Adeno-associated virus vector delivery of muscle specific micro-dystrophin to treat muscular dystrophy

Abstract

To provide gene therapy vectors, such as adeno-associated virus (AAV) vectors, expressing a miniaturized human micro-dystrophin gene, and a method of using these vectors to express micro-dystrophin in skeletal muscles including diaphragm and cardiac muscle and to protect muscle fibers from injury, increase muscle strength and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy.SOLUTION: The present invention relates to gene therapy vectors, e.g. AAV, expressing the micro-dystrophin gene to skeletal muscles including diaphragm and cardiac muscle to protect muscle fibers from injury, increase muscle strength and reduce and / or prevent fibrosis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 686,668, filed June 18, 2018, U.S. Provisional Patent Application No. 62 / 740,402, filed October 2, 2018, U.S. Provisional Patent Application No. 62 / 752,841, filed October 30, 2018, U.S. Provisional Patent Application No. 62 / 823,649, filed March 25, 2019, and U.S. Provisional Patent Application No. 62 / 860,220, filed June 11, 2018, each of which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials This application contains, as a separate part of this disclosure, a Sequence Listing in computer readable form, which is incorporated by reference in its entirety and identified as follows: Filename: 53169_Seqlisting.txt; Size: 60,056 bytes; Creation Date: June 17, 2019.

[0003] The present invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, that express a miniaturized human micro-dystrophin gene, and methods of using these vectors to express micro-dystrophin in skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy. [Background technology]

[0004] The importance of muscle mass and strength for daily activities such as locomotor activity and respiration, as well as for body metabolism, is evident. Deficits in muscle function result in muscular dystrophies (MDs), characterized by muscle wasting and atrophy, severely impacting quality of life. The most well-characterized MDs result from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs result from membrane fragility associated with loss of sarcolemma-cytoskeleton anchoring by DAPCs. Duchenne muscular dystrophy (DMD) is one of the most serious muscle diseases, affecting one in every 5,000 newborn boys.

[0005] DMD is caused by mutations in the DMD gene that result in reduced mRNA and the absence of dystrophin, a 427-kD sarcolemmal protein that associates with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51(6):919-28, 1987). The DAPC is composed of multiple proteins in the muscle sarcolemma that form structural links between the extracellular matrix (ECM) and the cytoskeleton via the actin-binding protein dystrophin and the laminin-binding protein α-dystroglycan. These structural links stabilize the sarcolemma during contraction and protect it from contraction-induced damage. Loss of dystrophin leads to membrane fragility, resulting in sarcolemmal tearing and calcium influx, which triggers calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol., 10(2):168-75, 1997). This uncontrolled cycle of muscle degeneration and regeneration ultimately depletes the muscle stem cell population (Sacco et al., Cell, 2010, 143(7):1059-71; Wallace et al., Annu Rev Physiol, 2009, 71:37-57), resulting in progressive muscle weakness, fasciitis, and fibrotic scarring.

[0006] Without membrane stabilization by dystrophin or microdystrophin, DMD manifests as an uncontrollable cycle of tissue damage and repair, ultimately replacing lost muscle fibers with fibrous scar tissue through connective tissue proliferation. Fibrosis is characterized by the excessive deposition of extracellular matrix proteins, including collagen and elastin. ECM proteins are primarily produced from cytokines, such as TGFβ, released by activated fibroblasts in response to stress and inflammation. While the primary pathological hallmark of DMD is muscle fiber degeneration and necrosis, fibrosis as a pathological consequence has comparable consequences. Excessive production of fibrous tissue limits muscle regeneration and contributes to progressive muscle weakness in DMD patients. In one study, the presence of fibrosis in early DMD muscle biopsies was highly correlated with poor motor outcomes at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol, 2009, 68(7):762-7). These results point to fibrosis as a major contributor to muscle dysfunction in DMD and emphasize the need for early intervention before overt fibrosis. There is a need for treatments that increase muscle strength and protect against muscle damage in patients with DMD. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Hoffman et al., Cell 51(6):919–28, 1987 [Non-patent document 2] Straub et al., Curr Opin. Neurol., 10(2):168-75, 1997 [Non-patent document 3] Sacco et al., Cell, 2010, 143(7):p.1059~71 [Non-patent document 4] Wallace et al., Annu Rev Physiol, 2009, 71:p.37~57 [Non-Patent Document 5] Desguerre et al., J Neuropathol Exp Neurol, 2009, 68(7):p.762~7 Summary of the Invention [Means for solving the problem]

[0008] The present invention relates to gene therapy vectors, e.g., AAV, that express the micro-dystrophin gene in skeletal muscle, including fascia and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis.

[0009] The present invention provides therapies and approaches for increasing muscle strength and / or increasing muscle mass using gene therapy vectors to deliver micro-dystrophin to address the genetic defect observed in DMD. Example 2 describes a systemic gene delivery clinical trial for Duchenne muscular dystrophy, in which subjects received 2 x 10 vg / kg AAVrh74.MHCK7.micro-dystrophin. The clinical trial described in Example 3 provides a novel core clinical protocol, including a randomized, double-blind, placebo-controlled design. At the start of the study, subjects were randomized and administered 2 x 10 14 vg / kg AAVrh74.MHCK7.microdystrophin or lactated Ringer's.

[0010] The present invention provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, 8, or 9. The present invention also provides an rAAV comprising the nucleic acid sequence of SEQ ID NO: 9, or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3, and an rAAV particle comprising the nucleic acid sequence of SEQ ID NO: 9, or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3.

[0011] Another aspect of the invention provides compositions comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, 8, or 9, an rAAV comprising the nucleic acid sequence of SEQ ID NO: 9 or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3, and an rAAV particle comprising the nucleic acid sequence of SEQ ID NO: 9 or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3. Any of the methods disclosed herein may be practiced using these compositions.

[0012] The present invention provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, wherein the rAAV is administered via a systemic route at a dose of about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 15 The muscular dystrophy may be Duchenne muscular dystrophy or Becker muscular dystrophy.

[0013] For example, the dose of rAAV administered is about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 13 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 13vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 5.0×1014 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg~6.0×10 14 , or approximately 1.25 × 10 14 vg / kg~5.0×10 14 , or approximately 1.25 × 10 14 vg / kg~4.0×10 14 , or approximately 1.25 × 10 14 vg / kg~1.0×10 15 , or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5 × 10 14 vg / kg ~ approx. 1.0×10 15vg / kg, or approximately 1.5 × 10 14 vg / kg~6.0×10 14 , or approximately 1.5 × 10 14 vg / kg~5.0×10 14 , or approximately 1.5 × 10 14 vg / kg~4.0×10 14 , or approximately 1.5 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg~approx. 3.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.75 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.75 × 10 14 vg / kg~6.0×10 14 , or approximately 1.75 × 10 14 vg / kg~5.0×10 14 , or approximately 1.75 × 10 14 vg / kg~4.0×10 14 , or approximately 1.75 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.75 × 10 14vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg~1.0×10 15 , or approximately 2.0 × 10 14 vg / kg~6.0×10 14 , or approximately 2.0 × 10 14 vg / kg~5.0×10 14 , or approximately 2.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg.

[0014] In one embodiment, the method of the invention comprises systemically administering rAAV, wherein the systemic administration route is intravenous, and the dose of rAAV administered is about 2.0 x 10 14 In another embodiment, the method of the present invention comprises systemically administering rAAV, wherein the systemic administration route is intravenous, and the dose of rAAV administered is 5.0 x 10 12 vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 10 12 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 10 13 vg / kg, or approximately 3.0 × 10 13 vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14 vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 10 14 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14 vg / kg, or approximately 1 × 10 15vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0015] In any of the methods of the invention, the dose of rAAV may be administered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the dose or rAAV is administered at about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin, nucleotides 56 to 4820 of SEQ ID NO:5.

[0016] In any of the methods of the present invention, the dose of rAAV may be administered by injection, infusion, or implantation. For example, the dose of rAAV may be administered by infusion over about 1 hour. Additionally, the dose of rAAV may be administered intravenously through a peripheral limb vein, such as a peripheral arm vein or a peripheral leg vein. Alternatively, the infusion may be administered over about 30 minutes, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin set forth in SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0017] The rAAV administered by any of the methods of the invention may comprise the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. Additionally, the rAAV administered by any of the methods of the invention may comprise the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. For example, the rAAV may comprise the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6.

[0018] In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0019] In any of the methods of the invention, the rAAV administered is of serotype AAVrh7.4.

[0020] In some embodiments, the methods of the invention treat Duchenne muscular dystrophy or Becker muscular dystrophy. An exemplary embodiment is a method of treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, comprising administering a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7 microdystrophin, wherein the route of administration is intravenous infusion, and the dose of rAAV administered is about 2 x 10 over about 1 hour. 14vg / kg, and the rAAV vector comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, or nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, or nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0021] In one embodiment, the present invention provides an rAAV comprising a muscle-specific regulatory element nucleotide sequence and a nucleotide sequence encoding a micro-dystrophin protein. For example, the nucleotide sequence encodes a functional micro-dystrophin protein, where the nucleotide sequence has, 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 at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, and the protein retains micro-dystrophin activity. The micro-dystrophin protein provides stability to muscle membranes during muscle contraction; for example, micro-dystrophin functions as a shock absorber during muscle contraction. In one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is SEQ ID NO:9, or AAVrh74.MHCK7.microdystrophin from nucleotides 55 to 5021 of SEQ ID NO:3, from nucleotides 1 to 4977 of SEQ ID NO:8, or from nucleotides 56 to 5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0022] The present invention also provides an rAAV, wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 1 or its complementary sequence, and encodes a functional micro-dystrophin protein.

[0023] In one embodiment, the rAAV is a non-repetitive AAVrh74.MHCK7.microdystrophin vector encoding SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. This vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeats (ITRs), microdystrophin, SV40 introns (SD / SA), and a synthetic polyadenylation (Poly A) signal, all under the control of the MHCK7 promoter / enhancer. A schematic diagram of the vector genome and expression cassette is shown in Figure 1. Using the AAVrh74 serotype, efficient gene transfer in skeletal and cardiac muscle following IV administration can be achieved.

[0024] The term "stringent" refers to conditions generally understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate (65°C-68°C) or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide (42°C). 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, or other denaturing agents) may be used, but the hybridization rate will be affected. In the case of deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include washing in 6xSSC, 0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos).

[0025] Other agents may be included in hybridization and wash buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4 (SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents may also be used. The concentration and type of these additives may be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4, although under typical ionic strength conditions, the rate of hybridization is nearly independent of pH. Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Chapter 4, IRL Press Limited, Oxford, England. Hybridization conditions can be adjusted by one skilled in the art to control for these variables and allow DNAs of different sequence relatedness to form hybrids.

[0026] The term "muscle-specific control element" refers to a nucleotide sequence that regulates the expression of a coding sequence that is specific for expression in muscle tissue. These control elements include enhancers and promoters. The present invention provides constructs that include the muscle-specific control elements MCKH7 promoter, MCK promoter, and MCK enhancer.

[0027] The term "operably linked" refers to a regulatory element nucleotide sequence, for example, a promoter nucleotide sequence, positioned in such a way that the regulatory element confers expression of the nucleotide sequence.

[0028] In one embodiment, the invention provides an rAAV wherein the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF), a muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), a hybrid alpha-myosin heavy chain enhancer / MCK enhancer-promoter (MHCK7), C5-12, a mouse creatine kinase enhancer element, a fast skeletal troponin c gene element, a slow cardiac troponin c gene element, a slow troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0029] For example, the muscle-specific control element is the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the muscle-specific control element is the MCK nucleotide sequence of SEQ ID NO:4. Furthermore, in any of the rAAV vectors of the invention, the muscle-specific control element nucleotide sequence, e.g., the MHCK7 or MCK nucleotide sequence, is operably linked to a nucleotide sequence encoding a microdystrophin protein. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO:2 or SEQ ID NO:7) is operably linked to the human microdystrophin coding sequence (SEQ ID NO:1) set forth in the construct provided in Figure 1 or Figure 2 (SEQ ID NO:3) or Figure 13 (SEQ ID NO:9). For example, the MCK promoter (SEQ ID NO:4) is operably linked to the human microdystrophin coding sequence (SEQ ID NO:1) set forth in the construct provided in Figure 5 or Figure 6 (SEQ ID NO:5). In another aspect, the invention provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:1 and SEQ ID NO:7. The invention also provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:4.

[0030] In a further aspect, the present invention provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8. For example, the AAVrh74.MHCK7.microdystrophin vector comprises the nucleotide sequence within the ITRs of SEQ ID NO:3 and is shown in Figure 2. The rAAV vector comprises the 5' ITR, the MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, polyA, and the 3' ITR. In one embodiment, the vector comprises nucleotides 55 to 5021 of SEQ ID NO:3. The plasmid set forth in SEQ ID NO:3 further comprises a pGEX plasmid backbone with ampicillin resistance and a pBR322 origin of replication.

[0031] In another aspect, the present invention provides an rAAV comprising the nucleotide sequence of SEQ ID NO:9. For example, the AAVrh74.MHCK7.microdystrophin vector comprises the nucleotide sequence of SEQ ID NO:9 and is shown in Figure 13. The rAAV vector construct comprises an MHCK7 promoter, a chimeric intron sequence, a coding sequence for the human microdystrophin gene, and a polyA. In one embodiment, the rAAV vector construct further comprises an ITR 5' to the promoter and an ITR 3' to the polyA. In one embodiment, the rAAV is AAVrh74.

[0032] In another aspect, the AAVrh74.MHCK7.microdystrophin vector comprises the nucleotide sequence within the ITRs of SEQ ID NO:8 and is shown in Figure 15. The rAAV vector comprises the 5' ITR, the MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, polyA, and the 3' ITR. In one embodiment, the vector comprises nucleotides 1-4977 of SEQ ID NO:9. The plasmid set forth in SEQ ID NO:3 further comprises a pGEX plasmid backbone with kanamycin resistance and a pBR322 origin of replication.

[0033] In another aspect, the present invention provides a plasmid comprising the AAVrh74.MHCK7.microdystrophin vector construct. In one embodiment, the plasmid comprises a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, a coding sequence of the human microdystrophin gene, polyA, and a 3' ITR. In one embodiment, the plasmid comprises a pGEX plasmid backbone containing kanamycin resistance and, optionally, a pBR322 origin of replication. In a specific embodiment, the plasmid is set forth in SEQ ID NO:8 and shown in Figures 14 and 15.

[0034] The present invention provides a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7. The rAAV vector is of the AAV serotype AAVrh.74.

[0035] The present invention also provides an rAAV comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence within the ITRs in SEQ ID NO: 3, the nucleotide sequence within the ITRs in SEQ ID NO: 8, or the nucleotide sequence set forth in SEQ ID NO: 9. This rAAV vector is of the AAV serotype AAVrh.74.

[0036] The rAAV vectors of the invention may be of any AAV serotype, such as serotypes AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0037] The present invention also provides pharmaceutical compositions (or sometimes simply referred to herein as "compositions") comprising any of the rAAV vectors of the present invention.

[0038] In another embodiment, the invention provides a method for producing rAAV vector particles, comprising culturing cells transfected with any of the rAAV vectors of the invention and recovering the rAAV particles from the supernatant of the transfected cells. The invention also provides viral particles comprising any of the recombinant AAV vectors of the invention.

[0039] In one of the methods for treating muscular dystrophy, the level of microdystrophin gene expression in a subject's cells is increased after administration of rAAV. Expression of the microdystrophin gene in cells is detected by measuring microdystrophin protein levels by Western blot in muscle biopsied before and after administration of rAAV. Specifically, the level of microdystrophin protein increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of rAAV, compared to the level of microdystrophin before administration of rAAV. For example, the level of microdystrophin protein is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the rAAV compared to the level of microdystrophin before administration of the rAAV.

[0040] Furthermore, expression of the microdystrophin gene in cells is detected by measuring microdystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of rAAV, and the level of microdystrophin protein increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of rAAV compared to the level of microdystrophin before administration of rAAV. For example, the level of microdystrophin protein is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the rAAV compared to the level of microdystrophin before administration of the rAAV.

[0041] In any of the methods for treating muscular dystrophy, the serum CK level in the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the rAAV. For example, the serum CK level in the subject is reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% by 60 days after administration of the rAAV compared to the serum CK level before administration of the rAAV. In particular, in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 87% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV; or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 72% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV; or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 73% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV; or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 78% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV; or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 95% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV. In any of the methods for treating muscular dystrophy, the number of microdystrophin-positive fibers in the muscle tissue of the subject increases after administration of rAAV compared with the number of microdystrophin-positive fibers before administration of rAAV.For example, the number of microdystrophin-positive fibers is detected by measuring the microdystrophin protein level in muscle biopsy before and after administration of rAAV by Western blot or immunohistochemistry.

[0042] In any of the methods for treating muscular dystrophy, administration of rAAV increases the expression of DAPC protein, such as α-sarcoglycan or β-sarcoglycan.For example, the level of α-sarcoglycan in a subject increases after administration of rAAV, compared with the level of α-sarcoglycan before administration of rAAV.Furthermore, the level of β-sarcoglycan in a subject increases after administration of rAAV, compared with the level of β-sarcoglycan before administration of rAAV.The level of α-sarcoglycan or β-sarcoglycan is detected by measuring the protein level of α-sarcoglycan or β-sarcoglycan by Western blot or immunohistochemistry in muscle biopsy before and after administration of rAAV.

[0043] In any of the methods of treating muscular dystrophy, disease progression in the subject is slowed after administration of the rAAV, as measured by any of the following: Six-Minute Walk Test, Latency to Stand, Latency to Climb Four Steps, Latency to Climb Four Steps, North Star Ambulation Assessment (NSAA), Timed 10-Meter Test, Timed 100-Meter Test, Handheld Dysfunction Test (HHD), Timed Up-and-Go, and / or Gross Motor Subtest Measure (Bayley-III) score.

[0044] For example, in any of the methods, the subject has an improvement of at least 6 points in NSAA score at least 270 days after administration of rAAV compared to the NSAA score before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 0.8 seconds in the time to stand at least 270 days after administration of rAAV compared to the time to stand before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 1.2 seconds in the time to climb four stairs test at least 270 days after administration of rAAV compared to the time to climb four stairs test before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 7 seconds in the timed 100-meter test at least 270 days after administration of rAAV compared to the timed 100-meter test before administration of rAAV.

[0045] In another embodiment, the present invention provides a method for expressing a micro-dystrophin gene in patient cells, comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, micro-dystrophin gene expression in patient cells is detected by measuring micro-dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. Micro-dystrophin gene expression is further measured in the patient by detecting the number of vector genomes per nucleus, where one vector genome per nucleus is approximately 50% micro-dystrophin expression and more than one copy per nucleus is consistent with a low level of micro-dystrophin expression. For example, a cell has 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0046] In a further embodiment, the invention provides a method of reducing serum CK levels in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, the serum CK level in the patient is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, by 60 days after administration of the rAAV, compared to the serum CK level before administration of the rAAV. In particular, serum CK levels in the subject are reduced by about 87% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 72% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 73% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 78% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 95% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV.

[0047] The present invention also provides a method for increasing microdystrophin-positive fibers in muscle tissue of a patient, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, the number of microdystrophin-positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV. Furthermore, microdystrophin gene expression is measured in the patient by further detecting the number of vector genomes per nucleus, where one vector genome per nucleus represents approximately 50% microdystrophin expression and more than one copy per nucleus corresponds to a low level of microdystrophin expression. For example, a cell has 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0048] In another embodiment, the present invention provides a method of increasing α-sarcoglycan expression in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, α-sarcoglycan levels are detected by measuring α-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.

[0049] The present invention further provides a method for increasing β-sarcoglycan expression in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, β-sarcoglycan levels are detected by measuring β-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0050] The present invention also provides a method of treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, so as to slow disease progression in the patient as measured by any of the following: Six-Minute Walk Test, Latency to Stand, Latency to Climb Four Steps, Latency to Climb Four Steps, North Star Ambulation Assessment (NSAA), Timed 10-Meter Test, Timed 100-Meter Test, Handheld Dynamics Assessment (HHD), Timed Up-and-Go, and / or Gross Motor Subtest Measure (Bayley-III) score.

[0051] For example, in any of the methods, the subject has an improvement of at least 6 points in NSAA score at least 270 days after administration of rAAV compared to the NSAA score before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 0.8 seconds in the time to stand at least 270 days after administration of rAAV compared to the time to stand before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 1.2 seconds in the time to climb four stairs test at least 270 days after administration of rAAV compared to the time to climb four stairs test before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 7 seconds in the timed 100-meter test at least 270 days after administration of rAAV compared to the timed 100-meter test before administration of rAAV.

[0052] "Fibrosis" refers to the excessive or uncontrolled deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues upon injury, including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas. Deposited ECM components include fibronectin and collagen, such as collagen 1, collagen 2, or collagen 3.

[0053] The present invention also provides methods for reducing or preventing fibrosis in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of an rAAV vector comprising the human micro-dystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, the AAVrh74.MHCK7.micro-dystrophin is AAVrh74.MHCK7.micro-dystrophin of nucleotides 55-5021 of SEQ ID NO:3. In another embodiment, the AAVrh74.MHCK7.micro-dystrophin is AAVrh74.MHCK7.micro-dystrophin of SEQ ID NO:9. In another embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin from nucleotides 1 to 4977 of SEQ ID NO: 8 or from nucleotides 56 to 5066 of SEQ ID NO: 6. In a further embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO: 5.

[0054] In another embodiment, the present invention also provides a method of preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAV74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, any of the rAAVs of the present invention can be administered to a subject suffering from muscular dystrophy to prevent fibrosis, e.g., administered before an rAAV of the present invention expressing a human microdystrophin protein is observed in the subject. Additionally, an rAAV of the present invention expressing a human microdystrophin gene can be administered to a subject at risk of developing fibrosis, such as those suffering from or diagnosed with muscular dystrophy, e.g., DMD. The rAAV of the present invention can be administered to subjects suffering from muscular dystrophy to prevent new fibrosis in these subjects.

[0055] The present invention contemplates administering rAAV before fibrosis is observed in a subject.Furthermore, rAAV can be administered to subjects at risk of developing fibrosis, such as those suffering from or diagnosed with muscular dystrophy (e.g., DMD).rAAV can be administered to subjects who have already developed fibrosis due to muscular dystrophy, in order to prevent new fibrosis in these subjects.

[0056] The present invention also provides a method for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0057] The present invention contemplates administering an rAAV vector to a subject diagnosed with DMD before fibrosis is observed in the subject, or before muscle strength is reduced, or before muscle mass is reduced.

[0058] The present invention also contemplates administering an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 to subjects with muscular dystrophy who have already developed fibrosis to prevent new fibrosis in these subjects or to reduce fibrosis in these subjects. The invention also provides for administering an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 to a subject suffering from muscular dystrophy who already has reduced muscle strength or reduced muscle mass to protect the muscle from further damage.

[0059] In any of the methods of the invention, the subject may be suffering from a muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0060] In other embodiments of any of the methods of the invention described herein, the serum CK level in the subject is increased after administration of the rAAV compared to the serum CK level before administration of the rAAV: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 85% by 270 days after administration; c) at least 72, 73, 74, or 95% by 180 days after administration; d) at least 87, 88, 93, or 95% by 90 days after administration; e) at least 70% by 270 days after administration; f) 70–95% by 90, 180, or 270 days after administration; g) at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration; and h) is reduced by a percentage level selected from the group consisting of at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% by 90, 180 or 270 days after administration.

[0061] In another embodiment, the present invention provides a composition for treating muscular dystrophy in a human subject in need thereof, comprising a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, wherein the composition is formulated for a systemic route of administration and the dose of rAAV is about 1 x 10 14 vg / kg ~ approx. 4×10 14 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0062] For example, the composition of the present invention may contain about 5.0×10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 13 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 1013 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 14vg / kg ~ approx. 5.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg~6.0×10 14 , or approximately 1.25 × 10 14 vg / kg~5.0×10 14 , or approximately 1.25 × 10 14 vg / kg~4.0×10 14 , or approximately 1.25 × 10 14 vg / kg~1.0×10 15 , or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5 × 10 14 vg / kg ~ approx. 1.0×10 15vg / kg, or approximately 1.5 × 10 14 vg / kg~6.0×10 14 , or approximately 1.5 × 10 14 vg / kg~5.0×10 14 , or approximately 1.5 × 10 14 vg / kg~4.0×10 14 , or approximately 1.5 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg~approx.3.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.75 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.75 × 10 14 vg / kg~6.0×10 14 , or approximately 1.75 × 10 14 vg / kg~5.0×10 14 , or approximately 1.75 × 10 14 vg / kg~4.0×10 14 , or approximately 1.75 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.75 × 10 14vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg~1.0×10 15 , or approximately 2.0 × 10 14 vg / kg~6.0×10 14 , or approximately 2.0 × 10 14 vg / kg~5.0×10 14 , or approximately 2.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 The rAAV comprises a dose of rAAV of 1000 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0063] In one embodiment, the compositions of the present invention are formulated for intravenous administration and contain approximately 2.0 x 10 14In another embodiment, the compositions of the invention are formulated for intravenous administration and comprise a dose of rAAV that is about 5.0 x 10 vg / kg. 12 vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 10 12 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13 vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 10 13 vg / kg, or approximately 3.0 × 10 13 vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14 vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 1014 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14 vg / kg, or approximately 1 × 10 15 The dose of rAAV is 0.01 mg / mL. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In another embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0064] In any of the compositions of the invention, the dose of rAAV is delivered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the composition comprises a dose of rAAV delivered at about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In another embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0065] The composition of the present invention is formulated for administration by injection, infusion or implantation.For example, the composition is formulated for administration by infusion over about 1 hour.In addition, the composition of the present invention is formulated for intravenous administration through peripheral limb veins, such as peripheral arm veins or peripheral leg veins.Alternatively, the infusion can be administered over about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0066] Any of the compositions of the invention comprises an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7, or an rAAV vector comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0067] In particular, the compositions of the present invention are for treating Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present invention provides a composition for treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, comprising a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7 microdystrophin, formulated for administration by intravenous infusion over about 1 hour, wherein the dose of rAAV administered is about 2×10 14 vg / kg, and the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6.

[0068] In another embodiment, the present invention also provides a composition comprising an rAAV vector for reducing fibrosis in a subject in need thereof. Additionally, the present invention provides a composition comprising an rAAV vector for preventing fibrosis in a subject suffering from muscular dystrophy.

[0069] The present invention also provides compositions comprising rAAVs for increasing muscle strength and / or muscle mass in subjects suffering from muscular dystrophy. In a further embodiment, the present invention provides compositions comprising any of the rAAVs of the present invention for treating muscular dystrophy.

[0070] In other embodiments of any of the compositions of the invention, after administration of the composition to a human subject in need of treatment for muscular dystrophy, the serum CK level in the subject, compared to the serum CK level before administration of the composition, is a) at least 78% by 90, 180, or 270 days after administration; b) at least 46, 55, 70, or 85% by 270 days after administration; c) at least 72, 73, 74, or 95% by 180 days after administration; d) at least 87, 99, 93, or 95% by 90 days after administration; e) at least 70% by 270 days after administration; f) 70–95% by 90, 180, or 270 days after administration; g) at least 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration; and h) is reduced by a percentage level selected from the group consisting of 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration.

[0071] In another embodiment, the present invention provides a use of a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin for the preparation of a medicament for the treatment of muscular dystrophy in a human subject in need thereof, wherein the medicament is formulated for a systemic route of administration and the dose of rAAV is about 1 x 10 14 vg / kg ~ approx. 4×10 14 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0072] For example, the drug is about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 13 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 13vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 5.0×1014 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or 1.0 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg~6.0×10 14 , or approximately 1.25 × 10 14 vg / kg~5.0×10 14 , or approximately 1.25 × 10 14 vg / kg~4.0×10 14 , or approximately 1.25 × 10 14 vg / kg~1.0×10 15 , or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5 × 10 14 vg / kg ~ approx. 1.0×10 15vg / kg, or approximately 1.5 × 10 14 vg / kg~6.0×10 14 , or approximately 1.5 × 10 14 vg / kg~5.0×10 14 , or approximately 1.5 × 10 14 vg / kg~4.0×10 14 , or approximately 1.5 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg~approx.3.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.75 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.75 × 10 14 vg / kg~6.0×10 14 , or approximately 1.75 × 10 14 vg / kg~5.0×10 14 , or approximately 1.75 × 10 14 vg / kg~4.0×10 14 , or approximately 1.75 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.75 × 10 14vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg~1.0×10 15 , or approximately 2.0 × 10 14 vg / kg~6.0×10 14 , or approximately 2.0 × 10 14 vg / kg~5.0×10 14 , or approximately 2.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 The rAAV comprises a dose of rAAV of 1000 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0073] In one embodiment, the medicament of the present invention is formulated for systemic administration of a dose of rAAV, the systemic administration route is intravenous, and the administered dose of rAAV is about 2.0 x 10 14 In another embodiment, the medicament of the present invention is formulated for systemic administration of a dose of rAAV, wherein the systemic administration route is intravenous, and the dose of rAAV is 5.0 x 10 vg / kg. 12 vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 10 12 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13 vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 10 13 vg / kg, or approximately 3.0 × 10 13 vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 10 14 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14 vg / kg, or approximately 1 × 10 15 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0074] In any of the uses of the invention, the medicament comprises a dose of rAAV of about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In a specific embodiment, the dose or rAAV is about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0075] In any of the uses of the present invention, the medicament is formulated for administration by injection, infusion or implantation.For example, the medicament is formulated for administration by infusion over about 1 hour.Furthermore, the medicament is formulated for intravenous administration through a peripheral limb vein, such as a peripheral arm vein or a peripheral leg vein.Alternatively, the infusion may be administered over about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0076] In any of the uses of the invention, the medicament comprises an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7, or an rAAV comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6.

[0077] A particular use of the present invention is for the preparation of a medicament for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present invention relates to the use of a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7 microdystrophin for the preparation of a medicament for treating a human subject with Duchenne muscular dystrophy or Becker muscular dystrophy in need thereof, wherein the medicament is formulated for administration by intravenous infusion over about 1 hour, and the dose of rAAV administered is about 2 x 10 14 vg / kg, and the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6.

[0078] In a further embodiment, the present invention provides use of rAAV for the preparation of a medicament for reducing fibrosis in a subject in need thereof, for example, the subject in need thereof is suffering from a muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0079] In another embodiment, the present invention provides the use of rAAV for the preparation of a medicament for preventing fibrosis in a subject suffering from muscular dystrophy.

[0080] Furthermore, the present invention provides the use of rAAV for the preparation of a medicament for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy.

[0081] The present invention also provides the use of rAAV for the preparation of a medicament for the treatment of muscular dystrophy.

[0082] The present invention also provides use of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7 for the preparation of a medicament for the treatment of muscular dystrophy, or an rAAV vector comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6 for the treatment of muscular dystrophy.

[0083] In other embodiments of any of the uses of the invention, the serum CK level in the subject is increased after administration of the rAAV to the subject compared to the serum CK level before administration of the rAAV: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46, 55, 70, or 95% by 270 days after administration; c) at least 72, 73, 74, or 95% by 180 days after administration; d) at least 87, 88, 93, or 95% by 90 days after administration; e) at least 70% by 270 days after administration; f) 70–95% by 90, 180, or 270 days after administration; g) at least 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration; and h) is reduced by a percentage level selected from the group consisting of 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration.

[0084] In either the use of the composition for treating muscular dystrophy or the medicament for treating muscular dystrophy, the level of microdystrophin gene expression in the subject's cells is increased after administration of the composition or medicament. Expression of the microdystrophin gene in the cells is detected by measuring microdystrophin protein levels by Western blot in muscles biopsied before and after administration of the composition or medicament. In particular, the level of microdystrophin protein is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of the composition or medicament, compared to the level of microdystrophin before administration of the composition or medicament. For example, the level of microdystrophin protein is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition compared to the level of microdystrophin before administration of the composition or medicament.

[0085] Furthermore, expression of the microdystrophin gene in cells is detected by measuring microdystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of the composition or medicament, and the level of microdystrophin protein increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of the rAAV compared to the level of microdystrophin before administration of the composition or medicament. For example, the level of microdystrophin protein is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition or medicament compared to the level of microdystrophin before administration of the composition or medicament.

[0086] In any of the compositions for treating muscular dystrophy, the serum CK level in the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the composition or medicament. For example, the serum CK level in the subject is reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament. In particular, in any of the compositions for treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 87% by 60 days after administration of the composition or medicament, compared to serum CK levels before administration of the composition or medicament; or in any of the compositions for treating muscular dystrophy of the present invention or uses of the medicament for treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 72% by 60 days after administration of the composition or medicament, compared to serum CK levels before administration of the composition or medicament; or in any of the compositions for treating muscular dystrophy of the present invention, serum CK levels in the subject are reduced by about 72% by 60 days after administration of the composition or medicament, compared to serum CK levels before administration of the composition or medicament. or in the use of either the compositions for treating muscular dystrophy of the present invention or the medicament for treating muscular dystrophy of the present invention, the serum CK level in the subject is reduced by about 73% by 60 days after administration of the composition or medicament, compared to the serum CK level before administration of the composition or medicament; or in the use of either the compositions for treating muscular dystrophy of the present invention or the medicament for treating muscular dystrophy of the present invention, the serum CK level in the subject is reduced by about 78% by 60 days after administration of the composition or medicament, compared to the serum CK level before administration of the composition or medicament; or in the use of either the compositions for treating muscular dystrophy of the present invention or the medicament for treating muscular dystrophy of the present invention, the serum CK level in the subject is reduced by about 95% by 60 days after administration of the composition or medicament, compared to the serum CK level before administration of the composition or medicament. In either the compositions for treating muscular dystrophy or the use of the medicament for treating muscular dystrophy, the number of microdystrophin-positive fibers in the muscle tissue of the subject is increased after administration of the composition or medicament, compared to the number of microdystrophin-positive fibers before administration of the composition or medicament.For example, the number of microdystrophin-positive fibers is detected by measuring microdystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or medicament.

[0087] In either the use of the composition for treating muscular dystrophy or the medicament for treating muscular dystrophy, the administration of the composition or the medicament upregulates the expression of DAPC protein such as α-sarcoglycan or β-sarcoglycan.For example, the level of α-sarcoglycan in a subject increases after the administration of the composition or the medicament, compared with the level of α-sarcoglycan before the administration of the composition or the medicament.Furthermore, the level of β-sarcoglycan in a subject increases after the administration of the composition or the medicament, compared with the level of β-sarcoglycan before the administration of the composition or the medicament.The level of α-sarcoglycan or β-sarcoglycan is detected by measuring the level of α-sarcoglycan or β-sarcoglycan protein by Western blot or immunohistochemistry in muscle biopsies before and after the administration of the composition or the medicament.

[0088] In either the composition for treating muscular dystrophy or the use of the medicament for treating muscular dystrophy, the progression of the disease in the subject is slowed after administration of the composition or medicament, as measured by any of the six-minute walk test, latency to stand, latency to climb four stairs, latency to climb and descend four stairs, North Star Gait Assessment (NSAA), timed 10-meter test, timed 100-meter test, handheld dysmetology (HHD), timed up-and-go, and / or gross motor subtest measure (Bayley-III) score.

[0089] For example, after administration of a composition for treating muscular dystrophy or use of a medicament for treating muscular dystrophy, the subject has an improvement of at least 6 points in NSAA score at least 270 days after administration of the composition or medicament, compared to the NSAA score before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 0.8 seconds in the time to stand at least 270 days after administration of the composition or medicament, compared to the time to stand before administration of the composition or medicament. Further, in any of the methods or uses of the present invention, the subject has an improvement of at least about 1.2 seconds in the time to climb four stairs test at least 270 days after administration of the composition or medicament, compared to the time to climb four stairs test before administration of the composition or medicament. Further, in any of the methods or uses of the present invention, the subject has an improvement of at least about 7 seconds in the time to climb 100 meters test at least 270 days after administration of the composition or medicament, compared to the time to climb 100 meters test before administration of the composition or medicament.

[0090] In another embodiment, the invention provides a composition for expressing a micro-dystrophin gene in patient cells, the composition comprising the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In a further embodiment, the invention provides use of a dose of the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, for the preparation of a medicament for expressing a micro-dystrophin gene in patient cells. For example, expression of the micro-dystrophin gene in patient cells is detected by measuring micro-dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. Furthermore, the expression of the micro-dystrophin gene can be measured in patients by further detecting the number of vector genomes per nucleus, where one vector genome per nucleus corresponds to approximately 50% micro-dystrophin expression, and more than one copy per nucleus corresponds to the micro-dystrophin expression level. For example, a cell may have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0091] In a further embodiment, the present invention provides a composition for reducing serum CK levels in a patient in need thereof, the composition comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. Additionally, the present invention provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for reducing serum CK levels in cells of a patient in need thereof. For example, the serum CK level in the patient is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, by 60 days after administration of the composition or medicament, compared to the serum CK level before administration of the composition or medicament. In particular, the serum CK level in the subject is reduced by about 87% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 72% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 73% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 78% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 95% by 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament.

[0092] The present invention also provides a composition for increasing microdystrophin-positive fibers in a patient's muscle tissue, the composition comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. The present invention further provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for increasing microdystrophin-positive fibers in a patient's muscle tissue. For example, the number of microdystrophin-positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or medicament. Furthermore, the expression of the micro-dystrophin gene can be measured in patients by further detecting the number of vector genomes per nucleus, where one vector genome per nucleus corresponds to approximately 50% micro-dystrophin expression, and more than one copy per nucleus corresponds to the micro-dystrophin expression level. For example, a cell may have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0093] In another embodiment, the present invention provides a composition for increasing α-sarcoglycan expression in a patient in need thereof, the composition comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. The present invention also provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, for the preparation of a medicament for increasing α-sarcoglycan expression in a patient in need thereof. For example, α-sarcoglycan levels are detected by measuring α-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or medicament.

[0094] The present invention further provides a composition for increasing β-sarcoglycan expression in a patient in need thereof, the composition comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. The present invention also provides use of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for increasing β-sarcoglycan expression in a patient in need thereof. For example, β-sarcoglycan levels are detected by measuring β-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or medicament.

[0095] The invention also provides use of a dose of the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, such that administration of the medicament results in slowing disease progression in the patient, as measured by any of the following: Six-Minute Walk Test, Latency to Stand, Latency to Climb Four Steps, Latency to Climb Four Steps, Latency to Climb Four Steps, North Star Ambulation Assessment (NSAA), Timed 10-Meter Test, Timed 100-Meter Test, Handheld Dynamics Test (HHD), Timed Up-and-Go, and / or Gross Motor Subtest Measure (Bayley-III) score.

[0096] For example, the subject has an improvement of at least 6 points in NSAA score at least 270 days after administration of the composition or medicament, compared to the NSAA score before administration of the composition or medicament.Furthermore, the subject has an improvement of at least about 0.8 seconds in the time to stand up at least 270 days after administration of the composition or medicament, compared to the time to stand up before administration of the composition or medicament.Furthermore, the subject has an improvement of at least about 1.2 seconds in the time to climb four stairs test at least 270 days after administration of the composition or medicament, compared to the time to climb four stairs test before administration of the composition or medicament.Furthermore, the subject has an improvement of at least about 7 seconds in the time to climb 100 meters test at least 270 days after administration of the composition or medicament, compared to the time to climb 100 meters test before administration of the composition or medicament. The present invention provides, for example, the following items. (Item 1) 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin; The rAAV is administered in an amount of about 5.0×1012 vg / kg ~ approx. 1.0×10 15 wherein the compound is administered at a dose of (Item 2) The systemic administration route is an intravenous route, and the dose of rAAV administered is about 2 x10 14 2. The method according to item 1, wherein the saturation level is 1000 mg / kg. (Item 3) 3. The method of item 1 or 2, wherein the dose of rAAV is administered at a concentration of about 10 mL / kg. (Item 4) 4. The method of any one of items 1 to 3, wherein the rAAV is administered by injection, infusion, or implantation. (Item 5) 5. The method of any one of items 1 to 4, wherein the rAAV is administered by infusion over about 1 hour. (Item 6) 6. The method of any one of items 1 to 5, wherein the rAAV is administered by intravenous route through a peripheral limb vein. (Item 7) 7. The method of any one of items 1 to 6, wherein the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1. (Item 8) 8. The method of any one of items 1 to 7, wherein the rAAV comprises an MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. (Item 9) 9. The method of any one of items 1 to 8, wherein the rAAV is of the serotype AAVrh.74. (Item 10) 10. The method of any one of paragraphs 1 to 9, wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9 or of nucleotides 55 to 5021 of SEQ ID NO: 3. (Item 11) 11. The method according to any one of items 1 to 10, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. (Item 12) The human subject has Duchenne muscular dystrophy, and the rAAV is administered intravenously over a period of about 1 hour at a dose of about 2 x 10 14 12. The method of any one of paragraphs 1 to 11, wherein the rAAV is administered at a dose of 1000 mg / kg, and wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9 or nucleotides 55 to 5021 of SEQ ID NO: 3. (Item 13) 13. The method of any one of items 1 to 12, wherein the level of microdystrophin gene expression in the cells of the subject is increased after administration of the rAAV compared to the level of microdystrophin gene expression before administration of the rAAV. (Item 14) 14. The method of claim 13, wherein expression of the microdystrophin gene in the cells is detected by measuring microdystrophin protein levels by Western blot in muscle biopsied before and after administration of the rAAV. (Item 15) 15. The method of claim 14, wherein the level of microdystrophin protein is increased by at least 72% after administration of the rAAV compared to the level of microdystrophin before administration of the rAAV. (Item 16) Expression of the microdystrophin gene in the cells is confirmed by immunohistochemistry of muscle biopsies before and after administration of the rAAV. Item 14. The method according to item 13, wherein the detection is performed by measuring protein levels. (Item 17) 17. The method of claim 16, wherein the level of micro-dystrophin protein is increased by at least 72% after administration of the rAAV compared to the level of micro-dystrophin before administration of the rAAV. (Item 18) 13. The method of any one of items 1 to 12, wherein the serum CK level in the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the rAAV. (Item 19) Item 20. The method of item 18, wherein the serum CK level in the subject is reduced by 87% by 60 days after administration of the rAAV compared to the serum CK level before administration of the rAAV. 13. The method of any one of items 1 to 12, wherein the number of microdystrophin-positive fibers in the muscle tissue of the subject is increased after administration of the rAAV compared to the number of microdystrophin-positive fibers before administration of the rAAV. (Item 21) 21. The method of claim 20, wherein the number of microdystrophin-positive fibers is detected by measuring the microdystrophin protein levels by Western blot in muscle biopsies before and after administration of the rAAV. (Item 22) 21. The method of claim 20, wherein the number of microdystrophin-positive fibers is detected by measuring the microdystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 23) 13. The method of any one of items 1 to 12, wherein the level of α-sarcoglycan in the subject is increased after administration of the rAAV compared to the level of α-sarcoglycan before administration of the rAAV. (Item 24) 24. The method of claim 23, wherein the level of α-sarcoglycan is detected by measuring the α-sarcoglycan protein level by Western blot in muscle biopsies before and after administration of the rAAV. (Item 25) 24. The method of claim 23, wherein the number of α-sarcoglycans is detected by measuring α-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 26) 13. The method of any one of items 1 to 12, wherein the level of β-sarcoglycan in the subject is increased after administration of the rAAV compared to the level of β-sarcoglycan before administration of the rAAV. (Item 27) 27. The method of claim 26, wherein the level of β-sarcoglycan is detected by measuring β-sarcoglycan protein levels by Western blot in muscle biopsies before and after administration of the rAAV. (Item 28) 27. The method of claim 26, wherein the number of β-sarcoglycans is detected by measuring the β-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 29) The progression of the disease in the subject is monitored by the six-minute walk test, the time to stand up, the time to climb four steps, the time to climb and descend four steps, the North Star Ambulatory Assessment (N 13. The method of any one of items 1 to 12, wherein the patient is slowed after administration of the rAAV as measured by any of the following: SAA, 10-meter timed test, 100-meter timed test, Handheld Dysfunction Test (HHD), Timed Up and Go, and / or Gross Motor Subtest Measure (Bayley-III) score. (Item 30) 26. The method of claim 25, wherein the subject has an improvement of at least 6 points in the NSAA score at least 270 days after administration of the rAAV compared to the NSAA score before administration of the rAAV. (Item 31) 26. The method of claim 25, wherein the subject has an improvement of at least 0.8 seconds in the time to stand at least 270 days after administration of the rAAV compared to the time to stand before administration of the rAAV. (Item 32) 26. The method of claim 25, wherein the subject has an improvement of at least about 1.2 seconds in a time to climb four stairs test at least 270 days after administration of the rAAV compared to a time to climb four stairs test before administration of the rAAV. (Item 33) 26. The method of claim 25, wherein the subject has an improvement of at least 7 seconds in the 100-meter timed test at least 270 days after administration of the rAAV compared to the 100-meter timed test before administration of the rAAV. (Item 34) 1. A method of expressing a microdystrophin gene in patient cells, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55 to 5021 of SEQ ID NO:3. (Item 35) 31. The method of claim 30, wherein expression of the micro-dystrophin gene in the patient's cells is detected by measuring the micro-dystrophin protein levels by Western blot in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. (Item 36) 31. The method of claim 30, wherein expression of the micro-dystrophin gene in the patient's cells is detected by measuring micro-dystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. (Item 37) 31. The method of claim 30, wherein expression of the micro-dystrophin gene is measured in the patient by detecting more than one rAAV vector genome copy per nucleus. (Item 38) 1. A method of reducing serum CK levels in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55 to 5021 of SEQ ID NO:3. (Item 39) 35. The method of claim 34, wherein the serum CK level in the patient is reduced by at least 87% by 60 days after administration of the rAAV compared to the serum CK level before administration of the rAAV. (Item 40) A method for increasing microdystrophin-positive fibers in muscle tissue of a patient, comprising: The method comprises administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55 to 5021 of SEQ ID NO:3. (Item 41) 37. The method of claim 36, wherein the number of microdystrophin-positive fibers is detected by measuring the dystrophin protein levels by Western blot in muscle biopsies before and after administration of the rAAV. (Item 42) 37. The method of claim 36, wherein the number of microdystrophin-positive fibers is detected by measuring the dystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 43) 37. The method of claim 36, wherein the number of microdystrophin-positive fibers is measured by detecting more than one rAAV vector genome copy per nucleus. (Item 44) 1. A method for increasing expression of alpha-sarcoglycan in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55 to 5021 of SEQ ID NO:3. (Item 45) 41. The method of claim 40, wherein the level of α-sarcoglycan is detected by measuring the α-sarcoglycan protein level by Western blot in muscle biopsies before and after administration of the rAAV. (Item 46) 41. The method of claim 40, wherein the number of α-sarcoglycans is detected by measuring the α-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 47) 1. A method for increasing expression of β-sarcoglycan in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55 to 5021 of SEQ ID NO:3. (Item 48) 44. The method of claim 43, wherein the level of β-sarcoglycan is detected by measuring the β-sarcoglycan protein level by Western blot in muscle biopsies before and after administration of the rAAV. (Item 49) 44. The method of claim 43, wherein the number of β-sarcoglycans is detected by measuring the β-sarcoglycan protein levels by immunohistochemistry in muscle biopsies before and after administration of the rAAV. (Item 50) 1. A method of treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55-5021 of SEQ ID NO:3, so as to slow disease progression in the patient as measured by any of the following: Six-Minute Walk Test, Latency to Stand, Latency to Climb Four Steps, Latency to Climb Four Steps, North Star Ambulation Assessment (NSAA), Timed 10-Meter Test, Timed 100-Meter Test, Handheld Dynamics Assessment (HHD), Timed Up-and-Go, and / or Gross Motor Subtest Measure (Bayley-III) score. (Item 51) Item 47. The method of item 46, wherein the subject has an improvement of at least 6 points in the NSAA score at least 90 days after administration of the rAAV compared to the NSAA score before administration of the rAAV. (Item 52) 47. The method of claim 46, wherein the subject has an improvement of at least 0.8 seconds in time to stand at least 90 days after administration of the rAAV compared to time to stand before administration of the rAAV. (Item 53) 47. The method of claim 46, wherein the subject has an improvement of at least about 1.2 seconds in a time to climb four stairs test at least 90 days after administration of the rAAV compared to a time to climb four stairs test before administration of the rAAV. (Item 54) 47. The method of claim 46, wherein the subject has an improvement of at least 7 seconds in the 100 meter timed test at least 90 days after administration of the rAAV compared to the 100 meter timed test before administration of the rAAV. (Item 55) 1. A composition for treating muscular dystrophy in a human subject in need thereof, comprising a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, formulated for a systemic route of administration, wherein the dose of the rAAV is about 5×10 12 vg / kg~approx.1.0×10 15 vg / kg of the composition. (Item 56) The systemic administration route is an intravenous route, and the dose of the rAAV is about 2×10 14 52. The composition according to item 51, wherein the saturation is 0.05 to 0.15 vg / kg. (Item 57) 53. The composition of item 51 or 52, wherein the dose of rAAV is about 10 mL / kg. (Item 58) 54. The composition of any one of items 51 to 53, wherein the composition is formulated for administration by injection, infusion or implantation. (Item 59) 54. The composition of any one of items 51 to 53, wherein the composition is formulated for administration by infusion over about 1 hour. (Item 60) 54. The composition of any one of items 51 to 53, wherein the dose of composition is formulated for intravenous administration via a peripheral limb vein. (Item 61) 57. The composition of any one of items 51 to 56, wherein the rAAV comprises a human microdystrophin nucleotide sequence of SEQ ID NO: 1. (Item 62) 58. The composition of any one of items 51 to 57, wherein the rAAV comprises an MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. (Item 63) 59. The composition of any one of items 51 to 58, wherein the rAAV is of the serotype AAVrh.74. (Item 64) 60. The composition of any one of paragraphs 51 to 59, wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9 or of nucleotides 55 to 5021 of SEQ ID NO: 3. (Item 65) 61. The composition according to any one of items 51 to 60, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. (Item 66) 1. A composition for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin; Approximately 2 × 10 administered intravenously over approximately 1 hour 14 formulated for administration at a dose of vg / kg, The composition, wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55 to 5021 of SEQ ID NO:3. (Item 67) 1. Use of a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin for the preparation of a medicament for the treatment of muscular dystrophy in a human subject in need thereof, wherein said medicament is formulated for systemic administration and administered in an amount of about 1 x 10 14 vg / kg ~ approx. 4×10 14 Use, including a dose of rAAV in vg / kg. (Item 68) The medicament is formulated for intravenous administration, and the dose of the rAAV is about 2×10 14 56. The use according to item 55, wherein the serotonin concentration is 1000 mg / kg. (Item 69) The use according to Item 63 or 64, wherein the dose of rAAV is about 10 mL / kg. 66. The use according to any one of items 63 to 65, wherein the medicament is formulated for administration by injection, infusion or implantation. (Item 71) 66. The use according to any one of items 63 to 65, wherein the medicament is formulated for administration by infusion over about 1 hour. (Item 72) 66. The use according to any one of items 63 to 65, wherein the medicament is formulated for intravenous administration via a peripheral limb vein. (Item 73) 69. The use of any one of items 63 to 68, wherein the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1. (Item 74) 70. The use of any one of items 63 to 69, wherein the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. (Item 75) 71. The use of any one of items 63 to 70, wherein the rAAV is of the serotype AAVrh.74. (Item 76) 71. The use of any one of paragraphs 63 to 70, wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9 or of nucleotides 55 to 5021 of SEQ ID NO: 3. (Item 77) 72. The use according to any one of items 63 to 71, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. (Item 78) 1. Use of a recombinant adenovirus-associated (rAAV) rAAV.MHCK7 micro-dystrophin for the preparation of a medicament for treating Duchenne muscular dystrophy in a human subject in need thereof, wherein the medicament is administered by intravenous infusion over a period of about one hour. It is formulated for approximately 2 x 10 14vg / kg of said rAAV, wherein said rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9 or nucleotides 55 to 5021 of SEQ ID NO:3. (Item 79) the serum CK level in the subject is higher after administration of the rAAV compared to the serum CK level before administration of the rAAV; a) at least 78% by 90, 180, or 270 days after said administration; b) at least 46, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 85% by 270 days after said administration; c) at least 72, 73, 74, or 95% by 180 days after said administration; d) at least 87, 88, 93, or 95% by 90 days after said administration; e) at least 70% by 270 days after said administration; f) 70 to 95% by 90, 180, or 270 days after said administration; g) at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after said administration; and h) The method of any one of items 1 to 50, wherein the level of IL-16 is reduced by a percentage level selected from the group consisting of at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% by 90, 180 or 270 days after said administration. (Item 80) After administration of the composition to a human subject in need of treatment for muscular dystrophy, the serum CK level in the subject is compared to the serum CK level before administration of the composition, a) at least 78% by 90, 180, or 270 days after said administration; b) at least 46, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 85% by 270 days after said administration; c) at least 72, 73, 74, or 95% by 180 days after said administration; d) at least 87, 88, 93, or 95% by 90 days after said administration; e) at least 70% by 270 days after said administration; f) 70 to 95% by 90, 180, or 270 days after said administration; g) at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after said administration; and h) The composition of any one of items 51 to 62, wherein the IL-16 expression level is reduced by a percentage level selected from the group consisting of at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% by 90, 180 or 270 days after said administration. (Item 81) the serum CK level in the subject is after administration of the rAAV to the subject, compared to the serum CK level before administration of the rAAV; a) at least 78% by 90, 180, or 270 days after said administration; b) At least 46, 55, 56, 57, 58, 59, 6 by 270 days after the administration 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or 85%; c) at least 72, 73, 74, or 95% by 180 days after said administration; d) at least 87, 88, 93, or 95% by 90 days after said administration; e) at least 70% by 270 days after said administration; f) 70 to 95% by 90, 180, or 270 days after said administration; g) at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after said administration; and h) The use of any one of items 63 to 74, wherein the level of urinary tract infection is reduced by a percentage level selected from the group consisting of at least 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95% by 90, 180 or 270 days after said administration. (Item 82) a) the nucleotide sequence of SEQ ID NO: 3; b) the nucleotide sequence of SEQ ID NO: 8; c) the nucleotide sequence of SEQ ID NO: 9; d) an rAAV comprising nucleotides 55 to 5021 of SEQ ID NO: 3; e) an rAAV comprising the nucleic acid sequence of SEQ ID NO: 9; f) an rAAV comprising nucleotides 1 to 4977 of SEQ ID NO: 8; g) rAAV particles comprising nucleotides 55 to 5021 of SEQ ID NO: 3; h) an rAAV particle comprising the nucleic acid sequence of SEQ ID NO: 9, or i) a composition comprising rAAV particles comprising nucleotides 1 to 4977 of SEQ ID NO: 8. [Brief explanation of the drawings]

[0097] [Figure 1] Figure 1 shows the rAAV.MHCK7.microdystrophin construct. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). The construct is characterized by an in-frame rod deletion (R4 to R23), while the hinges 1, 2, and 4 (H1, H2, and H4) and cysteine-rich domains still produce a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) is driven by the MHCK7 promoter (795 bp). The intron and 5' UTR are derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contained a consensus Kozak sequence immediately before the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contained (R4-R23 / Δ71-78) as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)).

[0098] [Figure 2-1] FIG. 2 provides the nucleic acid sequence (SEQ ID NO: 3) AAVrh74.MHCK7.microdystrophin. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above.

[0099] [Figure 3] Figure 3 provides the pNLREP2-Caprh74 AAV helper plasmid map.

[0100] [Figure 4] FIG. 4 provides the Ad helper plasmid pHELP.

[0101] [Figure 5]Figure 5 shows the rAAV.MCK.micro-dystrophin construct.

[0102] [Figure 6-1] Figure 6 provides the nucleic acid sequence (SEQ ID NO:5) rAAVrh74.MCK.microdystrophin. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0103] [Figure 7] FIG. 7 shows microdystrophin gene expression in muscle fibers of gastrocnemius muscle biopsies as measured by immunocytochemistry.

[0104] [Figure 8-1] Figures 8A-8C provide Western blots showing microdystrophin protein expression at the correct molecular weight. In Figure 8C, the sample from subject 4 (*) was diluted 1:4 (to the linear range) to account for excess ULDQ (>80%) in the starting analysis, and the mean values ​​were multiplied by a dilution correction factor for the final values ​​compared to normal. The mean microdystrophin expression relative to normal was 182.7% for Method 1 and 222.0% for Method 2. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0105] [Figure 9-1] Figures 9A-9C show that administration of rAAVrh74.MHCK7.microdystrophin upregulates the expression of DAPC protein, α-sarcoglycan, and β-sarcoglycan. [Figure 9-2] Same as above. [Figure 9-3] Same as above.

[0106] [Figure 10]FIG. 10 shows a sustained and dramatic reduction in creatine kinase (CK) with administration of rAAVrh74.MHCK7.microdystrophin.

[0107] [Figure 11] Figure 11 provides the mean CK change from baseline to day 270. The data demonstrated a significant decrease in CK over time following rAAVrh74.MHCK7.micro-dystrophin administration.

[0108] [Figure 12] Figure 12 provides the mean NSAA change and mean CK change from baseline to day 270. The data demonstrated a significant increase in NSAA over time following rAAVrh74.MHCK7.micro-dystrophin administration.

[0109] [Figure 13-1] Figure 13 provides the nucleic acid sequence (SEQ ID NO:9) AAVrh74.MHCK7.microdystrophin. [Figure 13-2] Same as above. [Figure 13-3] Same as above.

[0110] [Figure 14] Figure 14 shows the AAVrh74.MHCK7.microdystrophin plasmid construct.

[0111] [Figure 15-1] Figure 15 provides the nucleic acid sequence (SEQ ID NO:8) of the AAVrh74.MHCK7.microdystrophin plasmid construct, which includes a kanamycin resistance gene. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0112] The present invention provides a gene therapy vector, e.g., an rAAV vector, that overexpresses human microdystrophin, as well as a method for reducing and preventing fibrosis in patients with muscular dystrophy. Muscle biopsies taken at the earliest age of diagnosis of DMD reveal significant connective tissue proliferation. Muscle fibrosis is detrimental in multiple ways. It reduces the normal passage of intrathecal nutrients through the connective tissue barrier, reduces blood flow, deprives muscles of vascular nutrients, and is functionally responsible for the early loss of walking ability through limb contractures. Over time, the challenge of treatment increases as a result of significant fibrosis in the muscles. This can be observed in muscle biopsies comparing connective tissue proliferation at successive time points. This process continues to worsen, leading to loss of walking movement and accelerating loss of control, especially in wheelchair-bound patients.

[0113] Without early treatment, including parallel approaches to reduce fibrosis, the benefits of exon skipping, stop codon readthrough, or gene replacement therapy are unlikely to ever be fully realized. Even small molecule or protein replacement strategies are likely to fail without approaches to reduce muscle fibrosis. Previous studies in elderly mdx mice with fibrosis treated with AAV.microdystrophin showed that complete functional recovery could not be achieved (Liu, M. et al., Mol Ther 11, 245-256 (2005)). Progression of DMD cardiomyopathy is also known to be accompanied by scarring and fibrosis of the ventricular wall.

[0114] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a coinfecting helper virus. Currently, there are 13 characterized AAV serotypes. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York. However, since it is well known that various serotypes are very closely related structurally, functionally, and even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174, in Parvoviruses and Human Disease, edited by J.R.P.Tattison; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all produce three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome, and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns suggest that the replication functions in each serotype are under similar regulatory control.

[0115] "AAV vector," as used herein, refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by AAV inverted terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0116] An "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsulated polynucleotide AAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, production of an AAV vector particle necessarily involves production of an AAV vector, and thus the vector is contained within the AAV vector particle.

[0117] AAV Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length and contains 145 nucleotide inverted terminal repeats (ITRs). There are several AAV serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), as revised by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided under GenBank accession number NC_002077; the complete genome of AAV-3 is provided under GenBank accession number NC_1829; the complete genome of AAV-4 is provided under GenBank accession number NC_001829; the AAV-5 genome is provided under GenBank accession number AF085716; and the complete genome of AAV-6 is provided under GenBank accession number NC_00 1862, and at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 regarding AAV-8); the AAV-9 genome is provided in 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 is provided in Virology, 330(2):375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac et al., Journal of Translational Medicine 5, 45 (2007). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19) combine with differential splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227) to drive the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately drive viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites contribute to the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0118] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is asymptomatic. Furthermore, AAV infects many mammalian cell types, allowing it to target many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and nondividing cells and persist essentially for the lifespan of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome can be inserted as cloned DNA into plasmids, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, part or all of the approximately 4.3 kb internal genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another important property of AAV is that it is an extremely stable and viable virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV may also be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0119] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad. Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of the transgene product in the systemic circulation after intramuscular injection, as described by 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). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) showed that skeletal muscle fibers possess the cellular factors necessary for proper antibody glycosylation, folding, and secretion, indicating that muscle can stably express secreted protein therapeutics.

[0120] The recombinant AAV genome of the present invention comprises a nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA of the rAAV genome may be of any AAV serotype from which the recombinant virus can be derived, including, but not limited to, AAVrh.74, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle-specific expression, AAV1, AAV6, AAV8, or AAVrh.74 can be used.

[0121] The DNA plasmid of the present invention contains the rAAV genome of the present invention. The DNA plasmid is transferred into a cell permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus), allowing the rAAV genome to assemble into infectious viral particles. The technology for producing rAAV particles, in which the AAV genome to be packaged, the rep and cap genes, and helper virus functions are provided in the cell, is standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and the helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13, from which recombinant virus may be derived than the rAAV genome ITRs. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0122] The method for generating packaging cells involves creating a cell line that stably expresses all components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes isolated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the cell's genome. The AAV genome is introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (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 adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus, rather than a plasmid, to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0123] General principles of rAAV production are described, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various approaches are described in Ratschin et al., Mol. Cell. Biol., 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol., 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al., J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al., Vaccine 13:1244-1250 (1995); Paul et al., Human Gene Therapy 4:609-615 (1993); Clark et al., Gene Therapy 3:1124-1132 (1996); U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis being placed on those sections of the documents relating to the production of rAAV.

[0124] Thus, the present 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 (homologous 293 strains). In another embodiment, the packaging cells are non-transformed cancer cells such as low-passage 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey lung cells).

[0125] The recombinant AAV (i.e., infectious, encapsidated rAAV particles) of the present invention comprise a rAAV genome. In a typical embodiment, the genome of the rAAV lacks both AAVrep and capDNA, i.e., there is no AAVrep or capDNA between the ITRs of the genome. Examples of rAAVs that can be constructed to comprise the nucleic acid molecules of the present invention are described in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352), the entire contents of which are incorporated herein by reference.

[0126] In a typical embodiment, the recombinant AAV vector of the present invention is produced by the triple transfection method (Xiao et al., J Virol 72, 2224-2232 (1998)) using the AAV vector plasmid rAAV.MHCK7.microdystrophin pNLRep2-Caprh74 and pHelp. The rAAV contains a microdystrophin gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs), which are encapsulated in AAVrh74 virions. The plasmid contains the microdystrophin sequence and the MHCK7 enhancer and core promoter elements of the muscle-specific promoter, resulting in gene expression. The expression cassette contains an SV40 intron (SD / SA) to drive high-level gene expression, and the bovine growth hormone polyadenylation signal is used for efficient transcription termination.

[0127] pNLREP2-Caprh74 is an AAV helper plasmid encoding four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from serotype rh74. A schematic diagram of the pNLREP2-Caprh74 plasmid is shown in Figure 3.

[0128] The pHELP adenoviral helper plasmid is 11,635 bp and was obtained from Applied Viromics. The plasmid contains regions of the adenoviral genome important for AAV replication, namely, E2A, E4 ORF6, and VA RNA (the adenoviral E1 function is provided by 293 cells). The adenoviral sequences present in this plasmid represent only approximately 40% of the adenoviral genome and do not contain cis elements important for replication, such as the adenoviral terminal repeats. Therefore, no infectious adenovirus is expected to be generated from such a production system. A schematic map of the pHELP plasmid is shown in Figure 4.

[0129] rAAV may be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0130] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The composition of the present invention comprises rAAV and a pharmaceutically acceptable carrier. The composition may also contain other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to recipients, preferably inert at the dosages and concentrations used, and include buffers and surfactants such as Pluronics.

[0131] The titer of rAAV to be administered in the methods of the invention can vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the targeted cell type(s), and can be determined by standard methods in the art. The titer of rAAV is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 The dosage may range from 0.1 to 1.0 mg / mL of DNase-resistant particles (DRP) or more. Dosages may also be expressed in units of viral genomes (vg). One exemplary method for determining encapsidated vector genome titers uses quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25(4):855-869, 2017).

[0132] Methods of transducing target cells with rAAV in vivo or in vitro are contemplated by the present invention. In vivo methods involve administering an effective dose, or effective repeated doses, of a composition comprising an rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease condition, reduces the extent of the disease, results in remission (partial or total) of the disease, and / or prolongs survival. An example of a disease contemplated for prevention or treatment by the methods of the present invention is DMD.

[0133] Combination therapy is also contemplated by the present invention. As used herein, combination includes both simultaneous and sequential therapy. Combination of the methods of the present invention with standard medical treatments (e.g., corticosteroids) is specifically contemplated, as is combination with novel therapies.

[0134] Administration of an effective dose of the composition can be by any standard route in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The administration route(s) and serotype(s) of the AAV components of the rAAV (particularly the AAV ITRs and capsid proteins) can be selected and / or adapted by those skilled in the art, taking into account the infection and / or disease state to be treated and the target cell / tissue(s) that will express microdystrophin.

[0135] The present invention provides for local administration and systemic administration of effective doses of rAAV and compositions of the present invention.For example, systemic administration is administration to the circulatory system, which affects the entire body.Systemic administration includes absorption through the gastrointestinal tract and enteral administration, such as parenteral administration via injection, infusion, or implantation.

[0136] In particular, the actual administration of the rAAV of the present invention can be achieved using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, injection into the muscle and the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has been shown to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carrier or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in typical rAAV-based methods). The capsid protein of rAAV may be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, International Publication No. WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable or topical formulations to be delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0137] In one embodiment of the invention, the AAVrh74.MHCK7.microdystrophin described herein is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0138] The dose of rAAV to be administered in the methods disclosed herein can vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the targeted cell type(s), and can be determined by standard methods in the art. The titer of each rAAV administered is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×1013 , about 1×10 14 , about 2×10 14 , or ~1 × 10 15 Dosages may also be expressed in units of viral genomes (vg) (i.e., 1 x 10 ... 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 2 × 10 14 vg, 1×10 15 Dosages may be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1 x 10 10 vg / kg, 1 × 10 11 vg / kg, 1 × 10 12 vg / kg, 1 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.25 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.75 × 10 14 vg / kg, 2.0 × 10 14 vg / kg, 2.25 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.75 × 10 14 vg / kg, 3.0 × 10 14 vg / kg, 3.25 × 10 14 vg / kg, 3.5 × 10 14 vg / kg, 3.75 × 10 14 vg / kg, 4.0 × 10 14 vg / kg, 1 × 10 15 vg / kg). A method for determining the titer of AAV is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0139] In particular, the actual administration of the rAAV of the present invention can be achieved using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, injection into the muscle and the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has been shown to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carrier or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in typical rAAV-based methods). The capsid protein of rAAV may be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, International Publication No. WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable or topical formulations to be delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0140] For intramuscular injection, solutions in adjuvants such as sesame or peanut oil, or aqueous propylene glycol, as well as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered, if desired, and the liquid diluent can first be rendered isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxpropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed are all readily available by standard techniques well known to those skilled in the art.

[0141] Pharmaceutical carriers, diluents, or excipients 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 fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0142] Sterile injectable solutions are prepared by incorporating the required amount of rAAV in an appropriate solvent with various other ingredients listed above, and then, as needed, filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired optional ingredients from the previously filter-sterilized solution.

[0143] Transduction using rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from a subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used if these cells do not elicit an inappropriate immune response in the subject.

[0144] Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in an appropriate medium and screening those cells for DNA of interest using conventional techniques such as Southern blot and / or PCR, or using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, and the composition can be introduced into a subject by various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or injection into smooth muscle and cardiac muscle, for example, using a catheter.

[0145] Transduction of cells with the rAAV of the present invention results in sustained expression of microdystrophin protein. Thus, the present invention provides methods for administering / delivering rAAVs expressing microdystrophin protein to animals, preferably humans. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of the present invention. Transduction may be performed using a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present invention utilizes regulatory elements derived from the actin and myosin gene family, such as the myoD gene family (see Weintraub et al., Science 251:761-766 (1991)), muscle cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11:4854-4862 (1991)), regulatory elements derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol 7:4089-4099 (1987)), cardiac actin gene, muscle creatine kinase sequence element (Johnson et al., Mol Cell Biol 9:3393-3399 (1989)), and mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene, and the slow-twitch troponin I gene; hypoxia-inducible nuclear factor (Semenza et al., Proc Natl. Acad. Sci. USA 88:5680-5684 (1991)), steroid-inducible elements and promoters including glucocorticoid response elements (GREs) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), and other regulatory elements.

[0146] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present invention allows for sustained expression of micro-dystrophin from transduced muscle fibers.

[0147] By "muscle cell" or "muscle tissue" is meant a cell or group of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, e.g., gastrointestinal, bladder, vascular, or cardiac tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0148] The term "transduction" is used to refer to the administration / delivery of the coding region for microdystrophin to recipient cells via a replication-deficient rAAV of the present invention, either in vivo or in vitro, resulting in expression of microdystrophin by the recipient cells.

[0149] Thus, the present invention provides methods of administering to a subject in need thereof an effective amount (or doses administered essentially simultaneously or at regular intervals) of rAAV encoding micro-dystrophin.

[0150] The following examples are offered by way of illustration and not by way of limitation: Numerical ranges recited are inclusive of each integer value within each range, including the minimum and maximum stated integers. [Example]

[0151] Example 1 A) Generation of the AAVrh74.MHCK7.microdystrophin construct The AAVrh74.MHCK7.microdystrophin plasmid contains a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 1). The microdystrophin construct is characterized by an in-frame rod deletion (R4-R23), while hinges 1, 2, and 4 and the cysteine-rich domain remain, producing a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) was driven by the MHCK7 promoter (792 bp). The plasmid was constructed from the rAAV.MCK.microdystrophin plasmid by removing the MCK promoter and inserting the MHCK7 promoter. Following the core promoter, 53 bp of the endogenous mouse MCK exon 1 (untranslated) are present for efficient transcription initiation, followed by the SV40 late 16S / 19S splice signal (150 bp) and a small 5' UTR (61 bp). The intron and 5'UTR were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contained a consensus Kozak sequence immediately before the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contained (R4-R23 / Δ71-78) as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)). The complementary DNA was codon-optimized for human use and synthesized by GenScript (Piscataway, NJ) (Mol Ther 18, 109-117 (2010)). The only viral sequence contained in this vector is the AAV2 inverted terminal repeat, which is required for both viral DNA replication and packaging. The microdystrophin cassette contains a small 53-bp synthetic poly(A) signal for mRNA termination.

[0152] Previous studies have demonstrated cardiac expression using the MHCK7 promoter (Salva et al., Mol Ther 15:320-329 (2007)) and AAVrh.74 to achieve skeletal, diaphragm, and cardiac muscle expression (Sondergaard et al., Annals of Clinical and Transl Neurology 2:256-270 (2015)). The construct sequence in Figure 1 was encapsulated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a rhesus monkey lymph node and is discussed in Rodino-Klapac et al., Journal of Translational Medicine 5:45 (2007). Table 1 shows the molecular characteristics of the plasmid AAVrh74.MHCK7.microdystrophin (SEQ ID NO: 3). [Table 1]

[0153] B) Generation of the AAVrh74.MHCK7.microdystrophin construct from a kanamycin (Kan) resistance-encoding plasmid and a kanamycin (Kan) resistance-encoding plasmid Cloning of MHCK7.μDys.KAN was achieved by isolating the MHCK7.μDys fragment from the MHCK7.μDys.AMP plasmid and kanamycin backbone and annealing them using the NEBuilder cloning workflow. The MHCK7.μDys fragment was isolated via restriction enzyme digestion with SnaBI. Digestion was performed for 1 hour at 37°C in a 50 μL total reaction volume in 1× CutSmart Buffer (NEB) and 1 μL of SnaBI. The resulting fragment was isolated by electrophoresis on a 1% agarose gel run at 105 volts for 1.5 hours. The band corresponding to the MHCK7.μDys insert was excised and purified using a gel purification kit (Macherey-Nagel). The resulting fragment had a DNA concentration of 10 ng / μL. The Kan backbone fragment was isolated via XbaI restriction enzyme digestion in a 50 μL reaction containing 1× CutSmart buffer (NEB) and 1 μL XbaI at 37°C for 1 hour. The resulting fragment was isolated by electrophoresis on a 1% agarose gel run at 105 volts for 1.5 hours. The band corresponding to the Kan backbone was excised and purified using a gel purification kit (Macherey-Nagel). The resulting fragment had a DNA concentration of 8.1 ng / μL. The two fragments were annealed using the NEB Builder cloning workflow, which has the ability to join two fragments with overlapping sequences. The NEBuilder cloning reaction was performed according to the manufacturer's protocol at 50°C for 15 minutes in a total reaction volume of 20 μL, using a 1:1 ratio of MHCK7.μDys to kanamycin backbone in 1× NEBuilder HiFi DNA Assembly Master Mix. The resulting clones were transformed into NEB® Stable Competent E. coli (C3040) by adding 2.5 L of the cloning product to the cells, followed by 30 minutes on ice, then 30 seconds at 42° C., and an additional 5 minutes on ice. After transformation, 950 μL of growth medium was added to the cells and grown at 30° C. for 1.5 hours with shaking at 225 rpm.After growth, 450 μL of these cells were plated onto 50 μg / mL kanamycin LB agar plates and incubated overnight at 30°C in a dry incubator. Colonies were picked from the plates and grown overnight in LB containing 50 μg / mL kanamycin. DNA was isolated from 3 mL of this culture using a QIAprep® Spin Miniprep Kit (Qiagen). This DNA was used to verify the cloning product. The cloning product was confirmed via restriction enzyme digestion with PmeI, MscI, and SmaI followed by gel electrophoresis. The cloning product was further verified via sequencing. The resulting plasmid is set forth in SEQ ID NO:8 and shown in Figures 14 and 15. The sequence of the construct in Figure 13, corresponding to that of SEQ ID NO:9, and nucleotides 1-4977 of SEQ ID NO:8, were encapsulated into AAVrh.74 virions as described above.

[0154] Example 2 Systemic gene delivery clinical trial for Duchenne muscular dystrophy This is a single-dose controlled study using rAAVrh74.MHCK7.micro-dystrophin of SEQ ID NO: 3, nucleotides 55-5021, for DMD subjects. Cohort A includes six subjects aged 3 months to 3 years, and Cohort B includes six subjects aged 4-7 years. All subjects will receive the micro-dystrophin vector intravenously (2 x 10 in 10 mL / kg). 14 rAAVrh74.MHCK7.microdystrophin is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0155] In the study, rAAVrh74.MHCK7.micro-dystrophin was infused via a peripheral arm vein to reach all muscles in the body. Cohort A enrolled six subjects with DMD aged 3 months to 3 years, and Cohort B enrolled six subjects with DMD aged 4 to 7 years. All subjects received the micro-dystrophin vector intravenously (2 × 10 in 10 mL / kg).14 Encapsulated vector genomes for the administered dose were determined using quantitative PCR using a Prism 7500 Taqman detector system (PE Applied Biosystems) with primers to the MHCK7 promoter, compared to a supercoiled DNA plasmid standard (Pozsgai et al., Mol. Ther. 25(4):855-869, 2017).

[0156] Subjects received an hour-long infusion in the pediatric intensive care unit (PICU) at Nationwide Children's Hospital. A muscle biopsy was performed at the screening visit before gene therapy. Subjects underwent a second muscle biopsy 90 days after delivery to determine whether the missing dystrophin protein had been replaced. After gene transfer, patients were carefully monitored for any side effects of treatment. This monitoring included blood and urine tests and physical examinations during the screening visit and at days 0, 1, 7, 14, 30, 60, 90, and 180, and at months 9, 12, 18, 24, 30, and 36 to ensure there were no side effects from the gene injection.

[0157] Cohort A subjects (n=6) were aged 3 months to 3 years and received intravenous administration of the rAAVrh74.MHCK7.microdystrophin vector (2 x 10 in 10 mL / kg). 14 vg / kg). One day before gene transfer in cohort A, subjects were started on 1 mg / kg prednisone or deflazacort and maintained for 30 days while monitoring the immune response. If negative on day 30, steroids were withdrawn over a one-week period. If the T cell response to AAV or microdiscs was >125 SFC / 106 PBMC, steroids were maintained until levels fell below this threshold.

[0158] Cohort B subjects (n = 6) were 4-7 years old and received intravenous administration of the rAAVrh74.MHCK7.microdystrophin vector (2 × 10 in 10 mL / kg) 14These subjects were maintained on stable doses of corticosteroids throughout the trial, but could be escalated for short periods if T cell responses to AAV or micro-dystrophin were >125 SFC / 106 PBMC.

[0159] Eligibility Criteria The inclusion criteria for the trial were as follows: Enrollment age: Cohort A: 3 months to 7 years old, Cohort B: 4 to 7 years old (inclusive). · Molecular characterization of the DMD gene with frameshift (deletion or duplication) or premature stop codon mutations in exons 18–58. CK elevation > 1000U / L Cohort A subjects: below average on the Bayley-III Motor Assessment for gross movement, defined as an adjusted score ≤9. Cohort B: Below average on the 100-meter time test, defined as <80% predicted value. · Men of any ethnicity. ·Ability to cooperate with athletic evaluation testing. Cohort A subjects: Corticosteroid-naive. Cohort B subjects: Oral corticosteroids equivalent to a stable dose for at least 12 weeks prior to screening, with the dose expected to remain constant throughout the study (except for adjustments to accommodate changes in body weight).

[0160] The exclusion criteria for this study were as follows: · Active viral infection based on clinical findings. · Signs of cardiomyopathy, including an echocardiogram showing an ejection fraction less than 40%. · Serological evidence of HIV infection or hepatitis B or C infection. · Diagnosis (or ongoing treatment) of an autoimmune disease. · Abnormal laboratory values ​​that are considered clinically significant. · Coexisting illnesses or the need for chronic drug treatment, which in the PI's opinion, creates unnecessary risks for gene transfer. Subjects with AAVrh74 or AAV8 antibody titers >1:400 as determined by ELISA immunoassay. · A medical condition or extenuating circumstances that, in the investigator's opinion, may compromise the subject's ability to comply with protocol-compliant tests or procedures or may compromise the subject's health, safety, or clinical competence. Severe infection (e.g., pneumonia, renal vein nephritis, or meningitis) within 4 weeks prior to the gene transfer visit (enrollment may be postponed). -Receipt of any investigational or experimental drug (other than corticosteroids) or exon-skipping drug (including ExonDys 51®) in the 6 months prior to screening for this study. Have received any type of gene therapy, cell-based therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy. Family members do not want the patient's clinical trial participation to be disclosed to the patient's primary care physician and other health care providers.

[0161] Evaluation items The primary endpoint was safety based on the number of participants with adverse events (time frame: 3 years). Side effects were monitored and scored for severity and relevance to the study literature.

[0162] Secondary endpoints were as follows:

[0163] Gross Motor Subtest Measure (Bayley-III) Score (Time Frame: Screening, Day 30-3 Years): Gross Motor Scale scores measured motor development. The Bayley-III Gross Motor Subtest was scored for Cohort A at all follow-up visits beginning at Day 30 through Year 3. Any subject who was 43-47 months of age (inclusive) at screening had a scaled score calculated relative to normative data for 42-month-old children. The Bayley-III provides normative data for children aged 1-42 months.

[0164] Physical Therapy Assessment. Timed 100-meter test (100m) (Time Frame: Screening, Day 30-3 years): The 100m was the primary motor outcome for Cohort B. The timed 100m test was a finding that began for Cohort A when children turned 3 years old.

[0165] Physical Therapy Assessment. North Star Gait Assessment (NSAA) (Time Frame: Screening, Day 30-3 Years): The North Star Gait Assessment (NSAA) was an outcome that began for Cohort A and for Cohort B when the children turned 4 years of age. The NSAA measures the quality of gait in young boys with Duchenne muscular dystrophy.

[0166] Pediatric Physical Therapy Assessment Timed Up and Go (TUG) (Time Frame: Screening, Day 30–3 Years): Findings for Cohort B included a modified Timed Up and Go (TUG) for children.

[0167] Four-level increase and decrease in physical therapy assessment (time frame: screening, day 30-3 years): Findings for Cohort B include four-level increase and decrease.

[0168] Physical Therapy Assessment Handheld Dynamometry (HHD) (Time Frame: Screening, Day 30-3 Years): Findings for Cohort B included handheld dynamometry (HHD) of the knee extensors and flexors, and elbow flexors and extensors.

[0169] Quantification of microdystrophin gene expression by immunofluorescence staining (time frame: screening, day 90): Microdystrophin gene expression levels were quantified by immunofluorescence staining and compared before and after muscle biopsy.

[0170] Quantification of microdystrophin gene expression by immunofluorescence staining (time frame: screening, day 90): Microdystrophin gene expression levels were quantified by Western blot and compared before and after muscle biopsy.

[0171] CK reduction after gene therapy (time frame: 3 years): Reduction in circulating CK levels.

[0172] Cardiac magnetic resonance imaging (at 1 year).

[0173] Microdystrophin gene expression Changes in microdystrophin expression from baseline were analyzed and quantified via immunofluorescence staining (IF) fiber intensity. As shown in Figure 7, subject 1 (5 years old) showed 78% microdystrophin protein expression in muscle fibers of gastrocnemius muscle biopsies after administration of rAAVrh74.MHCK7.microdystrophin, subject 2 (4 years old) showed 73.5% microdystrophin protein expression in muscle fibers of gastrocnemius muscle biopsies after administration of rAAVrh74.MHCK7.microdystrophin, and subject 3 (6 years old) showed 77.0% microdystrophin protein expression in muscle fibers of gastrocnemius muscle biopsies after administration of rAAVrh74.MHCK7.microdystrophin. Subject 4 (4 years old) showed 96.2% microdystrophin expression in muscle fibers of gastrocnemius muscle biopsies after administration of rAAVrh74.MHCK7.microdystrophin. All patients showed strong expression of the transduced microdystrophin, which was properly localized to the sarcolemma as measured by immunohistochemistry. Mean gene expression, as measured by the percentage of microdystrophin-positive fibers, was 76.2%, and mean fiber intensity was 74.5% compared to normal controls. [Table 7-1] [Table 7-2]

[0174] Changes in microdystrophin gene expression from baseline to day 60 were also assessed by quantifying microdystrophin protein expression measured by Western blot of biopsied muscle tissue. As shown in Figures 8A and 8B, Western blot analysis detected microdystrophin protein expression in subject 1 (5 years old), subject 2 (4 years old), and subject 3 (6 years old). Figure 8C provides a Western blot analysis detecting microdystrophin protein expression in subject 4 (4 years old). All post-treatment biopsies showed healthy levels of microdystrophin as measured by Western blot, with an average of 74.3% for subjects 1-4 compared to normal using method 1 and an average of 95.8% for subjects 1-4 compared to normal according to method 2, adjusting for adipose and fibrous tissue.

[0175] For each subject, the vector genome copies per muscle fiber nucleus were measured. As shown in Table 2, the vector genome copies per nucleus was greater than 1 for each subject after administration of rAAVrh74.MHCK7.micro-dystrophin. One copy of vector represents approximately 50% expression of the micro-dystrophin gene. An average of 1.6 vector copies per cell nucleus was measured in subjects 1-3, consistent with the observed high micro-dystrophin expression levels. When the value for subject 4 was included, the average vector copies / μg of DNA was >10 5 , with an average of 3.3 vector copies per cell nucleus. [Table 2]

[0176] The protein levels of α-sarcoglycan and β-sarcoglycan in muscle biopsy tissue were measured by immunohistochemistry before and after administration of rAAVrh74.MHCK7.microdystrophin. Administration of rAAVrh74.MHCK7 also resulted in upregulation of DAPC protein in the subjects. As shown in Figure 9, the expression of α-sarcoglycan and β-sarcoglycan in muscle biopsy tissue was increased compared to the levels of these proteins in muscle biopsies before administration of rAAVrh74.MHCK7 in Subject 1 (Figure 9A), Subject 2 (Figure 9B), and Subject 3 (Figure 9C). Circulating serum CK levels

[0177] Blood samples were then transfected with rAAVrh74.MHCK7.microdystrophin vector (2 × 10 in 10 mL / kg). 14 Subjects were collected every 30 days after intravenous infusion of 100 mg / kg (vg / kg). CK levels were measured at each visit and compared to baseline levels obtained before rAAVrh74.MHCK7.micro-dystrophin administration (visit day 0). Baseline serum CK levels (units / liter) are provided in Table 3 below. As shown in Figure 10, circulating serum CK levels decreased by approximately 87% two months after administration of rAAVrh74.MHCK7.micro-dystrophin. All subjects showed a significant decrease in serum creatine kinase (CK) levels, with a mean decrease in CK of more than 87% after two months of treatment (n=3). CK is an enzyme associated with muscle damage, and patients with DMD consistently exhibit elevated levels of CK. Indeed, significantly elevated CK is often used as a preliminary diagnostic tool for DMD, followed by definitive genetic testing.

[0178] Table 4 and Figure 10 provide the CK levels for each subject. Figure 11 provides the average CK levels over time and shows that the average CK levels significantly decrease over time after administration of rAAVrh74.MHCK7.micro-dystrophin. The average baseline CK level of 27,064 U / L (mean value in Table 3) decreases by approximately 63% to an average of 9,982 U / L (mean, day 270, Table 4). [Table 3] [Table 4]

[0179] Efficacy evaluation In addition to microdystrophin and CK levels, efficacy was measured by the following functional tests: floor rise time, four-step climb time, North Star Ambulatory Assessment (NSAA), stand-up time test, four-step climb time, 10-meter timed test (10m), and 100-meter timed test (100m). The data are provided in Tables 5 and 6 below and demonstrate consistent and sustained improvement nine months after administration of rAAVrh74.MHCK7.microdystrophin. Improvement in NSAA over time is also provided in Figure 12. [Table 5-1] [Table 5-2] [Table 6]

[0180] Safety evaluation No serious adverse events (SAEs) were observed during the study. Three subjects had elevated gamma-glutamyltransferase (GGT), which resolved with increased steroids within 1 week and returned to baseline levels. There were no other clinically significant laboratory findings. Patients generally experienced transient nausea during the first week of therapy, concurrent with increased steroid administration. This did not correlate with liver enzyme elevations or any other abnormalities.

[0181] Example 3 Randomized, double-blind, placebo-controlled, systemic gene delivery phase I / IIa clinical trial This is a randomized, double-blind, single-dose study using rAAVrh74.MHCK7.micro-dystrophin in subjects with DMD. The study will include 24 subjects aged 4-7 years. Subjects will be randomized to treatment or placebo at enrollment. Twelve subjects will receive an intravenous injection of the rAAVrh74.MHCK7.micro-dystrophin vector (2 x 10 at approximately 10 mL / kg). 14 vg / kg), 12 subjects will receive 10 mL / kg placebo (lactated Ringer's solution). Placebo subjects will proceed to treatment in the same manner as the 12 previously treated subjects, one year after the last treatment was administered. Subjects will receive an infusion of micro-dystrophin-bearing rAAV or lactated Ringer's solution over approximately one hour. Pre- and post-treatment (day 90) needle muscle biopsies will be taken from the gastrocnemius muscle.

[0182] The primary objective of this study is to evaluate the safety of intravenous administration of rAAVrh74.MHCK7.micro-dystrophin via a peripheral limb vein to DMD subjects. Safety endpoints will be assessed by hematology, blood chemistry, and urinalysis changes, immune responses to rAAVrh.74 and micro-dystrophin, and observation of reported medical history and symptoms. Dystrophin gene expression will serve as the primary outcome measure, along with safety. Quantification will be performed using validated immunofluorescence and immunoblot assays. Reduction of CK after gene therapy will serve as a secondary outcome. Efficacy will be measured by the following functional tests: time to stand, time to climb four stairs, North Star Gait Assessment (NSAA), timed 10-meter test (10 m), and timed 100-meter test (100 m). Investigational measures will include handheld dystrophin testing (HHD) for knee extensors and flexors, and elbow flexors and extensors.

[0183] The inclusion criteria for this study were as follows: Registration age: 4-7 years old (inclusive). · Molecular characterization of the DMD gene with frameshift (deletion or duplication) or premature stop codon mutations in exons 18–58. · Signs of symptomatic muscular dystrophy: CK elevation >1000U / L and less than the mean percent predicted time in the 100 meter walk test. Men of any ethnic group are eligible. ·Ability to cooperate with athletic evaluation testing. Oral corticosteroids equivalent to a stable dose for at least 12 weeks prior to screening, with the dose expected to remain constant throughout the study (except for possible modifications to accommodate changes in body weight).

[0184] The exclusion criteria for this trial are as follows: · Active viral infection based on clinical findings. · Signs of cardiomyopathy, including an echocardiogram showing an ejection fraction less than 40%. · Serological evidence of HIV infection or hepatitis B or C infection. · Diagnosis (or ongoing treatment) of an autoimmune disease. · Abnormal laboratory values ​​considered clinically significant (GGT>3XULN, bilirubin≥3.0mg / dL, creatinine≥1.8mg / dL, Hgb<8 or >18g / Dl, WBC>18,500 per cmm), platelets ≤50,000. · Coexisting illnesses or the need for chronic drug treatment, which in the PI's opinion, creates unnecessary risks for gene transfer. Subjects with AAVrh74 or AAV8 antibody titers >1:400 as determined by ELISA immunoassay. If endpoint titers are positive at screening, testing may be repeated before exclusion. · Have a medical condition or extenuating circumstances that, in the investigator's opinion, may compromise the subject's ability to comply with protocol-compliant tests or procedures or may compromise the subject's health, safety, or clinical competence. Severe infection (e.g., pneumonia, renal vein nephritis, or meningitis) within 4 weeks prior to the gene transfer visit (enrollment may be postponed). -Receipt of any investigational or experimental drug (other than corticosteroids) or exon-skipping drug (including ExonDys 51®) in the 6 months prior to screening for this study. Have received any type of gene therapy, cell-based therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy. Family members do not want the patient's clinical trial participation to be disclosed to the patient's primary care physician and other health care providers.

[0185] Efficacy evaluation Dystrophin gene expression will serve as the primary outcome measure along with safety. Quantification will be performed using validated immunofluorescence and immunoblot assays. Reduction of CK after gene therapy will serve as a secondary outcome. In addition, efficacy will be measured by the following functional tests: floor rise time, four-step climb time, North Star Gait Assessment (NSAA), 10-meter timed test (10m), and 100-meter timed test (100m). Study measurements will include handheld dystrophin testing (HHD) for knee extensors and flexors, and elbow flexors and extensors.

[0186] Muscle biopsies using ultrasound guidance will be used to quantify transgene expression at baseline and day 90. Biopsies will be performed in the same muscle as the original biopsy, but on the opposite leg. One year after dosing all subjects, placebo crossover subjects will resume the study schedule at Visit 1. Placebo subjects will not undergo the following at the second baseline screening: cardiac MRI and muscle biopsy. Placebo subjects will undergo a muscle biopsy at day 90 (a total of three muscle biopsies). Frozen sections will be stained for dystrophin using indirect immunofluorescence (IF). Full slide scans will be performed, and micro-dystrophin intensity and percentage of positive fibers will be quantified using validated image scans and the MuscleMap™ analysis algorithm. Muscle morphometry, including fiber size histograms, will be performed in a blinded manner. Blinded frozen muscle biopsy shavings will be used to perform quantitative protein analysis for micro-dystrophin using a validated Western blot method.

[0187] Muscle needle biopsies of the gastrocnemius muscle (unless PI is contraindicated in a particular subject, in which case PI will choose another muscle to biopsy) will be used to quantify microdystrophin expression.

[0188] Efficacy analysis The primary efficacy endpoint is the change from baseline to day 90 in the amount of microdystrophin protein expression, as measured by Western blot of biopsied muscle tissue. Differences between treatment groups for the primary efficacy endpoint are assessed using an analysis of covariance (ANCOVA) model with treatment as the fixed factor and baseline value as the covariate. Wilcoxon rank-sum tests are performed as supplemental analyses. Changes from baseline in microdystrophin expression via immunofluorescence staining (IF) fiber intensity are similarly analyzed.

[0189] Supplementary efficacy endpoints include changes from baseline to each scheduled assessment in floor rise time, four-step climb time, NSAA, 10-meter timed test (10m), 100-meter timed test (100m), and CK change. Study measures include HHD for knee extensors and flexors, and elbow flexors and extensors. Differences between treatment groups are assessed using an ANCOVA model with treatment as a fixed factor and baseline values ​​as a covariate. Wilcoxon rank-sum tests are performed as supplementary analyses.

[0190] Example 4 The tests and studies described above in Examples 2 and 3 are alternatively performed using the rAAVrh74.MHCK7.microdystrophin construct set forth in SEQ ID NO:9, set forth in SEQ ID NO:8, nucleotides 1-4977, or set forth in SEQ ID NO:6, nucleotides 56-5022.

[0191] Example 5 Generation of pAAV.MCK.micro-dystrophin construct The pAAV.MCK.microdystrophin plasmid was constructed by inserting an MCK expression cassette carrying a codon-optimized human microdystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski et al., J. Virol. 61(10):3096-3101). A muscle-specific regulatory element was included in the construct to confer muscle-specific gene expression. This regulatory element contained the mouse MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal portion). Following the core promoter, the construct contained 53 bp of endogenous mouse MCK exon 1 (untranslated) for efficient transcription initiation, followed by the SV40 late 16S / 19S splice signal (97 bp) and a small 5' UTR (61 bp). The intron and 5' UTR were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contains a consensus Kozak site immediately before the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contains the (R4-R23 / Δ71-78) domain previously described by Harper et al., Nat. Med. 8(3):253-61, 2002.

[0192] The pAAV.MCK.microdystrophin plasmid contained a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 5). This sequence was encapsidated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a rhesus monkey lymph node and is described in Rodino-Klapac et al., Journal of Tran. Med. 45 (2007).

[0193] References [ka] [ka] [ka]

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[Claim 1] An object, method or system as described in this specification and drawings.

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