (i.v.) route in a volume of 460 μl, divided into two separate injections of 230 μl, morning and afternoon, at the age of 4 weeks. Six SGCG −/− mice served as untreated disease controls, and 5 C57BL/6 WT mice served as normal healthy controls (Table 6). Complete necropsies were performed on all mice to isolate six skeletal muscles (TA, GAS, QUAD, GLUT, PSOAS, and TRI) on both the left and right sides, along with the diaphragm and heart, as well as internal organs including lungs, kidneys, and liver. , spleen and gonads. To assess the safety of the vector of the inventors, hematoxylin and eosin staining was performed on cryosections of muscle tissue, and all selected organs were fixed in formaldehyde and also stained with hematoxylin and eosin. These sections were then formally tested for toxicity by an independent veterinary pathologist and no harmful effects were detected and the results are summarized below in Table 1. 7, and a detailed histopathological report was also obtained. Quantitative PCR was performed to assess the biodistribution of the vector, and these results are shown in Table 1 below. 7 and fig. 13.

Histopathological analysis of vector-transduced tissue.

To determine the safety and toxicology profile of scAAVrh.74.MHCK7.hSGCG using systemic delivery, all skeletal muscle, including the diaphragm, along with the heart and five other organs selected from a group of vector-dosed SGCG-/- mice and controls from This preclinical study was stained with H&E and sections of each tissue were formally reviewed by an independent veterinary pathologist. Details regarding groups and study design are shown in Table. 6.

Table 6

Summary of cohorts for histopathological overview of scAAVrh.74.MHCK7.hSGCG gene transfer

Genotype Cohort Dose (vg) Floor Age at injection Age at autopsy Time of processing 1 SGCG-/- Testable product 1,ΟχΊΟ ¹³ women sk II 1 month 4 months 3 months women sk II 1 month 4 months 3 months feminine II 1 month 4 months 3 months feminine II 1 month 4 months 3 months feminine II 1 month 4 months 3 months 2 BL6 WT Control media LRS male oh 1 month 4 months 3 months male oh 1 month 4 months 3 months male oh 1 month 4 months 3 months male oh 1 month 4 months 3 months male oh 1 month 4 months 3 months 3 SGCG-/- Disease control n.p. feminine II n.p. 4 months n.p. feminine II n.p. 4 months n.p. male oh n.p. 4 months n.p. male oh n.p. 4 months n.p.

- 35 045951

Briefly, v.v. injection of scAAVrh.74.MHCK7.hSGCG did not cause any microscopic changes in muscle fibers of any of the skeletal muscles tested (Table 7). In addition, no treatment-related focal lesions were observed in any of the tissues evaluated histologically, indicating that the test product was well tolerated, see Appendix J for the full report (Report No. AAVrh74-SGCG-MOUSE-001.1). Any observed changes were seen in both treated and control mice and were considered to be due to chance. In addition, an independent review found that, relative to reference samples from control mice, administration of test product scAAVrh.74.MHCK7.hSGCG substantially reduced muscle fiber atrophy, degeneration, and destruction, suggesting that the vector may improve the degree of myopathy associated with lack of SGCG in diseased mice.

Table 7

Histopathology results from the safety study of scAAVrh.74.MHCK7.hSGCG in SGCG-/- mice

Product tested (vector dose) Age at injection Processing time Analyzed tissues Official histopath. scAAVrh.74.MHCK7.hS GCG (total dose 1e13vg - 5e14vg/Kr) 5 analyzed animals 1 month 3 months Skeletal muscles, heart, lungs, kidney, liver, spleen, gonads No data

Biodistribution of the vector genome.

The presence of DNA sequences specific to the test product was examined using a quantitative real-time PCR (qPCR) assay. Biodistribution analysis was performed on tissue samples collected from two vector-dosed SGCG −/− animals. A positive signal is equal to or greater than 100 detected ssDNA copies/µg genomic DNA. Tissues were selected at autopsy, and vector-specific primer-probe sets specific to MHCK7 promoter sequences were used. In table 8 and FIG. 13 depicts copies of the vector genome detected in each tissue sample from mice injected with scAAVrh.74.MHCK7.hSGCG.

The scAAVrh.74.MHCK7.hSGCG transcript was detected at varying levels in all tissues sampled. As expected, while the vector was detected at high levels in the liver due to the nature of the intravenous delivery route, the highest levels were observed in skeletal muscle and heart. The lowest levels were detected in the lungs, kidney and spleen. These data indicate that the test product was effectively delivered to all tissues tested in vector-dosed mice.

Table 8

qPCR results after high dose systemic delivery of scAAVrh.74.MHCK7.hSGCG

SGCG-/- mice

Textile Vector genome copies/µg 5229 5231 Heart 1.56E+06 1.30E+06 Lung 1.15E+05 2.29E+05 Bud 1.74E+05 2.48E+05 Liver 9.23E+06 1.50E+07 Spleen 1.58E+05 9.05E+04 Diaphragm 4.63E+05 1.60E+06 TRI 3.35E+05 3.48E+05 TA 6.23E+05 6.70E+05

Biochemical analysis of serum.

To further evaluate liver function, the applicant assessed the levels of two liver enzymes that are normal serum biochemical assay parameters, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Evaluation of any of these enzymes may indicate hepatocyte damage and impaired liver function. Applicant analyzed sera from all 6 C57BL/6 WT mice, all 6 untreated SGCG-/- mice and all 5 scAAVrh.74.MHCK7.hSGCG-dosed mice. In fig. 14A shows an increase in ALT levels in untreated SGCG-/- mice with double the levels observed in healthy BL6 WT mice (BL6 WT: 44.20 U/L in

- 36 045951 compared with SGCG-/-: 89.00 units/l). V.v. delivery of scAAVrh.74.MHCK7.hSGCG to SGCG-/- mice resulted in a 32.02% decrease in ALT levels (SGCG-/-: 89.00 U/L vs. treated: 60.50 U/L). In fig. 14B shows AST levels in all three groups of mice, showing a significant 113.27% increase in untreated SGCG-/- mice (BL6 WT: 326.00 U/L vs. SGCG-/-: 695.25 U/L ). These AST levels were reduced by 41.10% after systemic delivery of scAAVrh.74.MHCK7.hSGCG (Fig. 14B). In general, while levels of liver enzymes, considered biomarkers of liver injury, are elevated in diseased SGCG −/− mice, systemic transfer of the hSGCG gene in diseased SGCG −/− mice normalizes both ALT and AST levels. Separate values for each enzyme were determined for all mice.

In conclusion, systemic delivery of two different doses of AAV virus carrying the hSGCB transgene was shown to be safe and nontoxic. The doses tested included a total dose of 1.2 x 10 ¹³ vg (6.0 x ^{10 14} vg/kg) and a total dose of 1.0 x 10 ¹³ vg (5.0 x ^{10 14} vg/kg). In particular, high dose systemic delivery (total dose 1.0x 10 ¹³ vg - 5.0x 10 ¹⁴ vg/kg) of scAAVrh.74.MHCK7.hSGCG via the tail vein of SGCG-/ is safe and effective in restoring γ-sarcoglycan expression and relief of dystophic histopathology in the muscle affected by the disease.

Sequence listing <110> RESEARCH INSTITUTE AT NATIONWIDE CHILDREN'S HOSPITAL <120> GENE THERAPY AGAINST PELOVIC-BREAKAL MUSCULAR DYSTROPHY TYPE 2C <130> 106887-7141 <140>

<141>

<150> 62/624,616 <151> 2018-01-31 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 876 <212> DNA <213> Artificial Sequence <220>

<223> Description of the artificial sequence: synthetic polynucleotide <400> 1

atggtgagg agcagtacac cacagcaacc gagggaatct gcatcgagag gccagagaac 60 cagtacgtgt aaagatcgg catctacggc tggcggaaga gatgtctgta tctgttcgtg 120 ctgctgctgc tgatcatcct ggtggtgaat ctggccctga ccatctggat cctgaaagtg 180 atgtggtttt ccccaggcagg aatggacac ctgtgcgtga caaaggacgg actgcggctg 240 gagggagagt ctgagttcct gtttcccctg tatgccaagg agatccacag cagagtggat 300 agctccctgc tgctgcagtc cacccagaac gtgacagtga acgcaaggaa tagcgaggga 360 gaggtgaccg gcagactgaa ggtcggcccc aagatggtgg aggtgcagaa tcagcagttc 420 cagatcaact ccaatgacgg caagcctctg tttacagtgg atgagaagga ggtggtggtg 480 ggcaccgaca agctgaggt gacaggacct gagggcgccc tgttcgagca ctctgtggag 540 accccactgg tgcgcgcaga cccttttcag gatctgaggc tggagagccc aacacgcagc 600

- 37 045951 ctgtccatgg ctgagccaga accgtgtgcc ctgtacgaga accacatgcc acgcacccag tggatatcct tgcctaagct tctgcgtgtg aggagcacaa aggcgtgcac gttccactct ggtgcagggc cccagatggc ccacatctgt atccaggcac agcgacggca a catggggcc aagctgtatc ctgtga acgcaggcaa tgctggtgct catctggctc tgtccgtggc gatcgaggcc ggatgccgag ctctcagagc cggcgtgtct

660

720

780

840

876 <210> 2 <211> 291 <212> PRT <213> Homo sapiens <400> 2 Met Val 1

Arg Glu

Gln Tyr 5

Thr Thr

Ala Thr

Glu Gly

Ile Cys

Arg Pro

Glu Asn

Gln Tyr

Val Tyr

Lys Ile 25

Gly Ile

Tyr Gly 30

Lys Arg

Cys Leu 35

Tyr Leu

PheVal

Leu Leu

Leu Leu

Ile Ile 45

Val Asn

Leu Ala

Leu Thr

Ile Trp 55

Ile Leu

Lys Val

Met Trp

Ile Glu 15

Trp Arg

Leu Val

Phe Ser

Pro Ala 65

Gly Met

Gly His 70

Leu Cys

Val Thr

Lys Asp 75

Gly Leu

Arg Leu

Glu Gly

Glu Ser

Glu Phe 85

Leu Phe

ProLeu

Tyr Ala

Lys Glu

Ile His 95

Ser Arg

Val Asp

100

Ser Ser

Leu Leu

Leu Gln 105

Ser Thr

Gln Asn

110

Val Thr

Val Asn

Ala Arg 115

Asn Ser

Glu Gly

120

Glu Val

Thr Gly

Arg Leu 125

Lys Val

Gly Pro

130

Lys Met

Val Glu

Val Gln 135

Asn Asp 145

Gly Lys

ProLeu

150

Phe Thr

Gly Thr

Asp Lys

Leu Arg 165

Val Thr

Asn Gln

Val Asp

Gly Pro

170

Arg Ala

Gln Phe

140

Gln Ile

Asn Ser

Glu Lys 155

Glu Val

Val Val

160

Glu Gly

Ala Leu

Phe Glu 175

His Ser

Val Glu

Thr Pro

Leu Val

Asp Pro

Phe Gln

Asp Leu

- 38 045951

180 185190

Arg Leu Glu Ser Pro Thr Arg Ser Leu Ser Met Asp Ala Pro Arg Gly

195 200205

Val His Ile Gln Ala His Ala Gly Lys Ile Glu Ala Leu Ser Gln Met

210 215220

Asp Ile Leu Phe His Ser Ser Asp Gly Met Leu Val Leu Asp Ala Glu

225 230 235240

Thr Val Cys Leu Pro Lys Leu Val Gln Gly Thr Trp Gly Pro Ser Gly

245 250255

Ser Ser Gln Ser Leu Tyr Glu Ile Cys Val Cys Pro Asp Gly Lys Leu

260 265270

Tyr Leu Ser Val Ala Gly Val Ser Thr Thr Cys Gln Glu His Asn His

275 280285

Ile Cys Leu

290 <210> 3 <211> 2225 <212> DNA <213> Artificial Sequence <220>

<223> Description of the artificial sequence: synthetic polynucleotide <400> 3

ctgcgcgctc gctcgctcac tgaggccgcc cggggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggggtt aaccaattgg 120 cgcggccgca agcttgcatg tctaagctag acccttcaga ttaaaaataa ctgaggtaag 180 ggcctgggta ggggaggtgg tgtgagacgc tcctgtctct cctctatctg cccacggcc 240 ctttggggag gaggaatgtg cccaaggact aaaaaaaggc catggagcca gagggcgag 300 ggcaacagac ctttcatgggg caaaccttgg ggccctgctg tctagcatgc cccactacgg 360 gtctaggctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc tggttataat 420 taaccagac atgtggctgc cccccccccc ccaacacctg ctgcctctaa aaataaccct 480 gtccctggtg gatcccctgc atgcgaagat cttcgaacaa ggctgtgggg gactgaggc 540 aggctgtaac aggcttgggg gccaggggctt atacgtgcct gggactccca aagtattact 600 gttccatgtt cccggcgaag ggccagctgt cccccgccag ctagactcag cacttagttt 660

- 39 045951 aggaaccagt gagcaagtca gcccttgggg cagcccatac aaggccatgg ggctgggcaa720 gctgcacgcc tgggtccggg gtgggcacgg tgcccgggca acgagctgaa agctcatctg780 ctctcagggg cccctccctg gggacagccc ctcctggcta gtcacaccct gtaggctcct840 ctatataacc caggggcaca ggggctgccc tcattctacc accacctcca cagcacagac900 agacactcag gagcagccag cggcgcgccc aggtaagttt agtctttttg tcttttattt960 caggtcccgg atccggtggt ggtgcaaatc aaagaactgc tcctcagtgg at gttgcctt1020 tacttctagg cctgtacgga agtgttactt ctgctctaaa agctgcggaa ttgtacccgg1080 taccaccatg gtgagggagc agtacaccac agcaaccgag ggaatctgca tcgagaggcc1140 agagaaccag tacgtgtata agatcggcat ctacggctgg cggaagagat gtctgtatct1200 gttcgtgctg ctgctgctga tcatcctggt ggtgaatctg gccctgacca tctggatcct1260 gaaagtgatg tggttttccc cagcaggaat gggacacctg t gcgtgacaa aggacggact1320 gcggctggag ggagagtctg agttcctgtt tcccctgtat gccaaggaga tccacagcag1380 agtggatagc tccctgctgc tgcagtccac ccagaacgtg acagtgaacg caaggaatag1440 cgagggaatag gtgaccggca gactgaaggt cggcccca ag atggtggagg tgcagaatca1500 gcagttccag atcaactcca atgacggcaa gcctctgttt acagtggatg agaaggaggt1560 ggtggtgggc accgacaagc tgaggtgac aggacctgag ggcgccctgt tcgagcactc1620 tgtggagacc ccactggtgc gcgcagaccc ttttcaggat ctgaggctgg agagcccaac1680 acgcagcctg tccatggacg cacccagagg cgtgcacatc caggcacacg caggcaagat1740 cgaggccctg agccagatgg atatcctgtt ccactctagc gacggcatgc tggtgctgga1800 tgccgag acc gtgtgcctgc ctaagctggt gcagggcaca tggggcccat ctggctcctc1860 tcagagcctg tacgagatct gcgtgtgccc agatggcaag ctgtatctgt ccgtggccgg1920 cgtgtctacc acatgccagg agcacaacca catctgtctg tgactcgagg gccgcaataa1980 aagatcttta ttttcattag atctgtgtgt tggttttttg tgtgtcctgc aggggcgcgc2040 ctaatctaga gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg2100 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg2160 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag2220 cgcgc2225 <210> 4 <211> 792 <212> DNA <213> Artificial Sequence <220>

<223> Description of artificial sequence: synthetic polynucleotide

- 40 045951 <400> 4 aagcttgcat gtctaagcta gacccttcag attaaaaata actgaggtaa gggcctgggt60 aggggaggtg gtgtgagacg ctcctgtctc tcctctatct gcccatcggc cctttgggga120 ggaggaatgt gcccaaggac taaaaaaagg ccatggagcc agaggggcga gggcaacaga180 cctttcatgg gcaaaccttg gggccctgct gtctagcatg ccccactacg ggtctaggct240 gcccatgtaa ggaggcaagg cctggggaca cccgagatgc ctggttataa ttaacccaga300 catgtggctg cccccccccc cccaacacct gctgcctcta a aaataaccc tgtccctggt360 ggatcccctg catgcgaaga tcttcgaaca aggctgtggg ggactgaggg caggctgtaa420 caggcttggg ggccagggct tatacgtgcc tgggactccc aaagtattac tgttccatgt480 tcccggcgaa gggccagctg tcccccgcca gctagactca gcacttagtt taggaaccag540 tgagcaagtc agcccttggg gcagcccata caaggccatg gggctgggca agctgcacgc600 ctgggtccgg ggtgggcacg gtgcccgggc aacgagctga aagctcatct gctctcaggg660 gcccctccct ggggacagcc cctcctggct agtcacaccc tgtaggctcc tctatataac720 ccaggggcac aggggctgcc ctcattctac caccacctcc acagcacaga cagacactca780 ggagcagcca gc792 <210 > 5 <211> 148 <212> DNA < 213> Artificial Sequence <220>

<223> Description of artificial sequence: synthetic polynucleotide <400> 5 aggtaagttt agtctttttg tcttttattt caggtcccgg atccggtggt ggtgcaaatc60 aaagaactgc tcctcagtgg atgttgcctt tacttctagg cctgtacgga agtgttactt120 ctgctctaaa agct gcggaa ttgtaccc148 <210> 6 <211> 53 <212> DNA <213> Artificial Sequence <220>

<223> Description of the artificial sequence: synthetic oligonucleotide <400> 6 ggccgcaata aaagatcttt attttcatta gatctgtgtg ttggtttttt gtg 53 <210> 7 <211> 110

- 41 045951 <212> DNA <213> Artificial Sequence <220>

<223> Description of artificial sequence: synthetic polynucleotide <400> 7 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggggtt

110 <210> 8 <211> 104 <212> DNA <213> Artificial Sequence <220>

<223> Description of artificial sequence: synthetic polynucleotide <400> 8

ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 60 gccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgc 104 <210> 9 <211> 876 <212> DNA <213> Homo sapiens <400> 9 atggtgcgtg agcagtacac tacagccaca gaaggcatct gcatagag gccagagaat 60 cagtatgtct acaaaattgg catttatggc tggagaaagc gctgtctcta cttgtttgtt 120 cttcttttac tcatcatcct cgttgtgaat ttagctctta caatttggat tcttaaagtg 180 atgtggtttt ctccagcagg aatggggccac ttgtgtgtaa caaaagatgg actgcgcttg 240 gaaggggaat cagaattttt attcccattg tatgccaaag aaatacactc cagagtggac 300 tcatctctgc tgctacaatc aacccagaat gtgactgtaa atgcgcgcaa ctcagaaggg 360 gaggtcacag gcaggttaaa agtcggtccc aaaatggtag aagtccagaa tcaacagttt 420 cagatcaact ccaacgacgg caagccacta tttactgtag atgagaagga agttgtggtt 480 ggtacagata aacttcgagt aactggggcct gaaggggctc tttttgaaca ttcagtggag 540 acaccccttg tcagagccga cccgtttcaa gaccttagat tagaatcccc cactcggagt 600 ctaagcatgg atgccccaag gggtgtgcat attcaagctc acgctgggaa aattgaggcg 660 ctttctcaaa tggatattct ttttcatagt agtgatggaa tgcttgtgct tgatgctgaa 720 actgtgtgct tacccaagct ggtgcagggg acgtggggtc cctctggcag ctcacagagc 780 ctctacgaaa tctgtgtgtg tccagatgggg aagctgtacc tgtctgtggc cggtgtgagc 840 accacgtgcc aggagcacag ccacatctgc ctctga 876

- 42 045951 <210> 10 <211> 535 <212> PRT <213> Artificial Sequence <220>

<223> Description of artificial sequence: synthetic polypeptide <400> 10

Met Ala Ala Gly Gly Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala

5 1015

Asp Gly Val Gly Ser Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp 20 2530

Leu Gly Asp Arg Val Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro 35 4045

Thr Tyr Asn Asn His Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly 50 5560

Gly Ser Thr Asn Asp Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly 65 70 7580

Tyr Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp 85 9095

Gln Arg Leu Ile Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn

100 105110

Phe Lys Leu Phe Asn Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly

115 120125

Thr Lys Thr Ile Ala Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr

130 135140

Asp Ser Glu Tyr Gln Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly

145 150 155160

Cys Leu Pro Pro Phe Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly

165 170175

Tyr Leu Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe

180 185190

Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn

195 200205

Phe Glu Phe Ser Tyr Asn Phe Glu Asp Val Pro Phe His Ser Ser Tyr

- 43 045951

210

215

220

Ala His Ser Gln Ser 225

Leu Asp Arg Leu Met 230

Asn Pro Leu Ile Asp 235

Tyr Leu Tyr Tyr Leu

245

Ser Arg Thr Gln Ser

250

Thr Gly Gly Thr Ala

255

Thr Gln Gln Leu Leu

260

Phe Ser Gln Ala Gly

265

Pro Asn Asn Met Ser

270

Gln Ala Lys Asn Trp 275

Leu Pro Gly Pro Cys 280

Tyr Arg Gln Gln Arg

285

Ser Thr Thr Leu Ser

290

Gln Asn Asn Asn Ser

295

Asn Phe Ala Trp Thr

300

Ala Thr Lys Tyr His 305

Leu Asn Gly Arg Asp 310

Ser Leu Val Asn Pro 315

Val Ala Met Ala Thr

325

His Lys Asp Asp Glu

330

Glu Arg Phe Phe Pro

335

Ser Gly Val Leu Met

340

Phe Gly Lys Gln Gly

345

Ala Gly Lys Asp Asn

350

Asp Tyr Ser Ser Val 355

Met Leu Thr Ser Glu

360

Glu Glu Ile Lys Thr 365

Asn Pro Val Ala Thr

370

Glu Gln Tyr Gly Val

375

Val Ala Asp Asn Leu

380

Gln Gln Asn Ala Ala 385

Pro Ile Val Gly Ala 390

Val Asn Ser Gln Gly 395

Leu Pro Gly Met Val

405

Trp Gln Asn Arg Asp

410

Val Tyr Leu Gln Gly

415

Ile Trp Ala Lys Ile

420

Pro His Thr Asp Gly

425

Asn Phe His Pro Ser

430

Leu Met Gly Gly Phe

435

Gly Leu Lys His Pro 440

Pro Pro Gln Ile Leu

445

Lys Asn Thr Pro Val

450

Pro Ala Asp Pro Pro 455

Thr Thr Phe Asn Gln

460

Gln 240

Gly

Ala

Val

Gly

Gly 320

Ser

Val

Thr

Gln

Ala 400

Pro

Pro

Ile

Ala

- 44 045951

Lys 465

Leu

Ala

Ser

Phe

Ile

470

Thr

Gln

Tyr

Ser

Thr

475

Gly

Gln

Val

Ser

Val

480

Glu

Ile

Glu

Trp

Glu

485

Leu

Gln

Lys

Glu

Asn

490

Ser

Lys

Arg

Trp

Asn

495

Pro

Glu

Ile

Gln

Tyr 500

Thr

Ser

Asn

Tyr

Tyr

505

Lys

Ser

Thr

Asn

Val

510

Asp

Phe

Ala

Val

Asn

515

Thr

Glu

Gly

Thr

Tyr 520

Ser

Glu

Pro

Arg

Pro

525

Ile

Gly

Thr

Arg

Tyr 530

Leu

Thr

Arg

Asn

Leu

535 <210> 11 <211> 2217 <212> DNA <213> Artificial Sequence <220>

<223> Description of artificial sequence: synthetic polynucleotide <400> 11

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg acctgaaacc tggagccccg aaacccaaag ccaaccagca aaagcaggac 120 aacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctcc aagcggtga caatccgtac ctgcggtata atcacgccga cgccgagttt 300 caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgcgc agtcttccag 360 gccaaaaagc gggttctcga acctctggggc ctggttgaat cgccggttaa gacggctcct 420 ggaaagaaga gaccggtaga gccatcaccc cagcgctctc cagactcctc tacggggcatc 480 ggcaagaaag gccagcagcc cgcaaaaaag agactcaatt ttggggcagac tggcgactca 540 gagtcagtcc ccgaccctca accaatcgga gaaccaccag caggcccctc tggtctggga 600 tctggtacaa tggctgcagg cggtggcgct ccaatggcag acaataacga aggcgccgac 660 ggagtgggta gttcctcagg aaattggcat tgcgattcca catggctggg cgacagagtc 720 atcaccacca gcacccgcac ctggggccctg cccacctaca acaaccacct ctacaagcaa 780 atctccaacg ggacctcgg aggaagcacc aacgacaaca cctacttcgg ctacagcacc 840 ccctggggggt attttgactt caacagattc cactgccact tttcaccacg tgactggcag 900 cgactcatca acaacaactg gggattccgg cccaagaggc tcaacttcaa gctcttcaac 960 atccaagtca aggaggtcac gcagaatgaa ggcaccaaga ccatcgccaa taaccttacc 1020

-

Claims (10)

agcacgattc aggtctttac ggactcggaa taccagctcc cgtacgtgct cggctcggcg 1080 caccagggct gcctgcctcc gttcccggcg gacgtcttca tgattcctca gtacgggtac 1140 ctgactctga acaatggcag tcaggctgtg ggccggtcgt ccttctactg cctggagtac 1200 tttccttctc aaatgctgag aacggggcaac aactttgaat tcagctacaa cttcgaggac 1260 gtgcccttcc acagcagcta cgcgcacagc cagagcctgg accggctgat gaaccctctc 1320 atcgaccagt acttgtacta cctgtcccgg actcaaagca cggggcggtac tgcaggaact 1380 cagcagttgc tattttctca ggccggggcct aacaacatgt cggctcaggc caagaactgg 1440 ctacccggtc cctgctaccg gcagcaacgc gtctccacga cactgtcgca gaacaacaac 1500 agcaactttg cctggacgggg tgccaccaag tatcatctga atggcagaga ctctctggtg 1560 aatcctggcg ttgccatggc tacccacaag gacgacgaag agcgattttt tccatccagc 1620 ggagtcttaa tgtttgggaa acagggagct ggaaaagaca acgtggacta tagcagcgtg 1680 atgctaacca gcgaggaaga aataaagacc accaaccag tggccacaga acagtacggc 1740 gtggtggccg ataacctgca acagcaaaac gccgctccta ttgtagggggc cgtcaatagt 1800 caaggagcct tacctggcat ggtgtggcag aaccgggacg tgtacctgca gggtccatc 1860 tggggccaaga ttcctcatac ggacggcaac tttcatccct cgccgctgat gggaggcttt 1920 ggactgaagc atccgcctcc tcagatcctg attaaaaaca cacctgttcc cgcggatcct 1980 ccgaccacct tcaatcaggc caagctggct tctttcatca cgcagtacag taccggccag 2040 gtcagcgtgg agatcgagtg ggagctgcag aaggagaaca gcaaacgctg gaacccag 2100 attcagtaca cttccaacta ctacaaatct acaaatgtgg actttgctgt caatactgag 2160 ggtacttatt ccgagcctcg ccccattggc acccgttacc tcacccgtaa tctgtaa 2217 CLAIM 1. Use of a recombinant vector based on adeno-associated virus (AAV) to obtain a drug for the treatment of γ-sarcoglycanopathy; increasing muscle strength, muscle endurance and/or muscle mass; or to reduce fibrosis or contraction-induced damage in a subject, wherein the rAAV vector contains a polynucleotide sequence that contains the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 2. Use of a recombinant adeno-associated virus (AAV) vector for the production of a medicament for the treatment of γ-sarcoglycanopathy, wherein the rAAV vector contains a polynucleotide sequence that contains a nucleotide sequence encoding a γ-sarcoglycan that contains the amino acid sequence of SEQ ID NO: 2. 3. Use of a recombinant adeno-associated virus (AAV) vector for the production of a medicament for reducing fatty infiltration or central nucleation in a subject, wherein the rAAV vector contains a polynucleotide sequence that contains the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 4. Use according to any one of claims 1 to 3, wherein the polynucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1. 5. Use according to any one of claims 1 to 4, wherein the rAAV vector contains a self-complementary genome of the AAV vector. 6. Use according to any one of claims 1 to 5, wherein the rAAV vector contains a genome free of rep and cap AAV DNA. 7. Use according to any one of claims 1 to 6, where the rAAV vector belongs to the serotype AAV1, AAV2, - 46 045951 AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV rh74 or a variant thereof. 8. Use according to claim 6, wherein said rAAV vector contains an AAV rh.74 capsid. 9. Use according to claim 8, wherein the AAV rh.74 capsid contains the amino acid sequence set forth in SEQ ID NO: 10. 10. Use according to any one of claims 1 to 9, wherein the genome of the rAAV vector contains a muscle-specific regulatory element, optionally selected from the group consisting of a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor element ( mef), muscle creatine kinase (MCK) promoter, truncated MCK promoter (tMCK), myosin heavy chain (MHC) element, MHCK7 promoter, C5-12, mouse creatine kinase enhancer element, fast twitch skeletal muscle troponin gene element, troponin gene element slow-twitch cardiac muscle, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factor element, steroid-inducible element, and