EA044609B1 - DELIVERY OF BETA-SARCOGLYCAN AND MICRORNA-29 BY VECTOR BASED ON ADEN-ASSOCIATED VIRUS AND TREATMENT OF MUSCULAR DYSTROPHY - Google Patents

Description

This invention claims priority to US Provisional Application No. 62/323333, filed April 2016, and US Provisional Application No. 62/433548, filed December 13, 2016, both of which are incorporated herein by reference in their entirety.

Incorporation by reference of materials submitted electronically

This application contains as a separate part of the disclosure a sequence listing in machine-readable form, which is incorporated herein by reference in its entirety and is identified as: file name - 50622A_SeqListing.txt; size - 21466 bytes; created - April 13, 2017.

Field of invention

Disclosed herein are therapeutic vectors, such as AAV (adeno-associated virus) vectors expressing β-sarcoglycan, and methods of using these vectors to reduce and prevent fibrosis in subjects suffering from muscular dystrophy. The present invention also provides combination gene therapy methods comprising administration of a first AAV vector expressing β-sarcoglycan and a second AAV vector expressing miR-29 to reduce and prevent fibrosis in patients suffering from muscular dystrophy.

State of the art

Limb-girdle muscular dystrophy (LGMD) type 2E (LGMD2E) is an autosomal recessive disorder caused by mutations in the gene encoding β-sarcoglycan (SGCB), causing loss of functional protein (1). LGMD2E is a relatively common and severe form of LGMD in the United States, with a worldwide incidence of 1/200,000 to 1/350,000 (2). The absence of β-sarcoglycan leads to progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis, leading to deterioration of muscle strength and function (3, 4). As a complex, sarcoglycans (α-, β, γ-, δ-), varying in size from 35 to 50 kDa (5), are all transmembrane proteins that provide stability to the sarcolemma, providing protection from mechanical stress during muscle activity ( 3). Loss of β-sarcoglycan in LGMD2E typically results in varying degrees of concomitant loss of other sarcoglycan proteins, rendering the muscle membrane weak, resulting in muscle fiber loss (1). Although the range of time to onset of the clinical phenotype of LGMD2E varies, diagnosis is usually made before age 10 years, and loss of ambulation occurs during mid to late adolescence (1, 6, 7). Patients have elevated serum creatine kinase (CK), proximal muscle weakness, difficulty getting up from the floor, and progressive loss of ambulation. Fifty percent of cases involve heart problems.

Adeno-associated virus (AAV) is a replication-defective parvovirus, a single-stranded DNA gene that is approximately 4.7 kb in length, including two 145 nucleotide inverted terminal repeats (ITRs). There are several serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. As other examples, the complete AAV-1 genome is provided under GenBank accession number NC_002077; the complete AAV-2 genome is provided under GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete AAV-3 genome is provided under GenBank accession number NC_1829; the AAV-4 genome is provided under GenBank accession number NC 001829; the AAV-5 genome was provided under GenBank accession number AF085716; the AAV-6 genome was provided under GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under 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 presented in US Patent No. 9434928, incorporated herein by reference. Cis-acting sequences that direct replication, encapsidation/packaging, and integration into host cell chromosomes of viral DNA (rep) are contained in AAV ITRs (inverted terminal repeats). Three AAV promoters (designated p5, p19, and p40 based on their relative locations on the genomic map) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) in combination with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the production of four recipe proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins have several enzymatic properties that are ultimately responsible for viral genome replication. The cap gene is expressed from the p4 0 promoter and encodes three capsid proteins VP1, VP2 and VP3. Alternative splice sites and non-consensus translation start codons are responsible for the production of the three related capsid proteins. The only consensus polyadenylation site is located at position 95 of the AAV genome map. 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 noncytopathic, while natural infection in humans and other animals is

- 1 044609 hidden and asymptomatic. In addition, AAV infects many mammalian cells, allowing for application in a variety of tissue types in vivo. In addition, AAV transduces slowly dividing and non-dividing cells, and can essentially persist as a transcriptionally active nuclear episome (an extrachromosomal element) throughout the life of those cells. The AAV provirus genome is inserted into cloned DNA into plasmids, making it possible to create recombinant genomes. In addition, because signals for AAV genome replication and encapsidation are contained in the ITR of the AAV genome, some or all of the internal approximately 4.3 kb. genome (encoding replication and structural capsid proteins, rep-cap) can be replaced by foreign DNA. To create AAV-based vectors, the rep and cap proteins can be presented in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold storage of AAV less critical. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

Numerous studies have demonstrated long-term (greater than 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8: 659669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 80988108 (1996). See also, Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Moreover, since muscle is highly vascularized, transduction with recombinant AAV results in the appearance of transgene products in the circulatory system after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci uSa, 94: 5804-5809 (1997) and Murphy et 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 fibers possess the necessary cellular factors for proper glycosylation, folding and secretion of antibodies, indicating that muscle is capable of sustained expression of secreted protein therapeutic agents.

An emerging form of therapy for LGMD2E is gene delivery via a virus to restore the wild-type protein to the affected muscle, resulting in restoration of muscle function. Given that a subset of patients may develop cardiomyopathy (8, 9, 10, 13), this should be considered in the long-term care of these patients. The Sgcb-null mouse has been well characterized in previous reports. Araishi et al. (3) produced a β-sarcoglycan-deficient mouse with concomitant loss of all sarcoglycans as well as sarcospan, with at least modest retention of merosin, dystroglycans, and dystrophin, reproducing the clinical picture observed in LGMD2E. The histological changes in this animal model were also prototypical of its clinical counterpart, including distinct skeletal muscle fibrosis (14). Dressman et al. (25) injected the transversus abdominis muscle using rAAV2.CMV.SGCB. Expression was maintained for 21 months, and muscle fibers were protected from recurrent necrosis. The use of self-complementary AAV to enhance transgene expression (16), as well as a muscle-specific promoter to improve skeletal muscle (20, 26), and optimize the human β-sarcoglycan gene (hSGCB) have also been described.

Functional improvement in patients suffering from LGMD and other muscular dystrophies requires both gene restoration and fibrosis reduction. There is a need for methods to reduce fibrosis that can be combined with gene repair methods to more effectively treat LGMD and other muscular dystrophies.

The essence of the invention

Disclosed herein are gene therapy vectors, eg AAV, expressing the β-sarcoglycan gene and methods for delivering β-sarcoglycan to muscle to reduce and/or prevent fibrosis; and/or to increase muscle strength and/or to treat β-sarcoglycanopathy in a mammalian subject suffering from muscular dystrophy.

In addition, this invention provides combination therapies and approaches using gene therapy vectors delivering β-sarcoglycan to compensate for the gene defect observed in LGMD2E, and gene therapy vectors delivering miR-29 to subsequently suppress fibrosis.

In one aspect, described herein is a recombinant AAV vector comprising a polynucleotide sequence encoding a β-sarcoglycan. In some embodiments, the polynucleotide sequence encoding a β-sarcoglycan comprises, 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 of SEQ ID NO: 1, and encodes a protein that maintains β-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding the β-sarcoglycan comprises the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the polynucleotide sequence encoding the β-sarcoglycan comprises the nucleotide sequence set forth in SEQ ID NO: 1 .

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In another aspect, the recombinant AAV vector described herein contains a polynucleotide sequence encoding a β-sarcoglycan that is 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 identical to the amino acid sequence of SEQ ID NO: 2, and the protein retains β-sarcoglycan activity.

In another aspect, described herein is a recombinant AAV vector containing a polynucleotide sequence encoding a functional β-sarcoglycan that contains a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or its complement.

The term stringent is used to denote conditions that are generally understood to be stringent in the art. The stringency of hybridization is mainly determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent hybridization and wash conditions are 0.015 M sodium chloride, 0.0015 M sodium citrate at 6568°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, NY 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide concentration, or other denaturing agent) may also be used, however the rate of hybridization will be affected. In cases where hybridization of deoxyoligonucleotides is involved, additional exemplary stringent hybridization conditions include a 6x SSC 0.05% sodium pyrophosphate wash at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-basic oligonucleotides) and 60°C (for 23-basic oligonucleotides).

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, sonicated salmon sperm DNA (or other non-complementary DNA) and dextran sulfate, although other suitable agents may be used. The concentration and types of these additives can be changed without significantly affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8–7.4, but under typical ionic strength conditions the rate of hybridization is essentially 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 those skilled in the art to accommodate these variables and allow DNA with varying degrees of sequence relatedness to form hybrids.

In another aspect, the recombinant AAV vectors described herein can be operably linked to a muscle-specific regulatory element. For example, the skeletal muscle-specific regulatory element is a human skeletal muscle actin gene element, a cardiac actin gene element, myocyte-specific enhancer binding factor MEF, muscle creatine kinase (MKK), tMKK (truncated MKK), 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 troponin C gene element, slow-twitch muscle troponin I gene element fibers, hypoxia-inducible nuclear factors, steroid-inducible element or glucocorticoid response element (GRE).

In some embodiments, the muscle-specific promoter is MHCK7 (SEQ ID NO: 4). An exemplary rAAV described herein is pAAV.MHCK7.hSCGB, which contains the nucleotide sequence SEQ ID NO: 3; wherein the MCHK7 promoter spans nucleotides 130-921, the chimeric SV40 intron spans nucleotides 9311078, the β-sarcoglycan sequence spans nucleotides 1091-2047, and the polyA sequence spans nucleotides 2054-2106.

In some embodiments, the muscle-specific promoter is tMCK (SEQ ID NO: 6). An exemplary rAAV described herein is pAAV.tMCK.hSCGB, which contains the nucleotide sequence SEQ ID NO: 5; with the tMCK promoter spanning nucleotides 141-854, the chimeric SV40 intron spanning nucleotides 886-1018, the β-sarcoglycan sequence spanning nucleotides 1058-2014, and the poly-A sequence spanning nucleotides 2021-2073.

AAV can be of any serotype, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. The production of pseudotyped rAAV is described, 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).

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Compositions containing 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. Also contemplated are viral particles containing any of the recombinant AAV vectors described herein.

Methods for reducing fibrosis in a mammalian subject in need thereof are also provided. In this regard, the method includes administering a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein) to a mammalian subject. In some embodiments, the mammalian subject suffers from muscular dystrophy. In some embodiments, administration of an AAV vector described herein (or a composition comprising an AAV vector described herein) reduces fibrosis in skeletal muscle or cardiac muscle of a subject. These methods may further include the step of introducing a second recombinant AAV vector containing a polynucleotide sequence containing miR29C.

The term muscular dystrophy, as used herein, refers to a disorder in which muscle strength and size gradually decrease. Non-limiting examples of muscular dystrophy diseases may include: Becker muscular dystrophy, tibialis muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreyfuss muscular dystrophy, glenoscapulofacial myopathy, sarcoglycanopathy, congenital muscular dystrophy, such as congenital muscular dystrophy due to partial LAMA2 deficiency , congenital muscular dystrophy due to merosin deficiency, congenital muscular dystrophy type 1D, Fukuyama congenital muscular dystrophy, limbal girdle muscular dystrophy 1A, limbal girdle muscular dystrophy 2A, limbal girdle muscular dystrophy 2B, limbal girdle muscular dystrophy 2C, muscular 2D dystrophy 2D, muscle dystrophy of the Lumba-rack 2, muscle dystrophy of the Limba-Poyas 2F, muscle dystrophy of the Limba-Poyas 2G, muscle dystrophy of the defense 2N, muscle dystrophy of the defeat 2I, muscle dystrophy of the deceased 2I, muscle dystrophic limbal girdle muscular dystrophy 2J, limbal girdle muscular dystrophy 2K, rigid spine syndrome with epidermolysis bullosa simplex, oculopharyngeal muscular dystrophy, Ullrich congenital muscular dystrophy, and Ullrich scleroatonic muscular dystrophy. In some embodiments, the subject suffers from limbal girdle muscular dystrophy. In some embodiments, the subject suffers from limbal girdle muscular dystrophy 2E (LGMD2E).

The term fibrosis, as used herein, refers to excessive or dysregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues following injury, including skeletal muscle, cardiac muscle, liver, lung, kidney and pancreas. ECM components that are fine-tuned include collagen 1, collagen 2 or collagen 3, and fibronectin.

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

In any of the methods of the present invention, the subject may suffer from a muscular dystrophy such as limbal girdle muscular dystrophy or any other dystrophin-related dystrophy.

Also provided is a method of treating muscular dystrophy in a mammalian subject, comprising administering a therapeutically effective amount of an AAV vector described herein (or a composition comprising an AAV vector described herein) to the mammalian subject. In some embodiments, the muscular dystrophy is limbal girdle muscular dystrophy. Any of the methods described herein may further include the step of introducing a second recombinant AAV vector containing a polynucleotide sequence containing miR29C.

Combination therapy is also being considered. In this regard, any of the above methods described herein may further include the introduction of a second recombinant AAV vector containing a polynucleotide sequence containing miR29C. In some embodiments, the miR29C-containing polynucleotide is operably linked to a muscle-specific regulatory element. For example, a muscle-specific regulatory element is a human skeletal muscle actin gene element, a cardiac actin gene element, myocyte-specific enhancer binding factor MEF, muscle creatine kinase (MKK), tMKK (truncated MKK), myosin heavy chain (MHC), MHCK7 (hybrid version of MHC and MCK), C5-12 (synthetic promoter), mouse creatine kinase enhancer element, fast twitch skeletal muscle fiber troponin C gene element, slow twitch troponin C gene element

- 4 044609 contractile cardiac muscle fibers, slow twitch muscle fiber troponin I gene element, hypoxia-inducible nuclear factors, steroid-inducible element or glucocorticoid response element (GRE). In some embodiments, the second recombinant vector comprises the polynucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 8, as described in US Provisional Application No. 62/323,163 (the disclosure of which is incorporated herein by reference in its entirety).

In the combination therapy methods described herein, in which both the rAAV vector expressing β-sarcoglycan and the rAAV vector expressing miR29c are administered to a mammalian subject, wherein the rAAV vectors may be administered simultaneously or administered sequentially, wherein the rAAV vector expressing β -sarcoglycan is administered immediately before or after rAAV expressing miR29c. Alternatively, an AAV vector expressing β-sarcoglycan is administered over about 1-24 hours (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours) after administration of rAAV expressing miR-29, or implement methods according to this invention in which an AAV vector expressing β-sarcoglycan is administered for about 1-24 hours (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23 or 24 h) before administration of rAAV expressing miR-29. In some embodiments, the β-sarcoglycan-expressing AAV vector is administered within about 1-5 hours (e.g., 1, 2, 3, 4, or 5 hours) after administration of the miR-29-expressing rAAV, or the methods of the present invention are performed. wherein the AAV vector expressing β-sarcoglycan is administered for about 1-5 hours (eg, 1, 2, 3, 4 or 5 hours) before the rAAV expressing miR-29c is administered.

In any of the methods according to this invention, the rAAV vector is administered by intramuscular injection or intravenous injection. In addition, in any of the methods according to this invention, rAAV is administered systemically, for example administered parenterally by injection, infusion or implantation.

The compositions of this invention are formulated for intramuscular injection or intravenous injection. Moreover, the compositions of the present invention are formulated for systemic administration, such as parenteral administration by injection, infusion or implantation.

In addition, any composition is formulated for administration to a subject suffering from a muscular dystrophy (such as limbal girdle muscular dystrophy or any other dystrophin-related dystrophy). In some embodiments, the composition may further comprise a second recombinant AAV vector comprising the polynucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 8.

In any of the uses of the invention, the medicament is formulated for intramuscular injection or intravenous injection. Moreover, in any of the uses of the invention, the drug is formulated for systemic administration, such as parenteral administration by injection, infusion or implantation. In addition, any drug may be formulated for administration to a subject suffering from a muscular dystrophy (such as limbal girdle muscular dystrophy, or any other dystrophin-related muscular dystrophy). In some embodiments, the drug may further comprise a second recombinant AAV vector comprising the polynucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 8.

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 of the Invention. It is intended that the entire document be linked 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 a single sentence or paragraph or section of this document. The invention includes, as a further aspect, all embodiments of the invention narrower in scope than the embodiments defined in the specific paragraphs above. For example, there are certain aspects of an invention that are described as a genus, in which case it should be understood that each member of the genus is separately an aspect of the invention.

Brief description of graphic materials

In fig. 1A-1D depict that AAV-mediated β-sarcoglycan expression restores dystrophin-associated proteins and maintains membrane integrity. (a) Self-complementary AAV vector containing the codon-optimized human β-sarcoglycan gene (hSGCB) driven by the muscle-specific yMKK promoter. The cassette also contains a chimeric intron to enhance processing and a polyadenylation signal for stability. (b) Immunofluorescent staining with anti-β-SG antibody demonstrates high levels of sarcolemmal SGCB transgene staining in 5-week-old mice at 6 and 12 weeks post-injection. Shown are x20 images. The percentage of fibers expressing beta-sarcoglycan for each TA muscle averaged 88.4±4.2% at 6 weeks (n=9, 4 males, 5 females) and 76.5±5.8% at 12 weeks ( n=6, 4 males, 2 females). Protein expression was checked by Western blotting with gamma-tubulin staining as a loading control, (c) Delivery

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AAV of β-sarcoglycan leads to the restoration of other members of the sarcoglycan complex; asarcoglycan, dystrophy. x20 images. (d) scAAVrh.74.hSGCB protects sgcb ^−/− membranes from damage. An image showing a large area of Evans Blue-stained fibers (red) was compared with a cluster of β-sarcoglycan-positive fibers that were protected from Evans Blue staining. Shown are x40 images.

In fig. 2A-2D depict histological analysis of β-SG-deficient skeletal muscle. Treatment with scAAVrh.74.hSGCB normalizes histological parameters in sgcb'' mice. Hematoxylin and eosin and picrosirius red staining were performed on TA muscles from sgcb'/' mice along with normal control C57/BL6 mice and scAAVrh.74.hSGCB-treated mice, followed by quantification of histological parameters and % collagen staining. (a) Hematoxylin and eosin staining shows the presence of centrally nucleated fibers, inflammatory cells, and wide variation in fiber diameter in β-SG-deficient muscle, and improved histopathology after gene transfer. (b) Picrosirius red staining shows a decrease in red staining of collagen in the treated muscle. (c) Quantification of centrally nucleated fibers showing reduction after treatment (P < 0.0005, one-way ANOVA) and (d) representation of fiber size distribution and increase in mean fiber size of TA muscle from control C57/BL6 mice and sgcb' mice /' compared with treated mice (P<0.0001, one-way ANOVA). (f) Quantification of % collagen in TA muscles from control C57/BL6 mice and sgcb'/' mice compared to treated sgcb ^-/ ' mice (P<0.0001, one-way ANOVA). The 100 µm scale bar is shown for x20 images. ***P<0.001;****P<0.0001.

In fig. 3A-3C depict that intramuscular delivery of scAAVrh.74.hSGCB corrects tetanic force and resistance to contractile injury. TA muscles of sgcb'/' mice treated with 3x10 ¹⁰ gv (vector genomes) scAAVrh.74.hSGCB by IM (intramuscular) injection were collected 6 weeks after gene transfer, and the technique was used to assess tetanic force, and the eccentric contraction technique was used to assesses resistance to injury caused by contraction. (a) AAVrh.74.hSGCB-treated TA muscles showed significant improvements in both absolute tetanic force (P<0.01, paired t-test), (b) normalized specific force (P<0.05, paired t-test), which was no different from the wild type (C57/BL6) strength. (c) AAVrh.74.hSGCB-treated TA muscles showed significant improvement in resistance to contraction-induced injury compared with untreated sgcb'/' controls (P<0.01, two-way ANOVA). Strength retention is shown after 10 contractions. *P<0.05;**P<0.01.

In fig. 4A-4C depict the results of analysis of aged mice treated intramuscularly with scAAVrh.74.tMCK.hSGCB. (a) Immunofluorescence staining of TA muscle from 6-month-old treated sgcb'/' mice 12 weeks post-injection (n=5, 5 males) shows sarcolemmal expression of the SGCB transgene in amounts averaging 80% in injected versus untreated mice (n=4, 4 males). (b) Picrosirius red staining of treated and untreated TA muscle, (c) Quantification of collagen presented in picrosirius red-stained tissue shows a significant reduction in collagen amount after treatment with rAAVrh.74.tMCK.hSGCB (P<0.0001, one-sided analysis of variance). The 100 µm scale bar is shown for x20 images. ****P<0.0001.

In fig. 5A-5C depict the results of vascular delivery of scAAVrh.74.hSGCB. Four-week-old (n=5, 5 males) and 5-week-old (n=4, 2 males, 2 females) β-SG-deficient mice were treated with vector via the femoral artery to deliver the vector to the muscles of the lower limb. At a dose of 5x1011 gv, β-SG expression was 90.6 ± 2.8% in the TA muscle and 91.8 ± 4.7% in the gastrocnemius muscle of treated mice, which was accompanied by improved histopathology, leading to a significant improvement in specific force compared to untreated animals even following the injury model. (a) Protein expression (β-SG in three representative mice. Muscle from an untreated (β-SG KO mouse is shown for comparison in the inset (bottom right). ×20 images are shown. Expression in treated muscle confirmed by Western blotting, and gamma is shown -tubulin as a loading control. (b) Histopathology is significantly improved after high dose treatment. Top panels are treated TA and gastrocnemius muscles. Bottom panels are untreated β-SG deficient control muscles. 100 μm scale bar is shown for x 20 images. (c) Percentage of specific force retained in the DRP (extensor digitorum longus) muscle after 10 cycles of eccentric contraction-induced injury. Treatment with 5x1011 gv AAVrh.74.hSGCB resulted in a significant improvement in strength that is equivalent to the control DT (normal) muscle. P<0.05, one-way analysis of variance) *P<0.05.

In fig. 6A, 6B depict decreased fibrosis in ILP (isolated limb perfusion) treated β-SG KO mice. (a) Picrosirius red staining shows decreased fibrosis in treated mice, indicating decreased collagen deposition compared to untreated sgcb ^−/− mice. (b) Quantification of collagen in TA and gastrocnemius muscles from BL6

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WT, untreated sgcb ^−/ − mice, and treated mice confirmed the reduction in collagen amount in treated mice (P < 0.001, one-way ANOVA). The 100 µm scale bar is shown for x20 images. ***P<0.001.

In fig. 7A and 7B depict the vectorial biodistribution and expression of the protein. (a) Histogram of average vector distribution in tissues collected from ILP-treated mice, reported in copies of transcript per microgram of DNA added to qPCR. The left limb was treated. (b) No protein expression is observed in non-target organs by Western blotting.

In fig. 8A-D depict histological and functional deficits in sgcb ^-/- mice at 7 months of age. Tricolor staining in the diaphragm (A) and heart (C) of SGCB'/' mice shows extensive fibrosis (red). The force produced by the diaphragm is significantly reduced in the diaphragm (B), and cardiac ejection fraction is also reduced in sgcb ^−/− mice (D).

In fig. Figure 9 shows a diagram of a β-sarcoglycan therapeutic transgene cassette. Self-complementary AAV vector containing the codon-optimized human β-sarcoglycan gene (hSGCB). Expression is driven by the muscle-specific MHCK7 promoter. The cassette also contains a chimeric intron to enhance processing and a polyadenylation signal for stability.

In fig. 10 depicts immunofluorescent staining of β-sarcoglycan in various skeletal muscles, which shows robust expression with occasional negative fibers after 1, 4, or 6 months of treatment (1 month shown).

In fig. Figure 11 shows β-sarcoglycan immunofluorescence staining in the diaphragm and heart, which shows robust expression with sparse negative fibers after 1, 4, or 6 months of treatment (1 month shown).

In fig. 12A-D depict restoration of SGCB expression following intravenous delivery of scAAVrh.74.MHCK7.hSGCB. (a) Cassette scAAVrh.74.MHCK7.hSGCB. (b) Immunofluorescence imaging 6 months after injection of skeletal muscle, diaphragm, and heart in sgcb −/− mice injected intravenously with a total dose of le12 gv scAAVrh.74.MHCK7.hSGCB. Representative images of skeletal muscle showing an average of 98.13±0.31% transduction. 20x images shown. Representative images of cardiac tissue showing high levels of hSGCB transgene expression. 10x images shown. (c) Western blot of all muscles from one sgcb-treated mouse. /’confirming the expression of the hSGCB transgene. (d) Western blot analysis of hSGCB expression in the hearts of five sgcb-treated mice.^-/- with density quantification showing hSGCB overexpression up to 72.0% of BL6 WT levels.

In fig. 13A-D depict the effect of systemic treatment with scAAVrh74.MHCK7.hSGCB on muscle pathology. (a) Hematoxylin and eosin staining of the diaphragm and QUAD muscle of C57BL/6 WT, sgcb ⁻ / ⁻ and scAAVrh.74.MHCK7.hSGCB-treated mice showing normalized histopathology. (b) Quantification of the reduction in centrally nucleated fibers in muscles of treated sgcb ^- / ^- compared to untreated sgcb'/' (TA, GAS, GLUT, diaphragm, p < 0.0001) (QUAD, PSOAS, TRI, p < 0 .05). (c) Normalization of fiber distribution in GAS, PSOAS and TRI, and (d) increase in mean fiber size in treated muscles compared to untreated sgcb'/' muscles (p<0.001) (one-way ANOVA) (n=5 per group) .

In fig. 14A and 14B depict decreased collagen deposition in intravenously treated mice (β-SG KO. (a) Picrosirius red staining showed decreased fibrosis in treated mice, as shown by decreased collagen deposition in the diaphragm and GAS compared to untreated sgcb ^-/- mice. ( b) Quantification of collagen levels in diaphragm and GAS muscles in C57BL/6 WT mice (n=4), untreated sgcb ^-/- mice (n=4), and treated sgcb ^-/ mice (n=5) confirms decreased collagen levels in both treated muscles (p < 0.0001, one-way ANOVA) 100 µm scale bar shown for 20x images.

In fig. 15 depicts that delivery of scAAVrh.74.MHCK7.hSGCB via the tail vein to sgcb' ^/ 'mice completely restores strength in the diaphragm after 6 months of treatment (IV administration (1e12gv)). Diaphragm muscle strips were collected from treated and control SGCB'/' and WT mice and subjected to force measurements; treatment restored strength to DT levels.

In fig. 16 shows a diagram of the rAAV vector scAACrh.74.CMV.miR29c and the nucleotide sequence of miR-29c in the natural miR-30 scaffold.

In fig. 17 depicts that after 3 months of treatment with AAVrh.74.CMV.miR29C, TA muscles were collected from treated and control SGCB ^-/- mice and WT mice, and analyzed for fibrosis (collagen amounts) (n=5 per group). Using Sirius Red staining and quantification, a decrease in collagen amounts was observed after treatment (see FIG. 18). The results showed that the amounts of Col1A, Col3A and Fbn transcripts were normalized and muscle fiber size increased.

In fig. Figure 18 shows representative images of scanned whole sections of untreated and AAVrh.74.CMV.miR29C-treated tibialis anterior muscles stained with Sirius Red, which stains collagen 1 and 3. Quantification is depicted in

- 7 044609 fig. 17.

In fig. 19A and 19B depict correction of kyphoscoliosis in the thoracic spine, (a) Kyphoscoliosis in sgcb ^−/− mice as detected by radiography. (b) Kyphotic index (KI) score of sgcb ^-/- mice (3.69) is low compared to C57BL/6 WT (6.01) (p < 0.01), but increases after scAAVrh.74 .MHCK7 treatment .hSGCB (5.39) (p<0.05 vs. sgcb ^- / ^- ) (one-way ANOVA) (n=6 per group).

In fig. 20A-D depict the assessment of cardiomyopathy in cardiac muscle. (a) Hematoxylin-eosin and picrosirius red staining of 7-month-old BL6 WT, sgcb-- and AAV.MHCK7.hSGCB sgcb-- treated hearts 6 months after treatment shows myocardial degeneration in untreated ssgcb-- muscles and improvement after treatment. (b) Cardiac MRI analysis showing reduction in stroke volume of sgcb ^- / ^- hearts (p<0.01), cardiac output and ejection fraction (p<0.05) (one-way ANOVA), and improvement 6 months after treatment (n=6 per group). (c) Western blot analysis of two C57BL/6 WT hearts, two sgcb-- hearts, and five AAV.MHCK7.hSGCB-treated sgcb-- hearts showing decreased amounts of cardiac troponin I in diseased mice. (d) Densitometric quantification showing reduction in cardiac troponin I (cTrpI) up to 60.38% compared to amounts in BL6 WT, and overexpression up to 135.8% compared to amounts in BL6 WT.

In fig. 21A-B depict corrected diaphragm function and increased activity in an open space cage. (a) Diaphragm muscle strips were collected to measure strength and fatigue resistance in BL6 WT mice (n=5), sgcb− mice (n=4), and AAV.MHCK7-treated mice. hSGCB sgcb- (n=5), all at 7 months of age. Six months of treatment restored strength to WT levels (p<0.01 vs. sgcb ^- / ^- ) and improved resistance to fatigue. (b) Overall locomotion ability in the x- and y-planes is significantly reduced in sgcb ⁻ / ⁻ mice (p < 0.0001) and slightly improved in MSNK7-treated mice (p < 0.05). Vertical activity, in the form of hindlimb locomotion, was also decreased in sgcb ^-/- mice (p < 0.01) and significantly increased in MSNK7-treated mice (p < 0.05) (one-way ANOVA) (n = 6 in group).

In fig. 22A, B depicts biodistribution and off-target expression analysis of the scAAVrh.74.MHCK7.hSGCB systemic delivery transgene. (a) Histogram of the distribution of the average number of gv transcript copies per microgram of DNA added to the qPCR reaction in various tissues from two sgcb ^−/− mice after IV (intravenous) delivery of scAAVrh.74.MHCK7.hSGCB at a total dose of 1e12 gv. (b) Western blot biodistribution in muscles and organs from sgcb ^−/− mice systemically injected with scAAVrh.74.MHCK7.hSGCB indicates a lack of hSGCB transgene expression in any non-muscle samples.

Detailed description of the invention

This invention is based on the discovery that administration of an AAV vector containing a β-sarcoglycan expressing polynucleotide results in a reduction or complete reversal of muscle fibrosis in an animal mouse model of limbal girdle muscular dystrophy. As shown in the Examples, administration of the AAV vector described herein resulted in a reversal of signs of degeneration including fewer reduced fibers, reduced inflammation, and improved functional recovery by protecting against eccentric contraction with increased force production.

As used herein, the term AAV is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that replicates only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen AAV serotypes that have been characterized. General information and reviews on 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). However, there is no doubt that these same principles will apply to additional AAV serotypes, since it is well known that the various serotypes are quite closely related both structurally and functionally, even at the 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 appear to have very similar replication properties mediated by homologous rep genes; and all have three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further predicted by heteroduplex analysis, which shows extensive cross-hybridization between serotypes along the entire 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.

An 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.

An 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 encapsidated AAV polynucleotide vector. If the particle contains a heterologous polynucleotide (that is, a polynucleotide different from the wild-type AAV genome, such as a transgene that is to be delivered into a mammalian cell), it is commonly referred to as an AAV vector particle or simply an AAV vector. Thus, producing an AAV vector particle necessarily involves producing an AAV vector, since such a vector is contained in the AAV vector particle. AAV

Recombinant AAV genomes of the present invention contain a nucleic acid molecule of the present invention, and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in rAAV genomes can be from any AAV serotype for which a recombinant virus can be produced, including but not limited to these AAV serotypes: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV12, AAV-13 and AAV rh.74. The production of pseudotyped rAAV is described, 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). As noted in the Background Art section above, 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 promote skeletal muscle-specific expression.

The DNA plasmids of the present invention contain rAAV genomes. DNA plasmids are transferred into cells suitable for infection with an AAV helper virus (eg, adenovirus, E1-deleted adenovirus, or herpes virus) to package the rAAV genome into infectious virus particles. Methods for producing rAAV particles, in which the AAV genome is introduced into a cell to be packaged, rep and cap genes, and accessory viral functions, are standard in the art. Production of rAAV requires that the following components be present in a single cell (referred to herein as a packaging cell): the rAAV genome, separated from (ie, not in) the rAAV genome, the AAV rep and cap genes, and helper virus functions. The AAV rep and cap genes can be from any AAV serotype for which a recombinant virus can be produced, and can be from an AAV serotype that is different from the ITR serotype of the rAAV genome, including but not limited to these AAV serotypes: AAV-1, AAV -2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV10, AAV-11, AAV-12, AAV-13 and AAV rh.74. The production of pseudotyped rAAV is described, for example, in WO 01/83692, which is incorporated herein by reference in its entirety.

The method for producing a packaging cell is to create a cell line that stably expresses all the necessary components for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing the rAAV genome without the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selection marker such as a neomycin resistance gene are integrated into the genome of the cell. AAV genomes have been introduced into bacterial plasmids using procedures such as GC attachment (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., 1983, Gene, 23:65-73) or direct blunt end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as an adenovirus. The advantages of this method are that cells can be selected and are suitable for large-scale production of rAAV. Other examples of suitable methods utilize adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells.

General principles for the production of rAAV are discussed, for example, 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. 5658776; 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 foregoing documents are hereby incorporated herein by reference in their entirety, with particular reference to those portions of the documents that relate to the production of rAAV.

Thus, the invention 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 (293 related lineage). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low passage number 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (fetal rhesus lung cells).

Recombinant AAVs (ie, infectious encapsulated rAAV particles) according to the present invention contain the rAAV genome. Embodiments include, but are not limited to: rAAV designated pAAV.MHCK7.hSCGB, which contains the polynucleotide sequence set forth in SEQ ID NO: 3; and pAAV.tMCK.hSCGB, which contains the polynucleotide sequence shown in SEQ ID NO: 5.

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

In another embodiment, the invention provides compositions containing rAAV according to this invention. The compositions described herein include rAAV in a pharmaceutically acceptable carrier. 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 doses 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; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

The titers of rAAV that will be administered in the methods of this invention will vary depending, for example, on the specific rAAV, the route of administration, the purpose of treatment, the individual and cell type(s) that will be targeted, and can be determined by methods in the art technology. 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 (DRPs) in ml. Dosages may also be expressed in viral genome units (vv).

The invention provides methods for transducing a target cell with rAAV, in vivo or in vitro. The in vivo methods include the step of administering an effective dose or multiple effective doses of a composition containing rAAV according to the present invention to an animal (including a human) in need thereof. If the dose is administered before the development 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 invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease condition being treated that slows or prevents progression to the disorder/disease condition that slows or prevents progression a disorder/disease state that reduces the severity of the disease, resulting in remission (partial or complete) of the disease and/or prolonging survival. Muscular dystrophy, such as limbal girdle muscular dystrophy, is an example of a disease that is considered suitable for prevention or treatment by the methods of this invention.

This invention also contemplates combination therapies. The combination used herein includes both simultaneous treatment and sequential treatment. Combinations of the methods of this invention with standard drug therapies (eg, corticosteroids) are especially contemplated, as are combinations with new therapies.

A therapeutically effective amount of rAAV vector is a dose of rAAV in the range of about 1e13 to about 5e14 gV/kg, or from about 1e13 to about 2e13 gV/kg, or from about 1e13 to about 3e13 gV/kg, or from about 1e13 to about 4e13 gv/kg, or from about 1e13 to about 5e13 gv/kg, or from about 1e13 to about 6e13 gv/kg, or from about 1e13 to about 7e13 gv/kg, or from about 1e13 to about 8e13 gv/kg, or from about 1e13 to about 9e13 gv/kg, or from about 1e13 to about 1e14 gv/kg, or from about 1e13 to about 2e14 gv/kg, or from 1e13 to about 3e14 gv/kg, or from about 1e13 to about 4e14 gv/kg kg, or from about 3e13 to about 4e13 gv/kg, or from about 3e13 to about 5e13 gv/kg, or from about 3e13 to about 6e13 gv/kg, or from about 3e13 to about 7e13 gv/kg, or from about 3e13 to about 8e13 gv/kg, or from about 3e13 to about 9e13 gv/kg, or from about 3e13 to about 1e14 gv/kg, or from about 3e13 to about 2e14 gv/kg, or from 3e13 gv/kg to about 3e14 gv/kg /kg, or from about 3e13 to about 4e14 gv/kg, or from about 3e13 to about 5e14 gv/kg, or from about 5e13 to about 6e13 gv/kg, or from about 5e13 to about 7e13 gv/kg, or from about 5e13 to about 8e13

- 10 044609 gv/kg, or from about 5e13 to about 9e13 gv/kg, or from about 5e13 to about 1e14 gv/kg, or from about 5e13 to about 2e14 gv/kg, or from 5e13 to about 3e14 gv/kg, or from about 5e13 to about 4e14 gv/kg, or from about

5e13 to about 5e14 gv/kg, or from about 1e14 to about 2e14 gv/kg, or from 1e14 to about 3e14 gv/kg, or from about 1e14 to about 4e14 gv/kg, or from about 1e14 to about 5e14 gv/kg . The invention also includes compositions containing these rAAV vector ranges.

For example, a therapeutically effective amount of rAAV vector is a dose of 1e13, about 2e13, about 3e13, about 4e13, about 5e13, about 6e13, about 7e13, about 8e13, about 9e13, about 1e14, about 2e14, about 3e14, about 4e14 and 5e14 gv /kg. The invention also includes compositions containing these dosages of the rAAV vector.

Administration of an effective dose of the compositions can be accomplished by standard routes, which include, but are not limited to: intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route(s) of administration and serotype(s) of the rAAV AAV components (particularly the AAV ITR and capsid protein) of the present invention may be selected and/or matched by those skilled in the art, taking into account the infection and/or disease state to be treated. treatment, and the target cell/tissue(s) that should express β-sarcoglycan.

The present invention provides topical administration and systemic administration of an effective dose of rAAV and compositions of the present invention. For example, systemic administration is administration into the circulatory system to affect the entire body. 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 rAAV according to the present invention can be accomplished using any physical method that will transfer the recombinant rAAV vector into the target tissue of the animal. Administration according to the invention includes, but is not limited to: injection into muscle, bloodstream and/or directly into the liver. It has been demonstrated that simply resuspending rAAV in phosphate-buffered saline is sufficient to obtain a transporter useful for expression in muscle tissue, and there are no known restrictions on the vehicles or other components that can be co-administered with rAAV (although rAAV follows the usual route avoid compositions that destroy DNA). The rAAV capsid proteins can be modified so that the rAAV is targeted to a 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 formulations for delivery to muscles via transdermal transport. Numerous formulations for intramuscular injection and transdermal transport have previously been developed and can be used in the practice of the invention. rAAVs 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 aqueous propylene glycol, as well as sterile aqueous solutions can be used. If desired, such aqueous solutions can be buffered and the liquid diluent first subjected to isotonic 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. The rAAV dispersion can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, the sterile aqueous media used are readily available for preparation by standard methods well known to those skilled in the art.

Dosage forms suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be administered by syringe. It must be stable under the conditions of production and storage, and must be protected from the contaminating effects 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. The desired fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by the use of surfactants. Prevention of exposure to microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it is preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved through the use of absorption delaying agents such as aluminum monostearate and gelatin.

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Sterile injection solutions are prepared by adding rAAV in the required amount to 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 the basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of injectable sterile solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient, plus any additional desired ingredient, from a pre-sterile-filtered solution thereof.

Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced to the subject. Alternatively, isogenic or xenogeneic muscle cells may be used as long as the cells will not elicit an inappropriate immune response in the subject.

Suitable methods for transduction and reintroduction of 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 an appropriate medium, and searching for cells carrying DNA of interest using conventional methods such as Southern blotting and/or PCR, or using selective markers. The transduced cells can then be formulated into pharmaceutical compositions and the composition administered to the subject by various routes, such as intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle using, for example, a catheter.

Transduction of cells with the rAAV of the invention results in sustained expression of β-sarcoglycan microRNA. Thus, the present invention relates to methods of administering/delivering rAAV that expresses β-sarcoglycan to an animal, preferably a human. 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 according to the present invention. Transduction can be accomplished using gene cassettes containing tissue-specific regulatory elements. For example, in one embodiment, the present invention provides methods for transducing muscle cells and muscle tissues controlled by muscle-specific regulatory elements, including, but not limited to: those derived from the actin and myosin gene families, e.g. , from 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)], regulatory elements derived from human skeletal muscle actin gene [Muscat et al., Mol Cell Biol, 7: 4089-4099 (1987)], cardiac actin gene, muscle creatine kinase element sequence [see Johnson et al., Mol Cell Biol, 9: 3393-3399 (1989)] and the mouse creatine kinase (mCK) enhancer element, regulatory elements derived from the fast-twitch skeletal muscle fiber troponin C gene, the slow-twitch cardiac muscle fiber troponin C gene, and slow twitch muscle fiber troponin I gene: 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 easily accessible. The invention addresses the sustainable expression of microRNAs from transduced myofibers.

By muscle cell or muscle tissue is meant 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.

The term transduction is used to refer to the introduction/delivery of a polynucleotide of interest (e.g., a polynucleotide sequence encoding a β-sarcoglycan) into a recipient cell, either in vivo or in vitro, by a replication-deficient rAAV of the invention, resulting in expression of the β-sarcoglycan by the cell -recipient.

Thus, the present invention provides methods for administering to a patient in need thereof an effective dose (or doses administered substantially simultaneously or at predetermined intervals) of rAAV that encodes a β-sarcoglycan.

All publications and patents mentioned herein are incorporated by reference in their entirety as if each individual publication or patent were specifically and separately identified for inclusion by reference. In the event of a conflict, this application, including any definitions herein, will control title.

- 12 044609

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

Examples

Materials and methods.

Animal models - All procedures were approved by the Institutional Animal Care and Use Committee of the National Children's Hospital Research Institute (protocol AR12-00040). Heterozygous B6.129-Sgcb ^tm1Kcam/1J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA, strain no. 006832). sgcb- mice were produced by crossing heterozygous mice. KO mice were bred and maintained as homozygous animals under standardized conditions at the Animal Resource Center at the National Children's Hospital Research Institute. Mice were maintained on a Teklad Global rodent diet (meal 3.8z5 fiber, 18.8% protein, 5% fat) on a 12:12-hour dark-light cycle. Identification of ^SGCB mice was performed by genotyping using PCR. All animals were housed in standard mouse cages with food and water in excess.

Construction of the beta-sarcoglycan gene.

Full-length human beta-sarcoglycan cDNA (GenBank accession number NM_0034994.3) was codon-optimized and synthesized at GenScript Inc, Piscataway, NJ, USA. Codon optimization with GenScript uses an algorithm that considers parameters that include transcription, mRNA processing and stability, translation, and protein folding to design the cDNA sequence that results in maximum expression in muscle tissue (www.genscript.com).

For the pAAV.tMCK.hSGCB construct, the cDNA was then cloned into a plasmid containing the AAV2 ITA, and the cassette contained the Kozak consensus sequence (CCASS), a chimeric SV40 intron, and a synthetic polyadenylation site (53 bp). The recombinant tMCK promoter was kindly provided by Dr. Xiao Xiao (University of North Carolina). It is a modification of the previously described CK627 promoter and contains a modification in the enhancer upstream of the promoter region containing transcription factor binding sites. The enhancer consists of two E-boxes (right and left). Modification of the tMCK promoter includes a mutation converting the left E-box to the right E-box (modification 2R) and a 6 bp insertion. (modification S5). The pAAV.tMCK.hSGCB vector was constructed by ligating the 1040 bp KpnI/XbaI fragment. from pUC57-BSG (Genscript Inc.) to the KpnI/XbaI sites of pAAV.tMCK.hSGCA ( 26 ).

The pAAV.MHCK7.hSGCB vector was constructed by removing the tMCK promoter and the chimeric SV40 intron at the NotI/KpnI sites and inserting a PCR-amplified fragment containing the MHCK7 promoter and the identical chimeric SV40 intron at the NotI/KpnI sites. MHCK7 is an MCK-based promoter that uses a 206-bp enhancer taken from the approximately 1.2 kb 5′ transcription start site in the endogenous muscle creatine kinase gene with a proximal promoter (enh358MCK, 584-bp) ^3,12 . The MHCK7 promoter itself contains this modified CK7 cassette from the MCK gene family ligated to 188 bp. α-MyHC (α-myosin heavy chain) enhancer of the 5' portion of CK to enhance expression in heart 12. The promoter portion of creatine kinase (CK) is 96% identical between tMCK and MHCK7. Finally, the pAAV.MHCK7.hSGCB vector was constructed by ligating the 960 bp fragment. NotI/KpnI MNSK7+intron from pAAV.MHCK7.DYSF5'DV44 to NotI/KpnI sites of pAAV.tMCK.hSGCB (Pozgai et al., Gene Ther. 23: 57-66, 2016).

Preparation of rAAV. To create the rAAV vector, a modified cross-packing approach previously described by Rodino-Klapac et al was used. (J. Trans. Med. 5:45, 2007). Here, a triple transfection method with CaPO ₄ precipitation of HEK293 cells allows the ITA of AAV2 to be packaged into the capsid of an AAV of a different serotype (28,29). The resulting plasmids were: (i) pAAV.tMCK.hSGCB or pAAV.MHCK7.hSGCB, (ii) modified AAV helper plasmids rep2-caprh.74 encoding the cap of isolate rh.74-like serotype 8, and (iii) type 5 adenovirus helper plasmid (pAdhelper) expressing the adenovirus genes E2A, E4 ORF6 and VA I/II RNA. Vectors were purified and the titer of encapsulated gV (using a Prism 7500 Taqman detection system, PE Applied Biosystems, Carlsbad, CA, USA) was determined as previously described ( 30 ). The primer and fluorescent probe were targeted to the tMCK promoter and were: tMCK forward primer, 5'-ACC CGA GAT GCC TGG TTA TAA TT-3' (SEQ ID NO: 10); reverse primer tMCK, 5'-TCC ATG GTG TAC AGA GCC TAA GAC-3' (SEQ ID NO: 11); and tMCK probe, 5'-FAM-CTG CTG CCT GAG CCT GAG CGG TTA C-TAMRA-3' (SEQ ID NO: 12). The primer and fluorescent probe were targeted to the MHCK7 promoter and were: MHCK7 forward primer, 5'-CCA ACA CCT GCT GCC TCT AAA-3' (SEQ ID NO: 16); reverse primer MHCK7, 5'-GTC CCC CAC AGC CTT GTT C-3' (SEQ ID NO: 17); and MNSC7 probe, 5'-FAM-TGG ATC CCC-Zen-TGC ATG CGA AGA TC-3IABKFQ-3' (SEQ ID NO: 18).

Intramuscular gene delivery. For intramuscular injection, mice were anesthetized and maintained under 1-4% isoflurane (in O ² ). The lower left anterior limb of 4-6 week old SGCB ⁻ / ⁻ mice was cleared with 95% EtOH and then into the transversus abdominis (TA) muscle.

- 13 044609 3x1011 gV scAAVrh.74.tMCK.hSGCB, diluted in saline, was administered in a volume of 30 μl using an ultra-thin 30-gauge insulin syringe. The contralateral muscle was left untreated to serve as a control. TA muscles from both limbs were removed either 6 (n=9, 4 males, 5 females) or 12 (n=6, 4 males, 2 females) weeks after injection to assess the efficiency of gene transfer. In experiments with 6-month-old mice (n=5, 5 males), treatment consisted of an intramuscular injection into the left TA of 3x1011 gv scAAVrh.74. tMCK.hSCGB. For isolated limb perfusion experiments, sgcb ^/ mice were injected at 4 (n=5, 5 males) and 5 (n=4, 2 males, 2 females) weeks of age with 5x1011 gv scAAVrh.74. tMCK.hSCBB by injection into the femoral artery as described previously (19). Animals were euthanized and muscles were analyzed 8 weeks after gene transfer.

Systemic gene delivery: Systemic delivery was achieved by injecting the vector into the tail vein of sgcb- - mice. Mice were injected with 1x10 ¹² gV scAAVrh.74.MHCK7.hSGCB diluted in saline in a volume of 212 μl using a 30-gauge ultrafine insulin syringe. Mice were immobilized in a holding tube by placing the tail back through the tail groove to warm it to dilate the blood vessels to facilitate injection. Once the artery was located along the central line of the tail, the injection was performed into one of the purple/blue lateral veins that run along the tail artery. All treated mice were injected at 4–5 weeks of age and euthanized 6 months after injection.

EDL force production and protection against eccentric contractions. Physiological analysis of EDL muscles in mice treated by insulated perfusion (ILP) was performed. EDL muscles from both lower hindlimbs of treated mice were dissected at the tendon and were subjected to a physiological protocol to assess function that has been previously described by our laboratory and others (19, 31) with some adaptations. During the eccentric contraction protocol, a 5% stretch-relengthening procedure was performed for 500 to 700 ms (5% stretched for 100 ms and then returned to optimal length in 100 ms). Following the tetanus and eccentric contraction protocol, the muscle was removed, wet weighed, placed in a tragacanth clamp, and then frozen in methylbutane cooled in liquid nitrogen.

TA force production and protection against eccentric contractions. A protocol for assessing the functional outcome of the TA muscle was performed on muscles isolated from mice receiving IM injection. This TA procedure has been described in several previous studies (32, 33). After eccentric contractions, mice were euthanized, and the NA muscle was excised, weighed, and frozen for analysis. Data analysis was performed in a blinded but not randomized manner.

Immunofluorescence. Cryosections (12 μm) were incubated with monoclonal anti-human beta-sarcoglycan primary antibody (Leica Biosystems, New Castle, UK; cat. no. NCL-LbSARC) at a dilution of 1:50 in blocking buffer (1xTBS (TRIS-buffered saline), 10 % goat whey, 0.1% Tween) for 1 hour at room temperature in a humid chamber. The sections were then washed with TBS three times, each wash for 20 min, and re-blocked for 30 min. AlexaFluor 594-conjugated goat anti-mouse IgG1 secondary antibody (Life Technologies, Grand Island, NY, USA; cat. no. A21125) was used at a dilution of 1:250 for 45 min. Sections were washed in TBS three times, each wash for 20 min, and mounted with mounting medium (Vector Laboratories, Burlingame, CA, USA). Four random x20 images covering four different muscle sectors were acquired using a Zeiss AxioCam MRC5 camera. The percentage of fibers positive for beta-sarcoglycan staining (450% muscle membrane staining intensity) was determined for each image and averaged for each muscle.

Western blot analysis. Tissue sections of the left treated TA muscle and the right contralateral TA muscle (20-20 microns thick) were collected in a microcentrifuge and homogenized with 100 μl of homogenization buffer (125 mM Tris-HC1, 4% SDS, 4 M urea) in the presence of 1 tablet of protease inhibitor mixture (Roche, Indianapolis, Indiana, USA). After homogenization, the samples were centrifuged at 10,000 rpm for 10 min at 4°C. Protein was quantified on a NanoDrop (Thermo Scientific, Waltham, MA, USA). Protein samples (20 μg) were electrophoresed on a 3–8% polyacrylamide Tris-acetate gel (NuPage, Invitrogen, Carlsbad, CA, USA) for 1 h 5 min at 150 V and then transferred to a PVDF membrane (Amersham Biosciences, Piscataway, New Jersey, USA) for 1 h 15 min at 35 V. The membrane was blocked in 5% nonfat dry milk in TBST (TBS with Tween 20) for 1 h and then incubated with rabbit polyclonal anti-human beta-sarcoglycan antibody (Novus Biologicals, Littleton, CO, USA; cat. no. NBP-190300 dilution 1:100 or 1:250) and 1:5000 monoclonal anti-mouse gamma-tubulin antibody (Sigma-Aldrich, St. Louis, MO, USA; cat. no. T6557 ) or a 1:5000 dilution of mouse monoclonal anti-mouse α-actinin antibody (Sigma-Aldrich, St. Louis, MO, USA; cat. no. A7811). A 1:500 dilution of rabbit polyclonal anti-mouse cardiac troponin I antibody (Abeam, Cambridge, MA; cat. no. ab47003) and a 1:1000 dilution of rabbit anti-mouse antibody were used.

- 14 044609 vinculin monoclonal antibody (Invitrogen, Frederick, MD; cat. no. 70062). For immunodetection of ECL (Enhanced chemiluminescence, enhanced chemiluminescence) we used anti-mouse (Millipore, Billerica, MA, USA, cat. no. AP308P) and anti-rabbit (Life Technologies, cat. no.

656120), secondary HRP (horseradish peroxidase) antibodies.

EBD analysis. A dose of 3x101° gv scAAVrh.74.tMCK.hSGCB was administered to 4-week-old sgcb- mice in the left TA by intramuscular injection. 4 weeks after injection, mice were injected into the intraperitoneal cavity on the right side with 5 μl/g body weight of filter-sterilized 10 mg/ml EBD (Evans blue dye) in 1x phosphate buffer solution. Mice were then sacrificed 24 h postinjection, and tissues were collected and sectioned. Sections were fixed in cold acetone for 10 min, and then an immunofluorescence protocol was used to stain human beta-sarcoglycan.

Morphometric analysis. Muscle fiber diameters and the percentage of muscle fibers with centrally located nuclei were determined from TA and GAS muscles stained with hematoxylin and eosin (H&E). Four random x20 images per slice for each animal were acquired using a Zeiss AxioCam MRC5 camera. Center-nucleated fibers were quantified using NIH ImageJ software (Bethesda, MD, USA). Fiber diameters were measured as the shortest diameter through the muscle fiber using Zeiss Axiovision LE4 software (Carl Zeiss Microscopy, Munich, Germany).

Biodistribution analysis using quantitative PCR. Taqman qPCR was used to determine the copy number of vector genomes present in contralateral target and nontarget muscle and nontarget organs as previously described ( 18 , 30 ). A vector-specific primer-probe set was used to amplify the sequence of the intronic region immediately downstream of the tMCK promoter, which is unique and located in the scAAVrh.74.tMCK.hSGCB transgene cassette. The following primers and probe were used in this study: forward primer for tMCK and MHCK7 5'-GTG AGG CAC TGG GCA GGT AA-3'(SEQ ID NO: 13); reverse primer to the intron of tMCK and MHCK7 5'-ACC TGT GGA GAG AAA GGC AAA G-3' (SEQ ID NO: 14); and a probe to the intron of tMCK and MHCK7 5'-6FAM-ATC AAG GTT ACA AGA CAG-GTT TAA GGA GAC CAA TAG AAA-tamra-3' (IDT) (SEQ ID NO: 15). Copy number is reported as the number of vector genomes per microgram of genomic DNA. Immunohistochemistry for staining immune cells. Immunohistochemistry was used to identify immune cells. Frozen tissue sections on charged Fisherbrand Superfrost microscope slides were incubated with rat monoclonal anti-mouse monoclonal antibodies using the HRP anti-rat Ig detection kit (BD Pharmagen, San Jose, CA, USA, cat. no. 551013): CD3 (cat. no. 555273) , CD4 (m 550280), CD8 (cat. no. 550281) and Mac-3 for macrophages (cat. no. 550292). All primary antibodies were diluted 1:20 with phosphate-buffered saline. Positive immunostaining was visualized using DAB chromagen diluted in DAB buffer using Streptavidin-HRP peroxidase Vexstatin ABC peroxidase. Ten random x40 images were acquired for each muscle and each was colored accordingly. The number of mononuclear cells was counted and expressed as the total number per ^mm2 .

Picrosirius red staining and collagen quantification. Frozen sections mounted on charged Fisherbrand Superfrost microscope slides were fixed in 10% neutral buffered formalin for 5 min and then washed in distilled water. The slides were then incubated in solution A (phosphomolybdic acid) from the Picrosirius Red staining kit (Polysciences Inc., Warrington, PA, USA, cat. no. 24901) for 2 min. After thorough rinsing in distilled water, the slides were placed in solution B (Direct Red 80/246 Trinitrophenol) for 15 min, followed by an additional rinse in distilled water, and then incubated in solution C (0.1 N hydrochloride) for 2 min. Slides were counterstained for 2.5 min with 1% Fast Green in 1% glacial acetic acid from Poly Scientific (cat. no. S2114) using a 1:10 dilution in deionized water. Finally, the slides were rinsed again in distilled water, dehydrated in serially diluted ethanol, cleared in xylene, and coverslipped using Cytoseal 60 media from Thermo-Scientific (Brotherham, MA, USA, cat. no. 8310). Images were taken using AxioVision 4.9.1 software (Carl Zeiss Microscopy). To analyze Sirius Red staining and calculate % collagen, the contrast between red and green colors was enhanced using Adobe Photoshop. The ImageJ color deconvolution plugin was selected and the RGB color deconvolution option was used. The Red image includes all connective tissues stained with Sirius Red. The Green image includes all muscles when counterstained with Fast Green. Only the Red image and the original image were used. A generation threshold was then applied to the images to produce black-and-white images with collagen-positive regions in black and negative regions in white. Using the measurement function, the collagen area was calculated. The total tissue area was then determined by converting the original image to 8-bit and adjusting the threshold to 254, which is one below the fully saturated image. The total area of the tissue was then measured as previously done and the total area was recorded. The percentage of collagen was then calculated by dividing the collagen area by the total tissue area. The average percentage was then calculated for each individual.

Tetanic contraction of the diaphragm for functional assessment: Mice were euthanized and the diaphragm was dissected from its rib attachments and the central tendon was left intact and placed in K-H buffer as previously described by Beastrom et al. (Am. J. Pathol. 179: 2464-74, 2011), Rafael-Forney et al. (Circulation 124: 582-8, 2011) and Moorwood et al. (J. Visualized Experiments 71:e50036, [year?]). A 2-4 mm wide diaphragm slice was obtained. The diaphragm strips were tightly woven with surgical silk (6/0, Surgical Specialties, Reading, PA) over the central tendon and sutured through a portion of the rib bone attached to the distal end of the strip. Each muscle was transferred to a water bath filled with an oxygen-containing K-H solution, which was maintained at 37°C. The muscles were aligned horizontally and tethered directly between a fixed pin and a dual-mode force transducer servomotor (305C; Aurora Scientific, Aurora, Ontario, Canada). Two platinum plate electrodes were placed in the organ bath to span the length of the muscle. The muscle was stretched to the optimal length for single-twitch measurements and then left for 10 min before the start of the tetanic protocol. Once the muscle had stabilized, the muscle was placed at the optimal length of 1 g and subjected to a warm-up, which consisted of three 1 Hz twitches every 30 s followed by three 150 Hz twitches every minute. After a 3-min rest period, the diaphragm was stimulated at 20, 50, 80, 120, 150, 180 Hz, allowing a 2-min rest period between each stimulus, each with a duration of 250 ms to determine maximum tetanic force. The length and mass of the muscles were measured. Strength was normalized by muscle weight and length.

Cardiac magnetic resonance imaging: Cardiac function was analyzed using a 9.4T magnetic resonance imaging (MRI) system and a mouse volumetric coil (Bruker BioSpin, Billerica, MA, USA). Mice were anesthetized with 2.5% isofluorane mixed with carbogen (1 L/min) for 3 min before being placed on the scanning bed. Once mice were placed in the scanning apparatus and imaging began, the isoflurane/carbogen mixture was reduced to 1.5% for the remainder of the study. ECG and respiration were monitored using an MRI-compatible system (Model 1025, Small Animal Instruments, Stonybrook, NY, USA). T1-weighted cine FLASH cardiac short-axis images were acquired for the entire mouse left ventricle (LV) (TR=8 ms, TE=2.8 ms, α=18°, matrix=256x256, FOV=3.0x3.0 cm , slice thickness=1 mm, number of slices=7, up to 20 frames per cardiac cycle). For image analysis, the end-diastolic and end-systolic time points of each minor axis image were identified, and the endocardial and epicardial cardiac boundaries were manually traced. Papillary muscles were excluded from the endocardial border of the LV (left ventricle). From these measured zones, end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), cardiac output (CO), ejection fraction (EF), and mean LV mass were calculated.

Immunofluorescence: Frozen sections (12 µm) of the tibialis anterior (TA), gastrocnemius (GAS), quadriceps (QUAD), psoas (PSA), gluteus (GLUT), triceps (TRI) and diaphragm along with the heart were subjected to immunofluorescence staining for the hSGCB transgene using our previously used protocol as described in Pozgai et al., Gene Therap. 23: 57–66, 2016. Sections were incubated with mouse monoclonal anti-human beta-sarcoglycan primary antibody (Leica Biosystems, New Castle, UK; cat. no. NCL-Lb-SARC) at a dilution of 1:100. Four random x20 images covering four different muscle sectors were acquired using a Zeiss AxioCam MRC5 camera. The percentage of fibers positive for beta-sarcoglycan staining (>50% muscle membrane staining) was determined for each image and averaged for each muscle.

Morphometric analysis: Hematoxylin and eosin (H&E) staining was performed on 12 μm muscle cryosections from 7-month-old C57BL6 WT mice (n=5), sgcb'/' mice (n=5), and 6-month-old rAAV.MHCK7.hSGCB-treated sgcb mice ^- / ^- (n=5) for analysis. The percentage of myofibers with central nuclei was determined in the TA, GAS, QUAD, PSOAS, GLUT, TRI muscles and diaphragm. In addition, muscle fiber diameters were measured in GAS, PSOAS, and TRI muscles. Four random x20 images for each muscle for each animal were acquired using a Zeiss AxioCam MRC5 camera. Center-nucleated fibers were counted using NIH ImageJ software, and fiber diameters were measured using Zeiss Axiovision LE4 software.

X-rays: Whole body X-rays were obtained on anesthetized 7 month old C57BL6 WT mice (n=6), untreated sgcb'/' mice (n=6) and 6 month treated rAAV.MHCK7.hSGCB sgcb'/' mice (n=6) using a Faxitron MX-20 digital X-ray system at 26 kV for 3 s (Faxitron X-Ray Corp, Lincolnshire, USA).

Laser activity monitoring in open space cages: An open space activity chamber was used to determine the overall activity of experimental mice. 7 month old mice from control C57BL6 WT (n=6) and untreated sgcb’/’ (n=6) groups together with

- 16 044609 6-month rAAV.MHCK7.hSGCB-treated sgcb ^-/ - mice (n=6) were analyzed according to a previously described protocol (Kobayashi et al., Nature 456: 511-5, 2008, Beastrom et al., Am J Pathol 179: 2464-74, 2011) with several modifications. All mice were tested at the same time of day in the early morning and then at the end of the night cycle, when the mice are most active. All mice were tested in an isolated room, under dim light, and were tested by the same person each time. To reduce anxiety and keep behavioral changes to a minimum, which could potentially interfere with the mice's normal activity and therefore assay results, test mice were not housed individually (Voikar et al., Genes Brain Behav. 4: 240-52, 2005). Mice activity was monitored using a Photobeam Activity System (San Diego Instruments, San Diego, CA). This system uses a grid of invisible infrared light beams that cross the camera from top to bottom, and from right to left, to observe the position and movement of the mouse in XYZ planes. Activity was recorded for 1 hour at 5-minute intervals. Mice were pre-acclimated to the activity testing room for an initial 1-h session several days before data collection began. Mice were tested in individual chambers, 4 at a time. Testing equipment was cleaned each time before use to reduce reactive behavioral abnormalities in mice that could alter our results. The collected data was converted into a Microsoft Excel worksheet and all calculations were performed in Excel. Individual ray interruptions for movement in the x and y planes were added for each mouse to reproduce overall locomotion ability, and ray interruptions in the z plane were added to obtain vertical activity over a 1-h time period.

Example 1: scAAVrh.74.tMCK.hSGCB construction and vector efficiency.

A transgene cassette containing the full-length codon-optimized human SCGB cDNA was constructed as shown in FIG. 1A. The cassette includes a Kozak consensus sequence (CCASS), a chimeric SV40 intron, a synthetic polyadenylation site, and a muscle-specific tMCK promoter (20) used to drive expression of the cassette. The cassette was packaged into the self-complementary (sc) AAV vector rh.74, which is 93% homologous to AAV8. AAVrh.74 has been shown to be safe and effective in mice and non-human primates, particularly in crossing the vascular barrier when delivered into muscle via the circulatory system (17, 18, 21). Vector efficacy was determined by intramuscular injection into the left TA of a Sgcb-null mouse. Delivery of 3x10 ¹⁰ gv transduced 70.5 ± 2.5% of muscle fibers, and 1x 10 11 gv transduced 89.0 ± 4.0% of muscle fibers, 3 weeks after gene transfer.

Example 2: Intramuscular delivery of scAAVrh.74.tMCK.hSGCB.

After testing the vector's effectiveness, the studies were expanded to analyze the effectiveness of the therapy 6 and 12 weeks after gene transfer. As a result of the high level of expression after the short 3-week efficacy study, the lowest effective total dose of 3x10 ¹⁰ gv was selected for use in subsequent studies. Five-week-old sgcb' ^/_ mice were treated with 3x10 ¹⁰ gv scAAVrh.74.tMCK.hSCGB intramuscularly into the left transverse abdominis (TA) muscle and β-sarcoglycan expression was demonstrated using immunofluorescence in 88.4±4.2% of muscle fibers after 6 weeks postinjection (n=9), and in 76.5±5.8% of muscle fibers 12 weeks postinjection (n=6), and expression was confirmed by Western blotting (Fig. 1B). Expression of β-sarcoglycan was accompanied by restoration of components of the dystrophin-associated protein complex (α-sarcoglycan and dystrophy) (Fig. 1C). Using Evans blue dye (EBD) as a marker of membrane permeability (22, 23), we found that all fibers expressing exogenous β-sarcoglycan were protected from leakage and EBD incorporation (Fig. 1D). Muscles from sgcb' ^/ ' mice showed severe muscular dystrophy with centrally nucleated fibers, frequent muscle fiber necrosis, fibrous tissue, and significant variability in fiber size, represented by both atrophic and hypertrophic fibers (3, 4). As shown in FIG. 2A, Hematoxylin and eosin staining demonstrates an overall improvement in the dystrophic phenotype of the affected muscle, including restoration of the central nuclei (untreated sgcb ^{- /} ' - 76.8 ± 2.3% vs. treated AAV.hSCGB - 38.86 ± 3.5%, P < 0.0001) (Fig. 2C). There was also a normalization of fiber size distribution with an increase in average fiber diameter after treatment (untreated sgcb --- 32.6 ± 0.31 µm compared to treated AAV.hSGCB - 35.56 ± 0.22 µm, P < 0.0001) (Fig. 2D).

The histopathological hallmark of the scgb- mouse is fibrosis, characterized by extensive replacement of muscle tissue primarily by collagens along with other extracellular matrix components such as fibronectin, elastin, laminin, and decorin (14). This replacement of muscle tissue with connective tissue challenges the potential value of gene replacement and may limit the extent of improvement (24). To test this, mice treated for 12 weeks were analyzed for reduced fibrosis. The TA muscle was specifically evaluated because its heritable degree of fibrosis was recapitulated in the KO model and because it represents a potential target for gene delivery by vascular ILV. Picrosirius red staining

- 17 044609 collagen types I and III TA muscles showed a significant decrease (52.74%) in the amount of collagen present in scAAVrh.74.tMCK.hSGCB-treated muscles compared to untreated muscles of scgb ^-/- mice (20.7 ± 0 .57% vs. 43.8±2.3%, AAV.hSGCB-treated vs. untreated sgcb ^−/− , respectively, P < 0.0001) (Fig. 2b and e). Untreated muscle from scgb −/− mice 5 weeks old at the time of injection had 24.05 ± 1.5% collagen deposition, indicating a small (14.0%) decrease in collagen amount after 12 weeks of treatment.

Example 3 Correction of function in skeletal muscle after scAAVrh.74.tMCK.hSGCB gene transfer.

To determine whether hSGCB gene transfer could improve muscle function, we assessed the functional properties of the TA muscle in scgb ^−/− mice treated with scAAVrh.74.tMCK.hSCGB. Following intramuscular delivery of 3x10 ¹⁰ gV scAAVrh.74.tMCK.hSCGB into the TA of 4-week-old scgb ^−/− mice, TA muscles were subjected to in situ force measurements 6 weeks after treatment (n=4). Treated muscles were compared with untreated contralateral muscles and those from C57BL/6 WT mice. scAAVrh.74.tMCK.hSCGB-treated muscle showed significant improvement in both absolute tetanic force and normalized specific force (Fig. 3A and B). Treated muscles had a mean absolute force of 1436.9 ± 199.5 mN compared with 770.9 ± 118.3 mN of untreated scgb ^−/− controls (P < 0.01). Similarly, treated TA muscles produced a mean specific force of 254.01 ± 6.9 mN/mm ² and untreated muscles produced 124.2 ± 13.9 mN/mm ² force (P < 0.01). Finally, muscles treated with scAAVrh.74.tMCK.hSCGB showed greater resistance to contraction-induced injury compared with untreated control muscles ( Fig. 3C ). The treated TA muscle lost 34.0 ± 5.1% of the force produced after the first contraction, whereas the untreated diseased muscle lost 54.1 ± 3.8% (P < 0.01) of force after performing the eccentric contraction protocol. These data indicate that hSGCB gene transfer does provide functional improvement in disease-prone β-sarcoglycan-deficient muscle.

Example 4: Treatment of old muscles with scAAVrh.74.tMCK. hSGCB.

Studies of disease progression in this LGMD2E mouse model have shown that although the most severe tissue remodeling in muscle occurs between 6 and 20 weeks, muscle histopathology continues to deteriorate with age, reminiscent of disease progression in patients (3, 4,14). Therefore, to simulate the clinical situation where treatment would occur at an older age with more advanced muscle damage and endomysial fibrosis, we treated 6-month-old sgcb ^/ mice (n=5) intramuscularly in the TA with 3x10 ¹⁰ gv scAAVrh.74.tMCK .hSCGB. After 12 weeks of treatment, at 9 months of age, 80.1 ± 4.8% of muscle fibers were transformed (Fig. 4A). Picrosirius red staining of collagen types I and III showed a significant 42.2% reduction in the amount of collagen present in treated mice compared to untreated scgb ^−/− mouse muscle (AAV.hSGCB treated - 20.0 ± 0.80% vs. untreated scgb ^−/− −34.6 ± 1.4%, P < 0.0001) ( Fig. 4B and C ). At treatment age, 6-month-old scgb −/− mice had 30.8 ± 2.0% collagen deposition (n = 4, 4 males); Thus, these results indicate that treatment with scAAVrh.74.tMCK.hSCGB not only prevents but also has the potential to reverse existing fibrosis.

Example 5. ILP scAAVrh.74.tMCK.hSGCB in sgcb ^-/- mice.

The ability to target multiple muscles in a single limb allows for a more clinically relevant delivery method for delivery to LGMD2E patients. Delivery of 5x1011 gV scAAVrh.74.tMCK.hSGCB ILP into 4-6 week old scgb ^−/− mice (n=9, 7 males, 2 females) was analyzed 2 months after gene transfer. β-sarcoglycan expression reached 91.8 ± 4.7% of fibers in the gastrocnemius muscle (GAS) and 90.6 ± 2.8% in the TA (Fig. 5A). ILP delivery of scAAVrh.74.tMCK.hSGCB resulted in a significant increase in protection from eccentric contraction-induced injury ( P < 0.05), which was not different from WT compared with untreated contralateral muscles ( Fig. 5C ). Vascular delivery also restored muscle histopathological parameters (Figure 5B).

Central nuclei were reduced in TA (untreated sgcb ^-/- 76.9 ± 2.8% vs. treated AAV.hSGCB - 23.2 ± 5.7%, P < 0.001) and GAS (untreated scgb ^-/- - 78.2±2.4% compared to those treated with AAV.hSGCB - 16.8±6.6%, P<0.001). Gene transfer also led to an increase in the average fiber size in the TA (untreated scgb ^-/- - 30.53 ± 0.52 μm compared to AAV.hSGCB - treated - 41.9 ± 0.46 μm, P < 0.0001) and GAS (untreated scgb ^-/- - 38.9 ± 0.37 μm vs. treated AAV.hSGCB - 33.3 ± 0.44 μm, P < 0.0001), with normalization of fiber diameter distribution. A significant decrease (approximately 60%) in the number of CD3 cells, CD4 cells and macrophages was observed (Table 1).

- 18 044609

Table 1

Immune response in ILP-treated scAAVrh.74.tMCK.hSGCB mice

Processed Raw Uninjected Cell type left TA right TA TA SGCB-/- cells/mm ² cells/mm ² cells/mm ² CD3 15.6 + 3.2 37.85 + 6.2 29.8 + 1.7 CD4 20.9 + 4.7 58.1 + 2.9 49.0 + 0.8 CD8 8.2 + 1.8 12.7 + 2.4 15.5 + 5.8 Macrophage 28.2 + 5.0 75.2 + 5.6 100.2 + 5.9

Abbreviations: ANOVA, analysis of variance; ILP, isolated limb perfusion; SGCB, βsarcoglycan; TA, tibialis anterior muscle. Enumeration of the number of immune cells present in uninjected SGCB'/' mice and scAAVrh.74.tMCK.hSGCB-treated and untreated muscles. Data are shown after ILP delivery of the virus and represent the mean number of cells/mm ² ±s.d.s., n=8 per group. One-way analysis of variance was used to compare values from the three different groups. The numbers of immune cells were reduced with a statistically significant difference (P<0.01) between treated left TA and untreated right TA and/or treated left TA and uninjected TA in all strains except for CD8.

Picrosirius red staining of TA and GAS muscles also showed a significant reduction in the amount of collagen compared to untreated sgcb ^-/ ' muscle after vascular delivery (Fig. 6A). TA collagen levels decreased to 21.6 ± 1.3% in treated muscle compared with 40.2 ± 1.5% in untreated scgb ^-/ ' mice at study endpoint age (P < 0.0001). As stated previously, scgb ^−/ ′ mice at injection age had 24.1 ± 1.5% collagen in the TA muscle, indicating a slight decrease (10.0%) in collagen deposition after 8 weeks of treatment. Similarly, GAS muscle staining showed that treated mice had 22.9±0.99% collagen compared to 37.9±1.3% in untreated scgb ^-/ ' mice at study endpoint (P<0.0001) . Qualitative PCR was performed to detect collagen transcript levels in muscle that correlated with Sirius Red staining results. Taken together, these data demonstrate that AAV-mediated delivery of human β-sarcoglycan reduces muscle fibrosis, improves muscle function, and reverses the dystrophic pathology of disease-prone scgb'/' muscle.

Example 6: Safety and biodistribution of rAAVrh.74.tMCK.hSGCB.

Initially, normal WT mice injected with 3x10 ¹⁰ g of scAAVrh.74.tMCK.hSGCB intramuscularly into the TA showed no signs of toxicity by H&E staining, indicating no adverse effects caused by the virus. Following intravascular ILP delivery of 5x1011 gv of a total dose of scAAVrh.74.tMCK.hSGCB as described in the previous section, safety was assessed in a small subset of mice in this cohort (n=4). First, target muscles with significant gene expression were analyzed histologically, as were non-target organs, including heart, lung, liver, kidney, spleen, gonads and diaphragm. Paraffin sections were reviewed accordingly by a veterinary pathologist and no evidence of toxicity was found in any designated organ (data not shown). Protein expression and vector biodistribution were also assessed in all the above tissues and organs using Western blotting and qPCR, respectively. Copies of the vector genome were found in all organs tested; however, protein expression was not detected in any samples other than the treated muscle (Fig. 7). Finally, analysis of treated and untreated wet muscle masses showed no significant differences or trends when comparing mean masses compared to the main group (data not shown). These data suggest that the muscle-specific tMCK promoter restricted expression to skeletal muscle and the vector is nontoxic.

Example 7: Histological and functional defects in the heart and diaphragm of SGCB'/' mice.

WT and 7-month-old SGCB'/' mice (n=6 per strain) that were not treated were analyzed with cardiac MRI and diaphragm physiology to look for defects. Following these analyses, animals were sacrificed and histopathology assessed (Fig. 8). Tricolor staining showed extensive fibrosis (red staining) in both the diaphragm (Fig. 8A) and the heart (Fig. 8C). This was accompanied by a functional deficiency of specific force in the diaphragm (116.24 mN/mm ² SGCB'/' vs. 236.67 mN/mm ² DT, Fig. 8B) and a significant deficiency of ejection fraction measured by MRI (DT, 78 % compared to SGCB'/' 65%, Fig. 8D).

Example 8. Construction of scAAVrh.74.MHCK7.hSGCB and vector efficiency.

A transgene cassette containing the full-length codon-optimized human SCGB cDNA was constructed as shown in FIG. 9A. The cassette includes the Kozak consensus sequence (CCASS), a chimeric SV40 intron, a synthetic polyadenylated site

- 19 044609 vaniya and muscle-specific MHCK7 used to control the expression of the cassette. This is an MCK-based promoter that uses a 206-bp enhancer taken from approximately 1.2 kb of the 5′ transcription start site in the endogenous muscle creatine kinase gene with a proximal promoter (enh358MCK, 584bp) ^3,12 . The cassette was packaged into the self-complementary (sc) AAV vector rh.74, which is 93% homologous to AAV8. AAVrh.74 has been shown to be safe and effective in mice and non-human primates, particularly in crossing the vascular barrier when delivered into muscle via the circulatory system (17, 18, 21). Vector efficacy was determined by intramuscular injection into the left TA of a Sgcb-null mouse. Delivery of 3x10 ¹⁰ gv transduced >90% of muscle fibers 4 weeks after gene transfer.

Example 9 Systemic delivery of scAAV.MHCK7.hSGC.

We delivered the vector via tail vein injection to 14 SGCB ⁻ / ⁻ mice at a dose of 1 x 10 ¹² gV total dose (5 x 10 ¹³ gV/kg) to evaluate transgene expression and the efficacy of our vector when delivered systemically with a long-term six-month control time point. Mice were injected at 4 weeks of age, and full necropsy was performed 6 months after injection (1 mouse was removed from the study at 1 month and 2 mice were removed at 4 months as interim expression assessments). All skeletal muscles discussed above, along with the diaphragm and heart, were removed for analysis. Organs were also removed for toxicology and biodistribution analysis.

Human beta-sarcoglycan immunofluorescence staining was used to determine hSGCB transgene expression in 5 limb muscles, both left and right, in addition to the diaphragm and heart of 6 KO mice that were given a systemic injection of the hSGCB vector. These muscles included the TA, gastrocnemius (GAS), quadriceps (QUAD), gluteus (GLUT) (not shown), psoas (PSOAS), and triceps (TRI) (Fig. 10). Qualitative analysis of cardiac tissue was also used to assess the relative level of transgene expression in cardiac muscle after delivery.

Four 20x images were acquired for each muscle, and the percentage of hSGCB positive fibers was determined for each image, resulting in an average percentage of transduction for each muscle from each mouse. The results shown in FIG. 10 and fig. 11 demonstrate >98% transduction in all muscles analyzed, including the diaphragm and heart. β-sarcoglycan-deficient mice completely lacked the protein as shown by immunofluorescence analysis. The therapeutic dose of a total dose of 1 x 10 ¹² gV resulted in an average vector transduction of 97.96 ± 0.36% (±SEM) in all skeletal muscles, including the diaphragm, and approximately 95% or greater in cardiac muscle (data not shown).

Example 10 Long-term systemic delivery of scAAVrh.74.MHCK7.hSGCB to SGCB-- mice.

Long-term (6-month) systemic delivery of the β-sarcoglycan cassette transgene was studied in sgcb ^- / ^- mice to obtain results from the one-month efficacy assay described in Example 9. 4-5 week old sgcb ^- / ^- mice were treated with a total dose of 1x10 ¹² gv scAAVrh.74.MHCK7.hSGCB intravenously through the tail vein (n=5). Mice were necropsied 6 months after injection, and hSGCB transgene expression was demonstrated using immunofluorescence in six skeletal muscles, both left and right, in addition to the diaphragm and heart, in all treated mice. Skeletal muscles analyzed included TA, GAS, QUAD, gluteal muscle (GLUT), PSOAS, and TRI. The average hSGCB expression induced by systemic delivery in treated mice was 98.13±0.31% (±SEM) for all skeletal muscle, including the diaphragm, with expression in the heart exceeding >95%. Representative images are shown in FIG. 12b. Expression levels in each individual muscle type, averaged across all treated mice, are shown in Table 1. 2. Western blotting as shown in FIG. 12c confirms transgene expression in all muscles. Expression values in Table 2 are presented for different muscles as the average of left and right muscles from systemically injected mice (n=5). Values are given as mean ±SEM. In addition, quantification of hSGCB transgene expression in hearts from treated mice by Western blotting and densitometry indicates hSGCB overexpression up to 72.0% above BL6 WT expression levels (Fig. 12d), which correlates with the high levels detected in skeletal muscle.

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table 2

Immunofluorescence of β-sarcoglycan expression

Dose Ultimate % Fiber, Muscle Delivery way IV (gv Total dose) dot (months) expressing SGCB TA (intravenously) IV 1e12 6 98.88 ± 0.55 G.A.S. (intravenously) IV 1e12 6 98.24 + 0.82 QD (intravenously) IV 1e12 6 99.32 ± 0.19 GLUT (intravenously) IV 1e12 6 97.50 ± 0.39 PSOAS (intravenously) IV 1e12 6 98.75 ± 0.23 TRI (intravenously) IV 1e12 6 97.21 ± 1.35 Diaphragm (intravenously) IV 1e12 6 97.00 ± 1.26 Heart (intravenously) 1e12 6 >95% An important characteristic of the sgcb muscle. described in previous reports (Araishi et al., Hum.

Mol. Genet 8: 1589-98, 1999, Durbeej et al., Mol. Cell. 5:141-51, 2000) and illustrated by hematoxylin and eosin staining of the GAS and diaphragm in FIG. 13a is a severe degenerative pathology, including central nucleation, necrosis, inflammatory infiltration and fibrosis. Gene transfer significantly improved the condition of this pathology, alleviating many of these dystrophic symptoms (Fig. 13a). Quantification of histological parameters showed a significant reduction in central nucleation in various skeletal muscles analyzed as a result of gene transfer (Fig. 13b). With the expected low levels of central nucleation in BL6 WT mice in all muscles averaging 1.89 ± 0.39%, as noted herein, taking into account all muscles analyzed, an average of 66.85 ± 1.86% central nuclei in untreated ssgcb ⁷ ' mice compared to 36.30±5.16% in AAV.MHCK7.hSGCB-treated sgcb ^- / ^- muscle (p < 0.0001) in Table. Figure 3 below shows the counts of central nuclei and fiber diameters for various muscles as the mean (±SEM) of left and right muscles from BL6 WT, sgcb ^-/- and systemically injected mice (n=5 per group). It should be noted that the most significant wave of destruction/repair occurs after 3 weeks in the sgcb ^-/- muscle, as evidenced by centrally located nuclei. The animals were treated after such damage and therefore complete recovery of the central nuclei was not expected. A more in-depth analysis of muscle histopathology revealed a normalization of fiber size distribution accompanied by an increase in mean fiber diameter in vector-treated diseased mice compared to untreated sgcb'/' mice in all three muscles examined (GAS: untreated sgcb'/' - 28.37± 0.23 µm compared to treated AAV.hSGCB - 36.04±0.17 µm, p<0.0001) (PSOAS: untreated sgcb' ^A - 24.75±0.23 µm compared to treated AAV.hSGCB - 38.43±0.28 µm, p<0.0001) (TRI: untreated sgcb' ^A - 28±0.31 µm compared to treated AAV.hSGCB - 35.56±0.22 µm, p<0 .0001) (Fig. 13c, 13d, Table 3).

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Table 3

Percent Central Nucleation Analysis

Group of animals Dose (guards Total dose) Muscle % Central cores (Mean± COC) Combined average % CN (± SEM) Fiber diameter µm (Mean± SEM) C57BL6 DT N/A TA 1.78 + 0.86 1.89 ± 0.39 N/A G.A.S. 0.83 + 0.41 39.69 ± 0.18 QD 0.98 + 0.31 N/A GLUT 2.50 + 0.68 N/A PSOAS 1.26 + 0.28 40.96 ± 0.22 TRI 2.13 + 0.36 41.53 ± 0.24 DIA 3.75 + 1.30 N/A ZdsN^ N/A T.A. 70.45+ 3, 04 66.85 ± 1.86 N/A G.A.S. 67.26+ 1.81 28.37 ± 0.23 QD 63.57 + 2.09 N/A GLUT 61.34 + 2.05 N/A PSOAS 62.73 + 5.20 24.75 ± 0.22 TRI 67.11 + 2.83 28.74 ± 0.22 DIA 75.47+ 3.79 N/A Processed AAV.MHCK7.hSG NE 1.00E+ 12 T.A. 43.85 + 3, 89 36, 30 ± 5.16 N/A G.A.S. 38.71 + 3.50 36.04 ± 0.18 QD 46, 10+ 6.26 N/A GLUT 42.11 + 5.48 N/A PSOAS 21.00 + 4, 69 38.43 ± 0.28

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TRI 48.39 ± 6.20 39.92 ± 0.27 DIA 11.59 ± 2.08 N/A

Due to the significant role of fibrosis in the pathogenesis of LGMD2E and the effectiveness of therapy, it was important to demonstrate the same effectiveness in reducing fibrosis. This was found with localized β-sarcoglycan gene transfer immediately following systemic delivery of scAAVrh.74.MHCK7.hSGCB. Using picrosirus red staining for collagen types I and III, we analyzed collagen levels in the gastrocnemius muscles and diaphragm of 7-month-old BL6 WT mice (n=4), untreated sgcb ^-/ mice (n=4), and treated sgcb' ^A mice ( n=5) 6 months after injection. Treated muscles showed significantly less collagen deposition compared to untreated sgcb'/' muscles (Fig. 14a). Vector-transduced GAS muscle contained 17.55 ± 0.59% collagen compared to 43.55 ± 3.33% collagen in untreated GAS sgcb' ^A muscle (p < 0.0001). Additionally, the treated diaphragm had 21.67±1.09% collagen compared to 44.05±2.39% in untreated ^sgcb'A muscle (p<0.0001) (Fig. 14b), demonstrating the ability to transfer the hSGCB gene reduce the fibrotic component of the LGMD2E phenotype.

Example 11: Restoration of diaphragm function after systemic delivery.

To determine whether hSGCB gene transfer could improve muscle function, we assessed the functional properties of the diaphragm muscle in SGCB ⁷ ' mice treated with scAAVrh.74.MHCK7.hSCGB (see Griffin et al. for methods). First, we established the presence of functional deficiency in the diaphragms of SGCB ⁷ ' mice. KO diaphragms showed a 50.9% decrease in specific force (116.24 mN/mm ² ) compared to BL6 WT mice (116.24 mN/mm ² versus 236.67 mN/mm ² ) and greater loss of strength after intensive fatigue protocol (23% loss in SGCB ⁷ '; 7% loss in BL6 DT). Delivery of scAAVrh.74.MHCK7.hSGCB via the tail vein resulted in almost 100% expression of hSGCB in the diaphragm, resulting in restoration of diaphragm function with specific force improved to 226.07 mN/mm ² and greater fatigue resistance with only 12% loss. strength (n=5) (Fig. 15).

Example 12: Delivery of scAAVrh.74.CMV.miR29C reduces fibrosis in SGCB'/' mice.

The extensive fibrosis that we identified in both skeletal muscle (Figs. 2, 4, and 6) and in the heart and diaphragm (Fig. 8) demonstrated the need to treat collagen deposition (fibrosis) in LGMD2E. We previously found that Mir29C (out of Mir29A, B and C) was most severely reduced in Duchenne muscular dystrophy. It was hypothesized that Mir-29C would also be reduced in beta-sarcoglycan-deficient mice (LGMD2E mouse model). We have proven that this is true (Fig. 15). Mir29C amounts were decreased, fibrosis (collagen) levels were increased, and three components of fibrosis (Co1A, Co13A, and Fbn) were increased at the RNA level. To test whether we could prevent fibrosis with Mir29C, the tibialis anterior muscle of 4-week-old SGCB'/' mice (n=5) was injected with the gene therapy vector scrAAVrh.74.CMV.miR29c (3x1011 gv). scrAAVrh.74.CMV.miR29c is shown in Fig. 16 and is described in US Provisional Application No. 62/323,163, the disclosure of which is incorporated herein by reference in its entirety. After 2 months of treatment with AAVrh.74.CMV.miR29C, TA muscles were harvested from treated, control, and WT SGCB'/' mice and analyzed for fibrosis (collagen levels) (n=5 per group). Using Sirius red staining and quantitation, collagen levels were found to decrease after treatment (see FIG. 17). The amounts of Col1A, Col3A and Fbn transcripts were normalized, and muscle fiber size increased. Representative images of scanned whole sections of untreated and AAVrh.74.CMV.miR29C-treated tibialis anterior muscles stained with Sirius red, which stains collagen 1 and 3, are shown in Figs. 18. This suggests that scAAVrh.74.CMV.miR29C reduces fibrosis in SGCB'/' mice and can be used in combination with gene replacement using scAAVrh.74.tMCK.hSGCB or scAAVrh.74.MHCK7.hSGCB.

Example 13: Intravenous gene transfer into SGCB'/' mice reduces kyphoscolysis of the thoracic spine.

Degeneration of the trunk muscles due to worsening histopathology in patients suffering from LGMD2E can be attributed to kyphosis. Kyphoscoliosis of the thoracic spine, due to weakening of the muscles that support the spinal column, can cause the diaphragm to be pushed forward, further impairing lung capacity and diaphragmatic function. Whole-body radiography was used to determine the degree of kyphosis in 7-month-old BL6 WT mice (n=6), sgcb ^-/- mice (n=6), and treated sgcb'/' mice 6 months post-injection (n=6). due to the severity of the phenotype in the sgcb'/' mouse with a common anatomical appearance of kyphoscoliosis. The Kyphotic Index (KI) score quantifies the degree of kyphoscolysis (Laws et al. J. Appl. Physiol. 97: 1970-7, 2004). As shown in the DT panel in FIG. 19a, the KI score is the ratio of the length from the forelimb to the hindlimb compared to the length of the midline to the apex of curvature in the spine. While the sgcb' ^/ 'mice had

- 23 044609 highly curved spine and lower KI score of 3.64±0.16 (n=6), BL6 WT mice have a significantly straighter spine, resulting in a higher KI score of 6.01±0.41 (n=6 ) (p<0.01) (Fig. 19b). Treated sgcb-/- mice showed a significant reduction in the degree of spinal kyphosis with an increase in KI score to 5.39±0.58 (n=6) (p<0.05) (Fig. 19b). These data showed that intravenous delivery of scAAVrh.74.MHCK7.hSGCB is beneficial to the overall integrity of the spine and may alleviate kyphosis and joint contractures present in this disease. These data demonstrated an improvement in kyphosis and an increase in physical activity in sgcb ^- / ^- mice after systemic delivery of the rAAV vector of the invention. These data provide further evidence that the gene therapy of the invention improves the quality of life of patients with LGMD2E.

Example 14: Evaluation of cardiomyopathy.

Histological destruction of the limb muscles and diaphragm was also detected in the myocardium of 7-month-old sgcb ^- / ^- mice, especially with the appearance of myocardial necrosis and fibrosis, as seen by HyE and Pyrcosirius red staining (Fig. 20a). Presentation of cardiac dysfunction is often in the form of dilated cardiomyopathy with reduced cardiac output and lower ejection fraction (Semplicini et al., Neurology 84: 1772-81, 2015, Fanin et al., Neuromuscul Disorder 13:303-9, 2003). Cardiac magnetic resonance imaging (MPT) was used to assess several cardiac functional parameters to establish functional deficits in the myocardium of sgcb ⁻ / ⁻ mice compared with BL6 WT mice for use as a measure of functionality. Imaging of control mice at 7 months of age showed a 29.4% decrease in stroke volume with 0.041 ± 0.0019 μl in sgcb ^- / ^- hearts compared to 0.029 ± 0.0024 ml in BL6 WT hearts (p < 0.01), 31.7% lower cardiac output with 14.70±0.74 µl/min in sgcb ^- / ^- hearts compared to 12.72±0.97 ml/min in BL6 WT hearts and, finally, 14, 3% lower ejection fraction, 66.21±3.83% in sgcb ^-/ hearts compared to 76.90±1.67% in BL6 WT hearts (p<0.05) (Fig. 20b). This indicates a moderate decline in overall cardiac function at this age and a tendency toward the development of cardiomyopathy. Restoring hSGCB expression in the hearts of KO mice through systemic delivery partially corrected these deficiencies, improving stroke volume to 0.032±0.0027 ml, cardiac output to 14.66±0.75 ml/min and ejection fraction to 68.16±2. 31% (Fig. 19b). Correlating with the histological and functional impairment of cardiac tissue described herein, expression of cardiac troponin I (cTrpI), an important regulator of cardiac function and indicator (biomarker) of cardiac injury, was reduced by up to 60 in the hearts of sgcb ^- / ^- patients, as demonstrated by Western blot analysis. .38% of the levels observed in BL6 WT mice (Fig. 20c). cTrpI levels are restored after treatment to 35.80% expression levels observed in WT hearts (Fig. 20d).

Example 15. Functional recovery in the diaphragm muscles with increased physical activity.

Because diaphragmatic dysfunction and respiratory failure are significant in LGMD2E, it is important to consider the functional benefit to the diaphragm when clinically evaluating systemic therapy. Using an ex vivo experimental protocol on strips taken from the diaphragm muscle, it was assessed whether β-sarcoglycan restoration provides a functional benefit in this severely impaired muscle. Consistent with the significant histopathology found in the diaphragms of 7-month-old diseased mice, sgcb ^- / ^- (n=4) diaphragms exhibited functional failure with a significant (51%) reduction in specific force compared to BL6 WT mice (n=5) (116,24 ±10.49 mN/mm ² compared to 236.67 ± 15.87 mN/mm ² , respectively, p < 0.001), as well as a greater loss of force compared to that produced after the first contraction after the intensive protocol fatigue (23±1.0% loss in sgcb ^- / ^- ; 7.0±3.0% loss in BL6 WT, p <0.05) (Fig. 6a). Six months after tail vein delivery of scAAVrh.74.MHCK7.hSGCB, a dramatic improvement in specific force was observed. Specific force increased to 226.07±27.12 mN/ ^mm2 (n=5) (p<0.05 compared with sgcb ^-/- ), and better protection of muscles from repeated fatigue was observed with loss of strength only at 12. 0±4.0% (p<0.05 compared to sgcb ^- / ^- ) (Fig. 21a). Overall, these data support our previous results in the TA muscle and indicate that restoration of β-sarcoglycan mediates functional recovery of the diaphragm muscle.

Symptoms of increased fatigue and decreased overall activity are often reported in many neuromuscular diseases, which is partly due to the occurrence of kyphosis. As a result, and given the LGMD2E phenotype, it was hypothesized that KO mice would naturally be less active compared to healthy WT mice, and furthermore, systemic delivery of rAAV.MHCK7.hSGCB to sgcb ^−/− mice would make the mice more physically active. To test this hypothesis and the additional potential functional benefits of gene transfer, all groups of mice were subjected to a laser activity monitoring protocol in open space cages similar to that described in Kobayashi et al., Nature 456: 511-5, 2008 and Beastrom et al., Am . J. Pathol. 179: 2464-74, 2011. Graphs in Figs. 21b demonstrate a significant reduction (55.5%) in KO mice compared to WT mice in both overall locomotion (horizontal movement in the x and y planes) and vertical hindlimb locomotion. Average number of laser beam interruptions horizontal

- 24 044609 active movement during 1 hour in WT mice was 7355±400.8 (n=6) compared to 3271±483.8 (n=6) in KO mice (p<0.0001). In addition, the mean number of vertical laser beam interruptions recorded in WT mice was 626.7 ± 53.76 compared to 264.5 ± 63.36 in KO mice (p < 0.01) (Fig. 21b). Consistent with the original hypothesis, rAAV.MHCK7.hSGCB-treated mice were markedly more active compared to KO, as illustrated by activity quantification where total movement increased by 22% to 5143±293.2 laser beam interruptions (p< 0.05) and vertical movement on the hind legs increased dramatically by 77% to 615.3±95.93 laser beam interruptions (p<0.05) in treated mice (n=6) (Fig. 21b).

Example 16: Safety and biodistribution analysis of rAAVrh.74.MHCK7.hSGCB.

Potential toxicity or safety concerns of hSGCB gene therapy were assessed in sgcb ^−/− mice 6 months after systemic delivery of scAAVrh.74.MHCK7.hSGCB at a total dose of 1.0 x 10 ¹² gV (5 x 10 ¹³ gV/kg). Vector biodistribution and off-target transgene expression were analyzed in tissue samples (TA, TRI, diaphragm, heart, gonads, lung, kidney, liver and spleen) from vector-dosed sgcb- - animals, using qPCR and Western blotting, respectively. Using vector-specific primer-probe sets, MHCK7.hSGCB vector genomes were detected in varying amounts in all tissues collected. As expected, the highest levels were observed in the liver, as well as in skeletal muscle and heart, indicating that the test article was effectively delivered to all intended muscles of vector-dosed mice (Fig. 22a). In addition, Western blot analysis for hSGCB protein expression confirmed the functionality of the muscle-specific MHCK7 promoter and transgene expression restricted to cardiac and skeletal muscle. Beta-sarcoglycan protein expression was observed at varying levels in all skeletal muscle samples as well as heart samples and, importantly, was not detected in any other non-muscle tissue (Fig. 22b), which is supported by the fact that beta-sarcoglycan is known as a muscle-specific protein. Finally, hematoxylin and eosin staining was performed on cryosections of muscle tissue and all non-target organs collected from five sgcb ^- / ^- mice and five C57BL6 WT mice treated systemically with our vector at the therapeutic dose used in this study. These sections were then respectively reviewed for toxicity by a veterinary pathologist and no harmful effects were found in any sample from either mouse. Taken together, these data indicate that the test product was well tolerated by the test animals.

The fact that such high levels of transduction in all muscles throughout the body were achieved without side effects using a relatively low dose (total dose 1 x 10 ¹² gV, 5 x 10 ¹³ gV/kg) holds great promise for practical use in LGMD2E patients. From a clinical perspective, the dose used in the experiments described herein is much lower than the dose used for systemic delivery of the SMN1-expressing AAV drug given to infants with SMA (spinal muscular atrophy), which is currently in clinical trials (Mendell et al., Mol. Ther. 24: S190, 2016). The highly effective restoration of β-sarcoglycan expression using the MHCK7 promoter, accompanied by functional improvements, is very encouraging at dosage levels that can be used clinically, and systemic delivery is of great benefit to these patients given the high cardiac involvement of β-sarcoglycan deficiency in patients with LGMD2E.

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Claims (11)

muscles and cardiac muscle of transgenic mice. Mol Cell Biol 1996; 16:5058-5068. 28 Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X et al. Cross-packaging of a single adeno-associated virus (M W) type 2 vector genome into multiple MW serotypes enables transduction with broad specificity. J Virol 2002; 76: 791-801. 29 Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno-associated viral vectors. Nat Protoc 2006; 1: 1412-1428. 30 Clark KR, Liu X, McGrath JP, Johnson PR. Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther 1999; 10: 1031-1039. 31 Liu M, Yue Y, Harper SQ, Grange RW, Chamberlain JS, Duan D. Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury. Mol Ther 2005; 11: 245-256. 32 Hakim CH, Grange RW, Duan D. The passive mechanical properties of the extensor digitorum longus muscle are compromised in 2- to 20-mo-old mdx mice. J Appl Physiol 2011; 110: 1656-1663. 33 Wein N, Vulin A, Falzarano MS, Szigyarto CA, Maiti B, Findlay A et al. Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nat Med 2014; 20: 992-1000. CLAIM 1. A recombinant AAV vector containing a polynucleotide sequence encoding a β-sarcoglycan, which contains the nucleotide sequence SEQ ID NO: 1. 2. A recombinant AAV vector containing a polynucleotide sequence encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2. 3. The recombinant AAV vector according to any one of claims 1 or 2, wherein the vector has a serotype of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAV rh.74. 4. Recombinant AAV vector according to any one of claims 1 to 3, wherein the polynucleotide sequence contains a muscle-specific regulatory element. 5. The recombinant AAV vector of claim 4, wherein the muscle-specific regulatory element is a human skeletal muscle actin gene element, a cardiac actin gene element, a myocyte-specific enhancer binding factor mef, a muscle creatine kinase (MCK), a truncated MCK ( tMCK), myosin heavy chain (MHC), MHCK7, C5-12, mouse creatine kinase enhancer element, fast-twitch skeletal muscle fiber troponin C gene element, slow-twitch cardiac muscle fiber troponin C gene element, slow-twitch muscle fiber troponin I gene element, nuclear hypoxia-inducible response element, steroid-inducible element, or glucocorticoid response element (gre). 6. The recombinant AAV vector according to claim 5, wherein the muscle-specific regulatory element is a truncated MCK promoter (tMCK). 7. The recombinant AAV vector according to claim 6, wherein the truncated MCK promoter (tMCK) contains the nucleotide sequence set forth in SEQ ID NO: 6. 8. The recombinant AAV vector according to claim 4, wherein the muscle-specific regulatory element is the MHCK7 promoter. 9. The recombinant AAV vector according to claim 8, wherein the MHCK7 promoter contains the nucleotide sequence set forth in SEQ ID NO: 4. 10. Recombinant AAV vector according to any one of claims 1 to 5, containing a nucleotide sequence at least 95% identical to SEQ ID NO: 3. 11. Recombinant AAV vector according to any one of claims 1-5, containing a nucleotide sequence - -