Adeno-associated viral vector delivery of muscle-specific minimal muscular dystrophy proteins for treatment of muscular dystrophy

Abstract

The present invention relates to adeno-associated viral vector delivery of muscle-specific minimal muscular dystrophy proteins for the treatment of muscular dystrophy. The present invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, that express miniaturized human Muscle Dystrophin genes, and methods of using these vectors to express minor muscular dystrophy proteins and to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy in skeletal muscles, including septal and cardiac muscles.

Description

[0001] This application is a divisional application of the invention patent with the application date of June 17, 2019, application number 201980054169.6, and invention title "Adeno-associated virus vector delivery of muscle-specific minidystrophin for the treatment of muscular dystrophy".

[0002] This application claims the priority benefits of U.S. Provisional Patent Application No. 62 / 686,668 filed on June 18, 2018; U.S. Provisional Patent Application No. 62 / 740,402 filed on October 2, 2018; U.S. Provisional Patent Application No. 62 / 752,841 filed on October 30, 2018; U.S. Provisional Patent Application No. 62 / 823,649 filed on March 25, 2019, and U.S. Provisional Patent Application No. 62 / 860,220 filed on June 11, 2018, and each of these provisional patent applications is incorporated herein by reference in its entirety. Technical Field

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

[0004] The importance of muscle mass and strength for daily activities such as movement and breathing, as well as whole-body metabolism, is obvious. Insufficient muscle function causes muscular dystrophy (MD), which is characterized by muscle weakness and wasting, and can severely affect the quality of life. The best-characterized MDs are the result of gene mutations encoding members of the dystrophin-associated protein complex (DAPC). These MDs are the result of membrane fragility associated with the loss of sarcolemmal cytoskeletal tethering caused by DAPC. Duchenne muscular dystrophy (DMD) is one of the most lethal muscle diseases, affecting 1 in 5000 newborn boys.

[0005] DMD is caused by mutations in the DMD gene that lead to reduced mRNA and 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 consists of multiple proteins at the muscle sarcolemma that form a structural link between the extracellular matrix (ECM) and the cytoskeleton via dystrophin, an actin-binding protein, and α-dystroglycan, a laminin-binding protein. These structural links are used to stabilize the muscle cell membrane during contraction and to prevent contraction-induced damage. With the loss of dystrophin, membrane fragility leads to 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): pp. 1059-71; Wallace et al., Annu Rev Physiol, 2009. 71: pp. 37-57), resulting in progressive muscle weakness, endomysial inflammation, and fibrotic scarring.

[0006] In the absence of membrane stabilization from dystrophin or utrophin, DMD will exhibit an uncontrolled cycle of tissue damage and eventual repair by fibrotic scar tissue replacement of lost muscle fibers through connective tissue proliferation. Fibrosis is characterized by the excessive deposition of ECM matrix proteins, including collagen and elastin. ECM proteins are mainly produced by cytokines such as TGF, which are released by activated fibroblasts in response to stress and inflammation. Although the primary pathological feature of DMD is muscle fiber degeneration and necrosis, fibrosis as a pathological consequence has the same outcome. The overproduction of fibrotic tissue limits muscle regeneration and contributes to progressive muscle weakness in DMD patients. In one study, the presence of fibrosis in initial 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): pp. 762-7). These results suggest that fibrosis is a major contributor to DMD muscle dysfunction and highlight the need for early intervention before significant fibrosis occurs.

[0007] For patients with DMD, treatments are needed to increase muscle strength and prevent muscle damage. SUMMARY OF THE INVENTION

[0008] The present invention relates to gene therapy vectors, such as AAV, for expressing microdystrophin gene in skeletal muscle (including diaphragm muscle 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 methods for using gene therapy vectors to deliver microdystrophin to address the gene defect observed in DMD, thereby increasing muscle strength and / or increasing muscle mass. Example 2 describes a clinical trial of systemic gene delivery for Duchenne muscular dystrophy, in which subjects received 2×1014 vg / kg AAVrh74.MHCK7.microdystrophin. The clinical study described in Example 3 provides a novel pivotal clinical protocol, which includes a randomized double-blind placebo-controlled design. At the start of the study, subjects were randomly assigned and received 2×10 14 vg / kg AAVrh74.MHCK7.microdystrophin or lactated Ringer's solution.

[0010] The present invention provides nucleic acid molecules comprising the nucleotide sequences of SEQ ID NO:3, 8, or 9. The present invention also provides rAAV comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 55-5021 of SEQ ID NO:3, and rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 55-5021 of SEQ ID NO:3.

[0011] Another aspect of the present invention provides a composition comprising a nucleic acid molecule, rAAV, and rAAV particles, the nucleic acid molecule comprising the nucleotide sequences of SEQ ID NO:3, 8, or 9, the rAAV comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 55-5021 of SEQ ID NO:3, and the rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9 or nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 55-5021 of SEQ ID NO:3. Any method disclosed herein can be carried out using these compositions.

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

[0013] For example, the dose of rAAV administered is about 5.0x10 12 vg / kg to about 1.0x10 14 vg / kg, or about 5.0x10 12 vg / kg to 1.0x10 14 vg / kg, or about 5.0x10 12 vg / kg to about 2.0x10 14 vg / kg, or about 5.0x10 12 vg / kg to about 1.0x10 14 vg / kg, or about 5.0x10 12 vg / kg to about 5.0x10 13 vg / kg, or about 5.0x10 12 vg / kg to about 2.0x10 13 vg / kg, or about 5.0x10 12 vg / kg to about 1.0x10 13 vg / kg, or 1.0x10 14 vg / kg to about 1.0x10 15 vg / kg, or 1.0x10 13 vg / kg to about 1.0x10 14 vg / kg, or about 1.0x10 13 vg / kg to 1.0x10 14 vg / kg, or about 1.0x10 13 vg / kg to about 2.0x10 14 vg / kg, or about 1.0x10 13 vg / kg to about 1.0x10 14 vg / kg, or about 1.0x10 13 vg / kg to about 5.0x10 13 vg / kg, or about 1.0x10 13 vg / kg to about 3.0x10 14 vg / kg, or about 1.0x10 13 vg / kg to about 5.0x10 14 vg / kg, or about 1.0x10 13 vg / kg to about 6.0x10 14 vg / kg, or 1.0x10 13 vg / kg to about 1.0x10 15vg / kg, or 5.0x10 13 vg / kg to approximately 1.0x10 14 vg / kg, or approximately 5.0x10 13 vg / kg to 1.0x10 14 vg / kg, or approximately 5.0x10 13 vg / kg to approximately 2.0x10 14 vg / kg, or approximately 5.0x10 13 vg / kg to approximately 1.0x10 14 vg / kg, or approximately 5.0x10 13 vg / kg to approximately 3.0x10 14 vg / kg, or approximately 5.0x10 13 vg / kg to approximately 5.0x10 14 vg / kg, or approximately 5.0x10 13 vg / kg to approximately 6.0x10 14 vg / kg, or 5.0x10 13 vg / kg to approximately 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg to approximately 6.0x10 14 vg / kg, or 1.0x10 14 vg / kg to approximately 5.0x10 14 vg / kg, or 1.0x10 14 vg / kg to approximately 4.0x10 14 vg / kg, or 1.0x10 14 vg / kg to approximately 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg to approximately 3.0x10 14 vg / kg, or approximately 1.0x10 14 vg / kg to approximately 2.5x10 14 vg / kg, or 1.0x10 14 vg / kg to approximately 2.0x10 14 vg / kg, or approximately 1.25x10 14 vg / kg to approximately 3.75x10 14 vg / kg, or approximately 1.25x10 14 vg / kg to 6.0x10 14 or approximately 1.25x10 14 vg / kg to 5.0x10 14 or approximately 1.25x10 14vg / kg to 4.0x10 14 、or about 1.25x10 14 vg / kg to 1.0x10 15 、or about 1.25x10 14 vg / kg to about 3.5x10 14 vg / kg、or about 1.25x10 14 vg / kg to about 3.0x10 14 vg / kg、or about 1.25x10 14 vg / kg to about 2.75x10 14 vg / kg、or about 1.25x10 14 vg / kg to about 2.5x10 14 vg / kg、or about 1.25x10 14 vg / kg to about 2.0x10 14 vg / kg、or 1.25x10 14 vg / kg to about 3.75x10 14 vg / kg、or about 1.25x10 14 vg / kg to about 3.5x10 14 vg / kg、or 1.5x10 14 vg / kg to about 1.0x10 15 vg / kg、or about 1.5x10 14 vg / kg to 6.0x10 14 、or about 1.5x10 14 vg / kg to 5.0x10 14 、or about 1.5x10 14 vg / kg to 4.0x10 14 、or about 1.5x10 14 vg / kg to about 3.75x10 14 vg / kg、or about 1.5x10 14 vg / kg to about 3.5x10 14 vg / kg、or about 1.5x10 14 vg / kg to about 3.25x10 14 vg / kg、or about 1.5x10 14 vg / kg to about 3.0x10 14 vg / kg、or about 1.5x10 14 vg / kg to about 2.75x10 14 vg / kg、or about 1.5x10 14 vg / kg to about 2.5x10 14vg / kg, or about 1.5x10 14 vg / kg to about 2.0x10 14 vg / kg, or 1.75x10 14 vg / kg to about 1.0x10 15 vg / kg, or about 1.75x10 14 vg / kg to 6.0x10 14 or about 1.75x10 14 vg / kg to 5.0x10 14 or about 1.75x10 14 vg / kg to 4.0x10 14 or about 1.75x10 14 vg / kg to about 3.75x10 14 vg / kg, or about 1.75x10 14 vg / kg to about 3.5x10 14 vg / kg, or about 1.75x10 14 vg / kg to about 3.25x10 14 vg / kg, or about 1.75x10 14 vg / kg to about 3.0x10 14 vg / kg, or about 1.75x10 14 vg / kg to about 2.75x10 14 vg / kg, or about 1.75x10 14 vg / kg to about 2.5x10 14 vg / kg, or about 1.75x10 14 vg / kg to about 2.25x10 14 vg / kg, or about 1.75x10 14 vg / kg to about 2.0x10 14 vg / kg, or about 2.0x10 14 vg / kg to 1.0x10 15 or about 2.0x10 14 vg / kg to 6.0x10 14 or about 2.0x10 14 vg / kg to 5.0x10 14 or about 2.0x10 14 vg / kg to about 4.0x10 14 vg / kg, or about 2.0x10 14 vg / kg to about 3.75x10 14 vg / kg, or about 2.0x10 14vg / kg to about 3.5x10 14 vg / kg, or about 2.0x10 14 vg / kg to about 3.25x10 14 vg / kg.

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

[0015] In any of the methods of the present invention, the dose of rAAV can 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 / k, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In a particular embodiment, the dose of rAAV is administered at about 10 mL / kg. In one embodiment, rAAV is AAVrh74.MHCK7.minidystrophin or AAVrh74.MCK.minidystrophin. In one embodiment, rAAV is AAVrh74.MHCK7.minidystrophin 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, rAAV is AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0016] In any of the methods of the present invention, the dose of rAAV can be administered by injection, infusion, or transplantation. For example, the dose of rAAV is administered by infusion over approximately one hour. In addition, the dose of rAAV is administered by an intravenous route through a peripheral limb vein (such as a peripheral arm vein or a peripheral leg vein). Alternatively, the infusion can be administered over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours, or approximately 3 hours. In one embodiment, the rAAV is AAVrh74.MHCK7.minidystrophin. In one embodiment, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0017] The rAAV administered by any of the methods of the present invention can comprise the human dystrophin nucleotide sequence of SEQ ID NO:1, the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7. In addition, the rAAV administered by any of the methods of the present invention comprises the human dystrophin 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 can comprise the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.MCK.minidystrophin. In one embodiment, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

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

[0020] In some embodiments, the methods of the present 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, the method comprising the step of administering a dose of recombinant adeno-associated virus (rAAV) associated with rAAV.MHCK7.minidystrophin, wherein the route of administration is intravenous infusion, and the dose of the administered rAAV is about 2×10 14 vg / kg over approximately one hour, and wherein the rAAV vector comprises the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct 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.minidystrophin. In one embodiment, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.MCK.minidystrophin. In one embodiment, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0021] In one embodiment, the present invention provides an rAAV comprising a muscle-specific control element nucleotide sequence and a nucleotide sequence encoding micro-dystrophin. For example, the nucleotide sequence encodes a functional micro-dystrophin, wherein the nucleotide 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%, even more typically at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:1, and wherein the protein retains micro-dystrophin activity. Micro-dystrophin provides stability to the muscle membrane during muscle contraction. For example, micro-dystrophin acts as a shock absorber during muscle contraction. In one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, the AAVrh74.MHCK7.micro-dystrophin is the AAVrh74.MHCK7.micro-dystrophin 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.MCK.micro-dystrophin. In one embodiment, the AAVrh74.MCK.micro-dystrophin is the AAVrh74.MCK.micro-dystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

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

[0023] In one embodiment, the rAAV is a non-replicating recombinant adeno-associated virus (AAV), namely the AAVrh74.MHCK7.micro-dystrophin 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 vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeats (ITRs), micro-dystrophin, the SV40 intron (SD / SA), and a synthetic polyadenylation (polyA) signal, all of which are under the control of the MHCK7 promoter / enhancer. Schematic diagrams of the vector genome and the expression cassette are shown in Figure 1As shown, after intravenous administration, AAVrh74 serotype can be used to achieve efficient gene transfer in skeletal and cardiac muscles.

[0024] The term "stringent" is used to refer to conditions that are generally understood to be stringent in the art. Hybridization stringency is mainly determined by temperature, ionic strength, and the concentration of denaturants (such as formamide). Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65 - 68 °C, or 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at 42 °C. See Sambrook et al., Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide or other denaturants) can also be used; however, the rate of hybridization will be affected. In the case of hybridization involving deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37 °C (for 14-base oligonucleotides), 48 °C (for 17-base oligonucleotides), 55 °C (for 20-base oligonucleotides), and 60 °C (for 23-base oligonucleotides).

[0025] To reduce non-specific and / or background hybridization, other reagents can be included in the hybridization and washing buffers. Examples are 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 reagents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically carried out at pH 6.8 - 7.4; however, under typical ionic strength conditions, the rate of hybridization is almost independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach , Chapter 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and to form hybrids of DNA with different sequence homologies.

[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 comprising the muscle-specific control elements MCKH7 promoter, MCK promoter, and MCK enhancer.

[0027] The term "operably linked" refers to the positioning of a regulatory element nucleotide sequence (e.g., a promoter nucleotide sequence) to confer expression of the nucleotide sequence through the regulatory element.

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

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

[0030] In another aspect, the present invention provides an rAAV construct contained in a plasmid, the plasmid comprising the nucleotide sequences of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:8. For example, the AAVrh74.MHCK7.minidystrophin vector contains such nucleotide sequences: the nucleotide sequences are within the ITR of SEQ ID NO:3 and include the ends, as Figure 2 shown. The rAAV vector contains a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human minidystrophin gene, a polyA, and a 3' ITR. In one embodiment, the vector contains nucleotides 55 - 5021 of SEQ ID NO:3. The plasmid shown in SEQ ID NO:3 also contains ampicillin resistance and a pGEX plasmid backbone with 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.minidystrophin vector construct contains the nucleotide sequence of SEQ ID NO:9, as Figure 13 shown. The rAAV vector construct contains an MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human minidystrophin gene, and a polyA. In one embodiment, the rAAV vector construct also contains an ITR 5' of the promoter and an ITR 3' of the polyA. In one embodiment, the rAAV is AAVrh74.

[0032] In another aspect, the AAVrh74.MHCK7.minidystrophin vector contains such nucleotide sequences: the nucleotide sequences are within the ITR of SEQ ID NO:8 and include the ends, as Figure 15As shown. The rAAV vector contains a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, a polyA, and a 3' ITR. In one embodiment, the vector contains nucleotides 1 - 4977 of SEQ ID NO:9. The plasmid shown in SEQ ID NO:3 also contains kanamycin resistance and a pGEX plasmid backbone with a pBR322 origin of replication.

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

[0034] The present invention provides a recombinant AAV vector, the 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 AAV serotype AAVrh.74.

[0035] The present invention also provides an rAAV, the rAAV comprising within the ITRs in SEQ ID NO:3 and including at the ends the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence, within the ITRs in SEQ ID NO:8 and including at the ends the nucleotide sequence, or the nucleotide sequence as shown in SEQ ID NO:9. The rAAV vector is AAV serotype AAVrh.74.

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

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

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

[0039] In any of the methods for treating muscular dystrophy, after administration of rAAV, the level of microdystrophin gene expression in the cells of the subject increases. Before and after administration of rAAV, the level of microdystrophin is measured by Western blot in a muscle biopsy to detect the expression of the microdystrophin gene in the cells. Specifically, compared with the level of microdystrophin before administration of rAAV, after administration of rAAV, the level of microdystrophin 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%. For example, compared with the level of microdystrophin before administration of rAAV, after administration of rAAV, the level of microdystrophin increases 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%.

[0040] In addition, the level of microdystrophin is measured by immunohistochemistry in a muscle biopsy before and after administration of rAAV to detect the expression of the microdystrophin gene in the cells. Compared with the level of microdystrophin before administration of rAAV, after administration of rAAV, the level of microdystrophin 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%. For example, compared with the level of microdystrophin before administration of rAAV, after administration of rAAV, the level of microdystrophin increases 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%.

[0041] In any of the methods of treating muscular dystrophy, the serum CK level of the subject decreases after administration of rAAV as compared to the serum CK level before administration of rAAV. For example, as compared to the serum CK level before administration of rAAV, the serum CK level of the subject decreases 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% 60 days after administration of rAAV. Specifically, in any of the methods of treating muscular dystrophy of the present invention, as compared to the serum CK level before administration of rAAV, the serum CK level of the subject decreases by about 87% 60 days after administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, as compared to the serum CK level before administration of rAAV, the serum CK level of the subject decreases by about 72% 60 days after administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, as compared to the serum CK level before administration of rAAV, the serum CK level of the subject decreases by about 73% 60 days after administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, as compared to the serum CK level before administration of rAAV, the serum CK level of the subject decreases by about 78% 60 days after administration of rAAV, or in any of the methods of treating muscular dystrophy of the present invention, as compared to the serum CK level before administration of rAAV, the serum CK level of the subject decreases by about 95% 60 days after administration of rAAV. In any of the methods of treating muscular dystrophy, the number of microdystrophin-positive fibers in the muscle tissue of the subject increases as compared to the number of microdystrophin-positive fibers before administration of rAAV. For example, the microdystrophin level is measured by Western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV to detect the number of microdystrophin-positive fibers.

[0042] In any of the methods of treating muscular dystrophy, administration of rAAV upregulates the expression of DAPC proteins such as α-sarcoglycan or β-sarcoglycan. For example, the level of α-sarcoglycan in the subject increases after administration of rAAV as compared to the level of α-sarcoglycan before administration of rAAV. In addition, the level of β-sarcoglycan in the subject increases after administration of rAAV as compared to the level of β-sarcoglycan before administration of rAAV. The α-sarcoglycan or β-sarcoglycan protein level is measured by Western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV to detect the level of α-sarcoglycan or β-sarcoglycan.

[0043] In any of the methods of treating muscular dystrophy, after administration of rAAV, disease progression in the subject is delayed, as measured by any of the following tests: six-minute walk test, time to rise, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment (NSAA), 10-meter timed test, 100-meter timed test, hand-held dynamometry (HHD), timed up-and-go, and / or Bayley-III gross motor subtest score.

[0044] For example, in any method, compared to the NSAA score before administration of rAAV, at least 270 days after administration of rAAV, the subject's NSAA score has an improvement of at least 6 points. In addition, in any method, compared to the time to rise before administration of rAAV, at least 270 days after administration of rAAV, the subject's time to rise has an improvement of at least about 0.8 seconds. In addition, in any method, compared to the time test for ascending 4 steps before administration of rAAV, at least 270 days after administration of rAAV, the subject's time test for ascending 4 steps has an improvement of at least about 1.2 seconds. In addition, in any method, compared to the 100m timed test before administration of rAAV, at least 270 days after administration of rAAV, the subject's 100m timed test has an improvement of at least about 7 seconds.

[0045] In another embodiment, the present invention provides a method for expressing the micro-dystrophin gene in a patient's cells, the method comprising administering to the patient 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 example, before and after administration of the rAAV.MHCK7.micro-dystrophin construct, micro-dystrophin levels are measured by Western blot or immunohistochemistry in muscle biopsies to detect expression of the micro-dystrophin gene in the patient's cells. In addition, expression of the micro-dystrophin gene in the patient is measured by detecting the number of vector genomes per nucleus, wherein 1 vector genome per nucleus is about 50% of micro-dystrophin expression, and greater than 1 copy per nucleus is consistent with the micro-dystrophin expression level. For example, the cells 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.

[0046] In another embodiment, the present invention provides a method of reducing serum CK levels in a patient in need thereof, the method comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct 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, compared to the serum CK level prior to administration of rAAV, at 60 days after administration of rAAV, 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%. Specifically, compared to the serum CK level prior to administration of rAAV, at 60 days after administration of rAAV, the serum CK level of the subject is reduced by about 87%, or in any of the methods for treating muscular dystrophy of the present invention, compared to the serum CK level prior to administration of rAAV, at 60 days after administration of rAAV, the serum CK level of the subject is reduced by about 72%, or in any of the methods for treating muscular dystrophy of the present invention, compared to the serum CK level prior to administration of rAAV, at 60 days after administration of rAAV, the serum CK level of the subject is reduced by about 73%, or in any of the methods for treating muscular dystrophy of the present invention, compared to the serum CK level prior to administration of rAAV, at 60 days after administration of rAAV, the serum CK level of the subject is reduced by about 78%, or in any of the methods for treating muscular dystrophy of the present invention, compared to the serum CK level prior to administration of rAAV, at 60 days after administration of rAAV, the serum CK level of the subject is reduced by about 95%.

[0047] The present invention also provides a method for increasing the number of micro-dystrophin positive fibers in the muscle tissue of a patient, the method comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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, before and after administering rAAV, the dystrophin level is measured by western blot or immunohistochemistry on a muscle biopsy to detect the number of micro-dystrophin positive fibers. In addition, the expression of the micro-dystrophin gene in the patient is measured by detecting the number of vector genomes per nucleus, wherein 1 vector genome per nucleus is about 50% of the micro-dystrophin expression, and more than 1 copy per nucleus is consistent with the micro-dystrophin expression level. For example, the cells 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.

[0048] In another embodiment, the present invention provides a method for increasing the expression of α-sarcoglycan in a patient in need thereof, the method comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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, before and after administering rAAV, the α-sarcoglycan protein level is measured by western blot or immunohistochemistry on a muscle biopsy to detect the level of α-sarcoglycan.

[0049] Furthermore, the present invention provides a method for increasing the expression of β-sarcoglycan in a patient in need thereof, the method comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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, before and after administering rAAV, the β-sarcoglycan protein level is measured by western blot or immunohistochemistry on a muscle biopsy to detect the level of β-sarcoglycan.

[0050] The present invention also provides a method for treating a patient suffering from Duchenne muscular dystrophy or Becker muscular dystrophy, the method comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct 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, such that the disease progression of the patient is delayed, as measured by any of the following tests: six-minute walk test, rise time, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment (NSAA), 10-meter timed test, 100-meter timed test, hand-held dynamometry (HHD), timed up-and-go, and / or Bayley-III scores for the gross motor subtest.

[0051] For example, in any method, the NSAA score of the subject has an improvement of at least 6 points at least 270 days after administration of rAAV, as compared to the NSAA score before administration of rAAV. Further, in any method, the rise time of the subject has an improvement of at least about 0.8 seconds at least 270 days after administration of rAAV, as compared to the rise time before administration of rAAV. Further, in any method, the time test for ascending 4 steps of the subject has an improvement of at least about 1.2 seconds at least 270 days after administration of rAAV, as compared to the time test for ascending 4 steps before administration of rAAV. Further, in any method, the 100m timed test of the subject has an improvement of at least about 7 seconds at least 270 days after administration of rAAV, as compared to the 100m timed test before administration of rAAV.

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

[0053] The present invention also provides a method for reducing or preventing fibrosis in a subject with muscular dystrophy, the method comprising administering a therapeutically effective amount of rAAV or an rAAV vector, the rAAV 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; the rAAV vector 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 one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, AAVrh74.MHCK7.micro-dystrophin is AAVrh74.MHCK7.micro-dystrophin of nucleotides 55-5021 of SEQ ID NO:3. In another embodiment, AAVrh74.MHCK7.micro-dystrophin is AAVrh74.MHCK7.micro-dystrophin of SEQ ID NO:9. In another embodiment, AAVrh74.MHCK7.micro-dystrophin is AAVrh74.MHCK7.micro-dystrophin of nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 56-5066 of SEQ ID NO:6. In yet another embodiment, the rAAV is AAVrh74.MCK.micro-dystrophin. In one embodiment, AAVrh74.MCK.micro-dystrophin is AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0054] In another embodiment, the present invention provides a method for preventing fibrosis in a subject in need thereof, the method comprising administering a therapeutically effective amount of 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 an rAAV vector comprising 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 with muscular dystrophy to prevent fibrosis, e.g., before fibrosis is observed in the subject, the rAAV of the present invention expresses the administered human microdystrophin. In addition, the rAAV of the present invention expressing the human microdystrophin gene can be administered to a subject at risk of developing fibrosis, such as those subjects with or diagnosed with muscular dystrophy (e.g., DMD). The rAAV of the present invention can be administered to a subject with muscular dystrophy to prevent new fibrosis in these subjects.

[0055] The present invention contemplates administering the rAAV before fibrosis is observed in the subject. In addition, the rAAV can be administered to a subject at risk of developing fibrosis, such as those subjects with or diagnosed with muscular dystrophy (e.g., DMD). The rAAV can be administered to a subject with muscular dystrophy who has developed fibrosis 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 with muscular dystrophy, the method comprising administering a therapeutically effective amount of 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 an rAAV 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.

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

[0058] The present invention also contemplates administering 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 rAAV, wherein the rAAV comprises 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 to dystrophic subjects who have developed fibrosis, to prevent new fibrosis in these subjects or reduce fibrosis in these subjects. The present invention also provides administering 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 an rAAV vector, wherein the rAAV vector comprises 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 to dystrophic subjects with reduced muscle strength or reduced muscle mass to protect the muscle from further damage.

[0059] In any of the methods of the present invention, the subject may have a muscular dystrophy (such as DMD) or any dystrophin-related muscular dystrophy.

[0060] In other embodiments of any of the methods of the invention described herein, the percentage level by which the serum CK level of the subject is reduced after administering rAAV compared to the serum CK level before administering rAAV is selected from the group consisting of:

[0061] a) at least 78% at 90, 180, or 270 days after administration;

[0062] b) at least 46%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, or 85% at 270 days after administration;

[0063] c) at least 72%, 73%, 74%, or 95% at 180 days after administration;

[0064] d) at least 87%, 88%, 93%, or 95% at 90 days after administration;

[0065] e) at least 70% at 270 days after administration;

[0066] f) 70% to 95% at 90, 180, or 270 days after administration;

[0067] 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% at 90, 180, or 270 days after administration; and

[0068] h) 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% at 90, 180, or 270 days after administration.

[0069] In another embodiment, the present invention provides a composition for treating muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of recombinant adeno-associated virus (rAAV)-related rAAV.MHCK7.minidystrophin, wherein the composition is formulated for systemic administration, and the dose of rAAV is about 1×10 14 vg / kg to about 4×10 14 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.minidystrophin. In one embodiment, AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0070] For example, the composition of the present invention comprises about 5.0×10 12vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 5.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 13 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 2.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 13 vg / kg, or about 1.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 13vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 5.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 5.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.0×10 14 vg / kg to about 2.5×10 14 vg / kg, or 1.0×10 14 vg / kg to about 2.0×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to 6.0×10 14 、or about 1.25×10 14 vg / kg to 5.0×10 14 、or about 1.25×10 14 vg / kg to 4.0×10 14 、or about 1.25×10 14 vg / kg to 1.0×10 15 、or about 1.25×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 3.0×1014 vg / kg, or about 1.25×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.0×10 14 vg / kg, or 1.25×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 3.5×10 14 vg / kg, or 1.5×10 14 vg / kg to about 1.0×10 15 vg / kg, or about 1.5×10 14 vg / kg to 6.0×10 14 , or about 1.5×10 14 vg / kg to 5.0×10 14 , or about 1.5×10 14 vg / kg to 4.0×10 14 , or about 1.5×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.25×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.0×10 14 vg / kg, or 1.75×10 14 vg / kg to about 1.0×10 15 vg / kg, or about 1.75×10 14 vg / kg to 6.0×10 14 , or about 1.75×10 14 vg / kg to 5.0×10 14, or about 1.75×10 14 vg / kg to 4.0×10 14 , or about 1.75×10 14 vg / kg to about 3.75×10 14 , or about 1.75×10 14 vg / kg to about 3.5×10 14 , or about 1.75×10 14 vg / kg to about 3.25×10 14 , or about 1.75×10 14 vg / kg to about 3.0×10 14 , or about 1.75×10 14 vg / kg to about 2.75×10 14 , or about 1.75×10 14 vg / kg to about 2.5×10 14 , or about 1.75×10 14 vg / kg to about 2.25×10 14 , or about 1.75×10 14 vg / kg to about 2.0×10 14 , or about 2.0×10 14 vg / kg to 1.0×10 15 , or about 2.0×10 14 vg / kg to 6.0×10 14 , or about 2.0×10 14 vg / kg to 5.0×10 14 , or about 2.0×10 14 vg / kg to about 4.0×10 14 , or about 2.0×10 14 vg / kg to about 3.75×10 14 , or about 2.0×10 14 vg / kg to about 3.5×10 14 , or about 2.0×10 14 vg / kg to about 3.25×10 14rAAV at a dose of vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7. micro-dystrophin. In one embodiment, the AAVrh74.MHCK7. micro-dystrophin is the AAVrh74.MHCK7. micro-dystrophin 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. micro-dystrophin. In one embodiment, the AAVrh74.MCK. micro-dystrophin is the AAVrh74.MCK. micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0071] In one embodiment, the composition of the present invention is formulated for intravenous administration and comprises approximately 2.0×10 14 vg / kg of rAAV. In another embodiment, the composition of the present invention is formulated for intravenous administration and comprises approximately 5.0×10 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 9.0×1×10 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 13vg / kg, or about 9.0×10 13 vg / kg, or about 1.0×10 14 vg / kg, or about 1.25×10 14 vg / kg, or about 1.5×10 14 vg / kg, or about 1.75×10 14 vg / kg, or about 2.25×10 14 vg / kg, or about 2.5×10 14 vg / kg, or about 2.75×10 14 vg / kg, or about 3.0×10 14 vg / kg, or about 3.25×10 14 vg / kg, or about 3.5×10 14 vg / kg, or about 3.75×10 14 vg / kg, or about 4.0×10 14 vg / kg, or about 5.0×10 14 vg / kg, or about 6.0×10 14 vg / kg, or about 1×10 15 of rAAV. In one embodiment, the rAAV is AAVrh74.MHCK7.minidystrophin. In one embodiment, AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0072] In any of the compositions of the present 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 a particular embodiment, 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 the 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 the AAVrh74.MCK.microdystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0073] The compositions of the present invention are formulated for administration by injection, infusion, or transplantation. For example, the compositions are formulated for administration by infusion over about one hour. Additionally, the compositions of the present invention are formulated for intravenous administration via a peripheral limb vein (such as a peripheral arm vein or a peripheral leg vein). 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.

[0074] Any of the compositions of the present invention comprises rAAV or an rAAV vector, the 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, the 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.

[0075] Specifically, the compositions of the present invention are for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present invention provides a composition for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of recombinant adeno-associated virus (rAAV)-related rAAV.MHCK7.minidystrophin, wherein the composition is formulated for administration by intravenous infusion over approximately one hour, and the dose of rAAV administered is about 2×10 14 vg / kg, and wherein the rAAV comprises the AAVrh74.MHCK7.minidystrophin 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.

[0076] In another embodiment, the present invention also provides a composition comprising rAAV for reducing fibrosis in a subject in need thereof. In addition, the present invention provides a composition comprising an rAAV vector for preventing fibrosis in a subject with muscular dystrophy.

[0077] The present invention also provides a composition comprising rAAV for increasing muscle strength and / or muscle mass in a subject with muscular dystrophy. In another embodiment, the present invention provides a composition comprising any one of the rAAVs of the present invention for the treatment of muscular dystrophy.

[0078] In other embodiments of any one of the compositions of the present invention, the serum CK level of the subject is reduced by a percentage level selected from the group consisting of: compared to the serum CK level before administration of the composition, after the composition is administered to a human subject in need of treatment for muscular dystrophy

[0079] a) at least 78% at 90, 180, or 270 days after administration;

[0080] b) at least 46%, 55%, 70%, or 85% at 270 days after administration;

[0081] c) at least 72%, 73%, 74%, or 95% at 180 days after administration;

[0082] d) at least 87%, 99%, 93%, or 95% at 90 days after administration;

[0083] e) at least 70% at 270 days after administration;

[0084] f) 70% to 95% at 90, 180, or 270 days after administration;

[0085] g) at 90, 180, or 270 days after administration, 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%; and

[0086] h) at 90, 180, or 270 days after administration, 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%.

[0087] In another embodiment, the present invention provides the use of a dose of recombinant adeno-associated virus (rAAV)-related rAAV.MHCK7.minidystrophin for the preparation of a medicament for treating dystrophy in a human subject in need thereof, wherein the medicament is formulated for systemic administration and the dose of rAAV is about 1×10 14 vg / kg to about 4×101 4 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.minidystrophin. In one embodiment, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0088] For example, the medicament comprises about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 14vg / kg, or about 5.0×10 12 vg / kg to about 5.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 13 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 2.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 13 vg / kg, or about 1.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 5.0×10 14vg / kg, or about 5.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 5.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.0×10 14 vg / kg to about 2.5×10 14 vg / kg, or 1.0×10 14 vg / kg to about 2.0×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to 6.0×10 14 、or about 1.25×10 14 vg / kg to 5.0×10 14 、or about 1.25×10 14 vg / kg to 4.0×10 14 、or about 1.25×10 14 vg / kg to 1.0×10 15 、or about 1.25×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.0×10 14vg / kg, or 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or 1.5×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or approximately 1.5×10 14 vg / kg to 6.0×10 14 , or approximately 1.5×10 14 vg / kg to 5.0×10 14 , or approximately 1.5×10 14 vg / kg to 4.0×10 14 , or approximately 1.5×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to approximately 3.25×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or 1.75×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or approximately 1.75×10 14 vg / kg to 6.0×10 14 , or approximately 1.75×10 14 vg / kg to 5.0×10 14 , or approximately 1.75×10 14 vg / kg to 4.0×10 14 , or approximately 1.75×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.75×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.75×1014 vg / kg to about 3.25×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.25×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to 1.0×10 15 、or about 2.0×10 14 vg / kg to 6.0×10 14 、or about 2.0×10 14 vg / kg to 5.0×10 14 、or about 2.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.25×10 14 vg / kg of rAAV. In one embodiment, the rAAV is AAVrh74.MHCK7.minidystrophin. In one embodiment, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0089] In one embodiment, the medicament of the present invention is formulated for systemic administration of a dose of rAAV, wherein the systemic administration route is the intravenous route, and the dose of the administered rAAV is about 2.0×10 14 vg / kg. 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 the intravenous route, and the dose of rAAV is about 5.0×10 12 vg / kg, or about 6.0×10 12 vg / kg, or about 7.0×10 12 vg / kg, or about 8.0×10 12 vg / kg, or about 9.0×10 12 vg / kg, or about 1.0×10 13 vg / kg, or about 1.25×10 13 vg / kg, or about 1.5×10 13 vg / kg, or about 1.75×10 13 vg / kg, or about 2.25×10 13 vg / kg, or about 2.5×10 13 vg / kg, or about 2.75×10 13 vg / kg, or about 3.0×10 13 vg / kg, or about 3.25×10 13 vg / kg, or about 3.5×10 13 vg / kg, or about 3.75×10 13 vg / kg, or about 4.0×10 13 vg / kg, or about 5.0×10 13 vg / kg, or about 6.0×10 13 vg / kg, or about 7.0×10 13 vg / kg, or about 8.0×10 13 vg / kg, or about 9.0×10 13 vg / kg, or about 1.0×10 14 vg / kg, or about 1.25×10 14 vg / kg, or about 1.5×10 14 vg / kg, or about 1.75×10 14 vg / kg, or about 2.25×10 14 vg / kg, or about 2.5×10 14 vg / kg, or about 2.75×10 14 vg / kg, or about 3.0×10 14 vg / kg, or about 3.25×10 14vg / kg, or about 3.5×10 14 vg / kg, or about 3.75×10 14 vg / kg, or about 4.0×10 14 vg / kg, or about 5.0×10 14 vg / kg, or about 6.0×10 14 vg / kg, or about 1×10 15 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.minidystrophin. In one embodiment, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0090] In any of the uses of the present 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 / k, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In a particular embodiment, the dose or rAAV is about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.minidystrophin. In one embodiment, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin 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.minidystrophin. In one embodiment, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0091] In any of the uses of the present invention, the drug is formulated for administration by injection, infusion, or transplantation. For example, the drug is formulated for administration by infusion over approximately one hour. Additionally, the drug is formulated for intravenous administration through a peripheral limb vein (such as a peripheral arm vein or a peripheral leg vein). Alternatively, the infusion can be administered over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours, or approximately 3 hours.

[0092] In any of the uses of the present invention, the drug comprises rAAV, which comprises 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 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.

[0093] A specific use of the present invention is the preparation of a drug for treating Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present invention provides the use of a certain dose of recombinant adeno-associated virus (rAAV) related rAAV.MHCK7.microdystrophin for the preparation of a drug for treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the drug is formulated for administration by intravenous infusion over approximately one hour, and the dose of rAAV administered is about 2×10 14 vg / kg, and wherein the rAAV comprises 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.

[0094] In another embodiment, the present invention provides the use of rAAV for the preparation of a drug for reducing fibrosis in a subject in need thereof. For example, the subject in need may have muscular dystrophy (such as DMD) or any other dystrophin-related muscular dystrophy.

[0095] In another embodiment, the present invention provides the use of rAAV for the preparation of a drug for preventing fibrosis in a subject with muscular dystrophy.

[0096] Furthermore, the present invention provides the use of rAAV for the preparation of a drug for increasing muscle strength and / or muscle mass in a subject with muscular dystrophy.

[0097] The present invention also provides the use of rAAV for the preparation of a medicament for treating muscular dystrophy.

[0098] The present invention provides the use of an rAAV vector for the preparation of a medicament for treating muscular dystrophy: the rAAV vector comprises 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 use of an rAAV vector for treating muscular dystrophy: the rAAV vector comprises the AAVrf74.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.

[0099] In other embodiments of any of the uses of the present invention, compared with the serum CK level before administration of rAAV, after rAAV is administered to a subject, the percentage level by which the serum CK level of the subject decreases is selected from the group consisting of:

[0100] a) at least 78% at 90, 180 or 270 days after administration;

[0101] b) at least 46%, 55%, 70% or 95% at 270 days after administration;

[0102] c) at least 72%, 73%, 74% or 95% at 180 days after administration;

[0103] d) at least 87%, 88%, 93% or 95% at 90 days after administration;

[0104] e) at least 70% at 270 days after administration;

[0105] f) 70% to 95% at 90, 180 or 270 days after administration;

[0106] 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% at 90, 180 or 270 days after administration; and

[0107] h) 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% at 90, 180 or 270 days after administration.

[0108] In any of the uses of a composition for treating muscular dystrophy or a medicament for treating muscular dystrophy, after administration of the composition or medicament, the level of micro-dystrophin gene expression in the cells of a subject increases. Before and after administration of the composition or medicament, the level of micro-dystrophin is measured by Western blotting in a biopsy muscle to detect the expression of the micro-dystrophin gene in the cells. Specifically, compared with the level of micro-dystrophin before administration of the composition or medicament, after administration of the composition or medicament, the level of micro-dystrophin 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%. For example, compared with the level of micro-dystrophin before administration of the composition or medicament, after administration of the composition, the level of micro-dystrophin increases 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%.

[0109] In addition, the level of micro-dystrophin is measured by immunohistochemistry in a muscle biopsy before and after administration of the composition or medicament to detect the expression of the micro-dystrophin gene in the cells. Compared with the level of micro-dystrophin before administration of the composition or medicament, after administration of rAAV, the level of micro-dystrophin 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%. For example, compared with the level of micro-dystrophin before administration of the composition or medicament, after administration of the composition or medicament, the level of micro-dystrophin increases 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%.

[0110] In any of the compositions for treating muscular dystrophy, the serum CK level of the subject is decreased after administration of rAAV as compared to the serum CK level before administration of the composition or the drug. For example, as compared to the serum CK level before administration of the composition or the drug, the serum CK level of the subject is decreased 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% 60 days after administration of the composition or the drug. Specifically, in any of the compositions for treating muscular dystrophy of the present invention, as compared to the serum CK level before administration of the composition or the drug, the serum CK level of the subject is decreased by about 87% 60 days after administration of the composition or the drug, or in any of the uses of the composition for treating muscular dystrophy of the present invention or the drug for treating muscular dystrophy, as compared to the serum CK level before administration of the composition or the drug, the serum CK level of the subject is decreased by about 72% 60 days after administration of the composition or the drug, or in any of the compositions for treating muscular dystrophy of the present invention, as compared to the serum CK level before administration of the composition or the drug, the serum CK level of the subject is decreased by about 73% 60 days after administration of the composition or the drug, or in any of the uses of the composition for treating muscular dystrophy of the present invention or the drug for treating muscular dystrophy, as compared to the serum CK level before administration of the composition, the serum CK level of the subject is decreased by about 78% 60 days after administration of the composition or the drug, or in any of the uses of the composition for treating muscular dystrophy of the present invention or the drug for treating muscular dystrophy, as compared to the serum CK level before administration of the composition or the drug, the serum CK level of the subject is decreased by about 95% 60 days after administration of the composition or the drug. In any of the uses of the composition for treating muscular dystrophy or the drug for treating muscular dystrophy, the number of micro-dystrophin positive fibers in the muscle tissue of the subject is increased as compared to the number of micro-dystrophin positive fibers before administration of the composition or the drug. For example, the level of micro-dystrophin is measured by Western blot or immunohistochemistry for muscle biopsy before and after administration of the composition or the drug, so as to detect the number of micro-dystrophin positive fibers.

[0111] In any of the uses of a composition for treating muscular dystrophy or a medicament for treating muscular dystrophy, the administration of the composition or medicament upregulates the expression of DAPC proteins such as α - dystroglycan or β - dystroglycan. For example, compared with the level of α - dystroglycan before the administration of the composition or medicament, the level of α - dystroglycan in the subject increases after the administration of the composition or medicament. In addition, compared with the level of β - dystroglycan before the administration of the composition or medicament, the level of β - dystroglycan in the subject increases after the administration of the composition or medicament. The levels of α - dystroglycan or β - dystroglycan proteins are measured by Western blotting or immunohistochemistry on muscle biopsies before and after the administration of the composition or medicament to detect the levels of α - dystroglycan or β - dystroglycan.

[0112] In any of the uses of a composition for treating muscular dystrophy or a medicament for treating muscular dystrophy, after the administration of the composition or medicament, the disease progression of the subject is delayed, as measured by any of the following tests: six - minute walk test, time to stand, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment (NSAA), 10 - meter timed test, 100 - meter timed test, hand - held dynamometry (HHD), timed up - and - go, and / or Bayley - III gross motor subtest scores.

[0113] For example, after the administration of any of the uses of a composition for treating muscular dystrophy or a medicament for treating muscular dystrophy, compared with the NSAA score before administration of rAAV, the NSAA score of the subject has an improvement of at least 6 points at least 270 days after the administration of the composition or medicament. In addition, in any method, compared with the time to stand before the administration of the composition or medicament, the time to stand of the subject has an improvement of at least about 0.8 seconds at least 270 days after the administration of the composition or medicament. In addition, in any of the methods or uses of the present invention, compared with the test of the time to ascend 4 steps before the administration of the composition or medicament, the test of the time to ascend 4 steps of the subject has an improvement of at least about 1.2 seconds at least 270 days after the administration of the composition or medicament. In addition, in any of the methods or uses of the present invention, compared with the 100 - m timed test before the administration of the composition or medicament, the 100 - m timed test of the subject has an improvement of at least about 7 seconds at least 270 days after the administration of the composition or medicament.

[0114] In another embodiment, the present invention provides a composition for expressing the micro-dystrophin gene in a patient's cells, the composition comprising the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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 present invention provides the use of a certain dose of the nucleotide sequence of the AAVrh74.MHCK7.micro-dystrophin construct 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 preparing a medicament for expressing the micro-dystrophin gene in a patient's cells. For example, micro-dystrophin levels are measured by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct to detect the expression of the micro-dystrophin gene in a patient's cells. In addition, the expression of the micro-dystrophin gene in a patient is measured by detecting the number of vector genomes per cell nucleus, where 1 vector genome per cell nucleus is approximately 50% of micro-dystrophin expression, and more than 1 copy per cell nucleus is consistent with the micro-dystrophin expression level. For example, the cells have 1.2 vector copies per cell nucleus, or 1.3 vector copies per cell nucleus, or 1.4 vector copies per cell nucleus, or 1.5 vector copies per cell nucleus, or 1.6 vector copies per cell nucleus, or 1.7 vector copies per cell nucleus, or 1.8 vector copies per cell nucleus, or 1.9 vector copies per cell nucleus.

[0115] In another embodiment, the present invention provides a composition for reducing serum CK levels in a patient in need thereof, the composition comprising the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct 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 the use of a dose of the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct 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 a patient in need thereof. For example, compared to the pre-administration serum CK level of the composition or medicament, at 60 days after administration of the composition or medicament, 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%. Specifically, the serum CK level of the subject is reduced by about 87% at 60 days after administration of the composition or medicament compared to the pre-administration serum CK level of the composition or medicament, or is reduced by about 72% at 60 days after administration of the composition or medicament compared to the pre-administration serum CK level of the composition or medicament, or is reduced by about 73% at 60 days after administration of the composition or medicament compared to the pre-administration serum CK level of the composition or medicament, or is reduced by about 78% at 60 days after administration of the composition or medicament compared to the pre-administration serum CK level of the composition or medicament, or is reduced by about 95% at 60 days after administration of the composition or medicament compared to the pre-administration serum CK level of the composition or medicament.

[0116] The present invention also provides a composition for increasing microdystrophin-positive fibers in a patient's muscle tissue, the composition comprising the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct 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 addition, the present invention provides the use of a dose of the nucleotide sequence of the AAVrh74.MHCK7.microdystrophin construct 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, before and after administration of the composition or medicament, the dystrophin level is measured by Western blot or immunohistochemistry on a muscle biopsy to detect the number of microdystrophin-positive fibers. In addition, the expression of the microdystrophin gene in a patient is measured by detecting the number of vector genomes per nucleus, wherein 1 vector genome per nucleus is about 50% of microdystrophin expression, and more than 1 copy per nucleus is consistent with the microdystrophin expression level. For example, the cells 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.

[0117] In another embodiment, the present invention provides a composition for increasing the expression of α - sarcoglycan in a patient in need thereof, 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. The present invention also provides the 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 increasing the expression of α - sarcoglycan in a patient in need thereof. For example, the level of α - sarcoglycan is detected by measuring the α - sarcoglycan protein level by Western blotting or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament.

[0118] In addition, the present invention provides a composition for increasing the expression of β - sarcoglycan in a patient in need thereof, 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. The present invention also provides the use 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 increasing the expression of β - sarcoglycan in a patient in need thereof. For example, the level of β - sarcoglycan is detected by measuring the β - sarcoglycan protein level by Western blotting or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament.

[0119] The present invention also provides the use of a nucleotide sequence of a certain dose of the AAVrh74.MHCK7.minidystrophin construct 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 suffering from Duchenne muscular dystrophy or Becker muscular dystrophy, such that administration of the medicament causes a delay in the progression of the patient's disease, as measured by any of the following tests: six-minute walk test, time to stand, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment (NSAA), 10-meter timed test, 100-meter timed test, hand-held dynamometry (HHD), timed up-and-go, and / or Bayley-III scores for the gross motor subtest.

[0120] For example, compared to the NSAA score before administration of the composition or medicament, at least 270 days after administration of the composition or medicament, the NSAA score of the subject has an improvement of at least 6 points. In addition, compared to the time to stand before administration of the composition or medicament, at least 270 days after administration of the composition or medicament, the time to stand of the subject has an improvement of at least about 0.8 seconds. In addition, compared to the test of the time to ascend 4 steps before administration of the composition or medicament, at least 270 days after administration of the composition or medicament, the test of the time to ascend 4 steps of the subject has an improvement of at least about 1.2 seconds. In addition, compared to the 100m timed test before administration of the composition or medicament, at least 270 days after administration of the composition or medicament, the 100m timed test of the subject has an improvement of at least about 7 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figure 1Shows the rAAV.MHCK7.minidystrophin 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 domain deletion (R4-R23), while hinges 1, 2, and 4 (H1, H2, and H4) and the cysteine-rich domain can still produce a 138 kDa protein. The expression of minidystrophin (3579 bp) is directed by the MHCK7 promoter (795 bp). The intron and 5' UTR are derived from plasmid pCMVβ (Clontech). The minidystrophin cassette has a consensus Kozak sequence before the ATG start codon and a small 53 bp synthetic polyA signal for mRNA termination. The human minidystrophin cassette contains the (R4-R23 / Δ71-78) domain as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)).

[0122] Figure 2 Provides the nucleic acid sequence of AAVrh74.MHCK7.minidystrophin (SEQ ID NO:3).

[0123] Figure 3 Provides a map of the pNLREP2-Caprh74 AAV helper plasmid.

[0124] Figure 4 Provides the Ad helper plasmid pHELP.

[0125] Figure 5 Shows the rAAV.MCK.minidystrophin plasmid construct.

[0126] Figure 6 Provides the nucleic acid sequence of rAAVrh74.MCK.minidystrophin (SEQ ID NO:5).

[0127] Figure 7 Shows minidystrophin gene expression in gastrocnemius muscle fiber biopsies measured by immunocytochemistry.

[0128] Figure 8A - 8C Provides a Western blot showing minidystrophin expression at the correct molecular weight. In Figure 8C the sample of subject 4 (*) was diluted 1:4 (to the linear range) because the ULDQ (>80%) exceeded the standard in the initial analysis, and the average value was multiplied by the dilution correction factor of the final value relative to the normal value. The average minidystrophin expression relative to the normal value was 182.7% in method 1 and 222.0% in method 2.

[0129] Figure 9A - 9C Administration of rAAVrh74.MHCK7.microdystrophin was shown to upregulate the expression of DAPC proteins, α-sarcoglycan, and β-sarcoglycan.

[0130] Figure 10 A persistent and significant decrease in creatine kinase (CK) was shown in the presence of rAAVrh74.MHCK7.microdystrophin administration.

[0131] Figure 11 The mean change in CK from baseline to day 270 was provided. This data indicated that CK decreased significantly over time following administration of rAAVrh74.MHCK7.microdystrophin.

[0132] Figure 12 The mean change in NSAA and the mean change in CK from baseline to day 270 were provided. This data indicated that NSAA increased significantly over time following administration of rAAVrh74.MHCK7.microdystrophin.

[0133] Figure 13 The nucleic acid sequence of AAVrh74.MHCK7.microdystrophin (SEQ ID NO:9) was provided.

[0134] Figure 14 The AAVrh74.MHCK7.microdystrophin plasmid construct was shown.

[0135] Figure 15 The nucleic acid sequence of the AAVrh74.MHCK7.microdystrophin plasmid construct containing the kanamycin resistance gene (SEQ ID NO:8) was provided. Detailed Description

[0136] The present invention provides gene therapy vectors (such as rAAV vectors) that overexpress human microdystrophin and methods for reducing and preventing fibrosis in dystrophic patients. Muscle biopsy samples taken early in the diagnosis of DMD showed significant connective tissue hyperplasia. Muscle fibrosis has deleterious effects in multiple ways. It reduces the normal transport of endomysial nutrients through the connective tissue barrier, decreases blood flow, deprives the muscle of vasogenic nutrients, and functionally causes early loss of ambulation due to limb contractures. Over time, the challenges of treatment increase due to significant muscle fibrosis. This can be observed in muscle biopsies of connective tissue hyperplasia at consecutive time points. The process continues to exacerbate, leading to loss of ambulation and accelerating out of control, especially in wheelchair-dependent patients.

[0137] The beneficial effects of exon skipping, nonsense codon readthrough, or gene replacement therapy cannot be fully realized without early treatment, including parallel approaches to reduce fibrosis. Even small molecule or protein replacement strategies may fail without methods to reduce muscle fibrosis. Previous studies of aged mdx mice that were already fibrotic and treated with AAV. microdystrophin demonstrated that we were unable to achieve full functional recovery (Liu, M. et al., Mol Ther 11, 245 - 256 (2005)). It is also known that the progression of DMD cardiomyopathy is accompanied by scar formation and fibrosis of the ventricular wall.

[0138] 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 co - infecting helper virus. Thirteen AAV serotypes have been identified to date. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Volume 1, pp. 169 - 228 and Berns, 1990, Virology, pp. 1743 - 1764, Raven Press, (New York). However, it is fully anticipated that these same principles will also apply to other AAV serotypes because it is well known that, even at the genetic level, the various serotypes are very closely related structurally and functionally. (See, for example, Blacklowe, 1988, Parvoviruses and Human Disease pp. 165 - 174, ed. J.R. Pattison; 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 carry three related capsid proteins, such as those expressed in AAV2. Heteroduplex analysis further demonstrates the degree of relatedness, showing extensive cross - hybridization between serotypes along the length of the genome; and the presence of similar self - annealing segments at the termini corresponding to the "inverted terminal repeats" (ITRs). Similar infectious patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0139] As used herein, an "AAV vector" refers to a vector that contains one or more polynucleotides (or transgenes) of interest flanked by AAV terminal repeats (ITRs). When such AAV vectors are present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products, they can be replicated and packaged into infectious virus particles.

[0140] "AAV viral particle", "AAV virion", or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector encapsulated by the capsid. 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 mammalian cells), the particle is generally referred to as an "AAV vector particle" or simply an "AAV vector". Thus, the production of an AAV vector particle necessarily includes the production of an AAV vector, as this vector is contained within the AAV vector particle.

[0141] AAV

[0142] Adeno-associated virus (AAV) is a replication-defective parvovirus with a single-stranded DNA genome approximately 4.7 kb in length, which includes inverted terminal repeats (ITRs) of 145 nucleotides. There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is provided in Srivastava et al., J Virol, 45:555-564 (1983), and was modified by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided in GenBank accession number NC_002077; the complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the AAV-5 genome is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least a portion of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Patent 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). The cis-acting sequences that direct viral DNA replication (rep), capsid encapsidation / packaging, and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 due to their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19), in combination with differential splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins have multiple enzymatic properties that ultimately are responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3.Alternative splicing and non-canonical translation start sites are responsible for the production of three related capsid proteins. A single canonical 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).

[0143] AAV has unique features that make it an attractive vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. In addition, AAV infects many mammalian cells, potentially targeting many different tissues in vivo. Furthermore, AAV transduces both slowly dividing and non-dividing cells and can persist essentially as a transcriptionally active nuclear episome (extrachromosomal element) throughout the life cycle of these cells. The AAV proviral genome is infectious as cloned DNA in a plasmid, which enables the construction of recombinant genomes. Additionally, because the signals that direct 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 the 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 can be provided in trans. Another remarkable feature of AAV is that it is an extremely stable and robust virus. It can easily withstand conditions used to inactivate adenoviruses (56°C to 65°C for several hours), making cryopreservation of AAV no longer a critical factor. AAV can even be lyophilized. Finally, cells infected with AAV are not tolerant to superinfection.

[0144] Multiple studies have shown that recombinant AAV-mediated proteins can be expressed in muscle for a long time (>1.5 years). 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). In addition, because muscle is highly vascularized, after intramuscular injection, recombinant AAV transduction causes the appearance of transgenic products in the systemic circulation, 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). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers have the cytokines necessary for correcting antibody glycosylation, folding, and secretion, indicating that muscle is capable of stably expressing secretory protein therapeutics.

[0145] The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be from any AAV serotype from which a recombinant virus can be derived, including but not limited to AAV serotypes 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 are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described 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.

[0146] The DNA plasmid of the present invention contains the rAAV genome of the present invention. The DNA plasmid is transferred to cells that are permissive to infection by an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) to assemble the rAAV genome into infectious virus particles. Techniques for producing rAAV particles are standard techniques in the art, where the AAV genome to be packaged, the rep and cap genes, and the helper virus functional elements are provided to the cells. Production of rAAV requires the provision of the following components within a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes that are separate from the rAAV genome (i.e., not within the rAAV genome), and helper virus functional elements. The AAV rep and cap genes can be from any AAV serotype from which a recombinant virus can be derived, and can be from an AAV serotype different from the rAAV genome ITR, 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. Production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0147] A method for generating packaging cells is to generate a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or plasmids) containing an rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker (such as the neomycin resistance gene) is integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid by procedures such as the GC-tailing method (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or by 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 advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus instead of plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0148] The general principles of rAAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129. Various methods 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., Mo1. 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 hereby incorporated by reference in their entirety, and in particular, those portions of the documents that relate to rAAV production are emphasized.

[0149] Accordingly, the present invention provides packaging cells for producing infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (a homologous 293 cell line). In another embodiment, the packaging cells are untransformed 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 embryonic lung cells).

[0150] The recombinant AAV of the present invention (i.e., an infectious capsid-wrapped rAAV particle) contains an rAAV genome. In an exemplary embodiment, the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAV that can be constructed to contain the nucleic acid molecule of the present invention are shown in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352), which is incorporated herein by reference in its entirety.

[0151] In an exemplary embodiment, the recombinant AAV vector of the present invention is produced by a 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). This sequence is encapsulated into AAVrh74 virions by the capsid. The plasmid contains the microdystrophin sequence and the MHCK7 enhancer and core promoter elements of a muscle-specific promoter driving gene expression. The expression cassette also contains an SV40 intron (SD / SA) to promote high-level gene expression, and a bovine growth hormone polyadenylation signal for efficient transcriptional termination.

[0152] pNLREP2-Caprh74 is an AAV helper plasmid that encodes 4 wild-type AAV2 rep proteins and 3 wild-type AAV VP capsid proteins from serotype rh74. A schematic map of the pNLREP2-Caprh74 plasmid is as Figure 3 shown.

[0153] The pHELP adenovirus helper plasmid is 11,635 bp and is obtained from Applied Viromics. The plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4 ORF6, and VA RNA (the adenovirus E1 functional element is provided by 293 cells). The adenovirus sequences present in this plasmid represent only ~40% of the adenovirus genome and do not contain replication-critical cis-elements such as adenovirus terminal repeats. Therefore, it is expected that infectious adenoviruses will not be produced from this production system. A schematic map of the pHELP plasmid is as Figure 4 shown.

[0154] rAAV can be purified by methods standard in the art, such as by column chromatography or cesium chloride gradient methods. Methods for purifying rAAV vectors from helper viruses are known in the art and include those disclosed in, for example, 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.

[0155] 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 comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to the recipient, preferably inert at the doses and concentrations employed, and include buffers and surfactants (such as pluronics).

[0156] The titer of rAAV to be administered in the methods of the present invention will vary depending on, for example, the specific rAAV, the mode of administration, the treatment objective, the individual, and the one or more cell types targeted, and can be determined by methods standard in the art. The titer of rAAV can range from about 1×10 6 to about 1×10 7 to about 1×10 8 to about 1×10 9 to about 1×10 10 to about 1×10 11 to about 1×10 12 to about 1×10 13 to about 1×10 14One or more DNAse-resistant particles (DRPs). The dose can also be expressed in terms of viral genomes (vg). An exemplary method for determining the titer of the capsid-encapsulated vector genome uses quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25(4):855-869, 2017).

[0157] The present invention contemplates methods for transducing target cells with rAAV in vivo or in vitro. In vivo methods include the step of administering to an animal (including a human) in need thereof an effective dose or multiple effective doses of a composition comprising the rAAV of the present invention. If the dose is administered prior to the development of the disorder / disease, the administration is prophylactic. If the dose is administered after the development of the disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents the progression of the disorder / disease state, reduces the extent of the disease, causes remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease contemplated to be prevented or treated by the methods of the present invention is DMD.

[0158] The present invention also contemplates combination therapies. Combinations as used herein include both concurrent therapy and sequential therapy. Combinations of the methods of the present invention with standard pharmaceutical therapies (e.g., corticosteroids) are specifically contemplated, as are combinations with novel therapies.

[0159] Administration of an effective dose of the composition can be effected by routes standard in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal routes. One of ordinary skill in the art can select and / or match one or more administration routes and one or more serotypes of the AAV components (in particular, AAV ITRs and capsid proteins) of the rAAV of the present invention, taking into account the infection and / or disease state being treated and the target cell(s) / tissue(s) in which minidystrophin is to be expressed.

[0160] The present invention provides for local and systemic administration of an effective dose of rAAV and the compositions of the present invention. For example, systemic administration is administration to the circulatory system, thereby affecting the entire body. Systemic administration includes enteral administration (such as absorption through the gastrointestinal tract) and parenteral administration (by injection, infusion, or transplantation).

[0161] Specifically, the actual administration of the rAAV of the present invention can be accomplished by using any physical method that transports the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection and injection into the bloodstream. Simply resuspending rAAV in phosphate buffered saline has been shown to provide a vehicle sufficient for expression in muscle tissue, and there are no known limitations on carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the conventional manner of using rAAV). The capsid protein of rAAV can be modified such that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or a topical formulation for transdermal delivery to muscle. Many formulations for intramuscular injection and transdermal delivery have been developed previously and can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0162] In one embodiment of the present invention, the AAVrh74.MHCK7. microdystrophin described herein is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl2), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0163] The dose of rAAV to be administered in the methods disclosed herein will vary depending on, for example, the particular rAAV, the mode of administration, the treatment objective, the individual, and the one or more cell types targeted, and can be determined by methods standard in the art. The titer of each rAAV administered can range from about 1×10 6 to about 1×10 7 to about 1×10 8 to about 1×10 9 to about 1×10 10 to about 1×10 11 to about 1×10 12 to about 1×10 13 to about 1×10 14 to about 2×10 14 or up to about 1×10 15 or more DNase resistant particles (DRP). The dose can also be expressed in terms of viral genomes (vg) (i.e., 1×10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12vg, 1×10 13 vg, 1×10 14 vg, 2×10 14 vg, 1×10 15 vg). The dose can also be expressed in terms of viral genomes (vg) / kilogram (kg) of body weight (i.e., 1×10 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). Methods for titrating AAV are described in Clark et al., Hum. Gene Ther., 10:1031 - 1039 (1999).

[0164] Specifically, the actual administration of the rAAV of the present invention can be accomplished by using any physical method that transports 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 muscle and injection into the bloodstream. Simply resuspending the rAAV in phosphate buffered saline has been shown to be sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations on carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the conventional manner of using rAAV). The capsid protein of the rAAV can be modified so that the rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or a topical formulation for transdermal delivery to muscle. Many formulations for intramuscular injection and transdermal delivery have been developed previously and can be used in the practice of the present invention. The rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0165] For intramuscular injection, solutions dissolved in adjuvants such as sesame oil or peanut oil, or solutions dissolved in aqueous propylene glycol, as well as sterile aqueous solutions, can be employed. If desired, such aqueous solutions can be buffered solutions and the liquid diluent is first isotonicized with saline or glucose. Solutions of the rAAV as the free acid (DNA contains acidic phosphate groups) or a pharmaceutically acceptable salt can be prepared by suitable mixing in water with a surfactant such as hydroxypropylcellulose. Dispersions of the rAAV can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, as well as in oils. Under ordinary storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed can be readily obtained by standard techniques well known to those skilled in the art.

[0166] Pharmaceutical carriers, diluents or excipients suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must have a fluidity that allows for easy injection. It must be stable under the conditions of manufacture and storage and must be able to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions by maintaining the desired particle size, and by the use of surfactants. The prevention of microbial action can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferred to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of the injectable composition can be achieved by the use of agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0167] Sterile injectable solutions are prepared by incorporating the desired amount of rAAV into a suitable solvent having the various other ingredients enumerated above, followed by filtration sterilization as required. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other components enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered solution.

[0168] Transduction using rAAV can also be carried out in vitro. In one embodiment, the desired target muscle cells are removed from a subject, transduced with rAAV and reintroduced into the subject. Alternatively, allogeneic or xenogeneic muscle cells can be used that do not elicit an inappropriate immune response in the subject.

[0169] Suitable methods for transducing and reintroducing the transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells, for example, in a suitable culture medium, and screening for those cells having the DNA of interest using conventional techniques such as Southern blotting and / or PCR or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition and introduced into the subject by various techniques such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection or by injection into smooth and cardiac muscle using, for example, a catheter.

[0170] Transduction of cells with the rAAV of the present invention results in the sustained expression of micro-dystrophin. Accordingly, the present invention provides methods for administering / delivering rAAV expressing micro-dystrophin to an animal (preferably a human). These methods include using one or more rAAV of the present invention to transduce tissues (including but not limited to tissues such as muscle, organs such as liver and brain, and glands such as salivary glands). Transduction can be carried out using a gene cassette containing tissue-specific control elements. For example, one embodiment of the present invention provides a method for transducing muscle cells and muscle tissue directed by a muscle-specific control element, the muscle-specific control element including but not limited to those derived from the actin and myosin gene families, such as those from the myoD gene family (see Weintraub et al., Science, 251:761-766 (1991)), the muscle cell-specific enhancer-binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11:4854-4862 (1991)), control elements derived from the human skeletal muscle actin gene (Muscat et al., MolCell Biol, 7:4089-4099 (1987)), the cardiac actin gene, the muscle creatine kinase sequence element (see Johnson et al., Mol Cell Biol, 9:3393-3399 (1989)) and the murine creatine kinase enhancer (mCK) element, control elements derived from the fast skeletal muscle troponin C gene, the slow cardiac troponin C gene and the slow skeletal 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 (GRE)) (see Maderand White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)) and other control elements.

[0171] 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 contemplates the sustained expression of micro-dystrophin from transduced muscle fibers.

[0172] The so-called "muscle cells" or "muscle tissue" means cells or cell populations derived from any type of muscle (e.g., skeletal muscle and smooth muscle, e.g., from the digestive tract, bladder, blood vessels or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, muscle cells, myotubes, cardiomyocytes and cardiomyoblasts.

[0173] The term "transduction" is used to refer to the in vivo or in vitro administration / delivery of the coding region of minidystrophin to recipient cells, resulting in minidystrophin expression in the recipient cells via the replication-defective rAAV of the present invention.

[0174] Accordingly, the present invention provides a method of administering to a subject in need thereof an effective dose (or doses administered substantially simultaneously, or doses administered at intervals) of rAAV encoding minidystrophin.

[0175] The following examples are provided by way of illustration and not limitation. The described numerical ranges include the end values of each integer value within the range, and include the end values of the smallest and largest of the said integers.

[0176] Examples

[0177] Example 1

[0178] A) Generation of the AAVrh74.MHCK7.minidystrophin construct

[0179] The AAVrh74.MHCK7.minidystrophin plasmid contains a human minidystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 1)。The micro-dystrophin construct is characterized by an in-frame rod domain deletion (R4-R23), while hinge 1, 2, and 4 and the cysteine-rich domain can still produce a 138 kDa protein. The expression of micro-dystrophin (3579 bp) is directed by the MHCK7 promoter (792 bp). The plasmid was constructed by removing the MCK promoter from the rAAV.MCK.micro-dystrophin plasmid and inserting the MHCK7 promoter. After the core promoter, a 53 bp endogenous mouse MCK exon 1 (untranslated) for efficient transcriptional initiation was provided, 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 micro-dystrophin cassette has a consensus Kozak sequence before the ATG start codon and a small 53 bp synthetic polyA signal for mRNA termination. The human micro-dystrophin cassette contains the (R4-R23 / Δ71-78) domain 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 the vector is the inverted terminal repeat of AAV2, which is necessary for viral DNA replication and packaging. The micro-dystrophin cassette has a small 53 bp synthetic polyA signal for mRNA termination.

[0180] Previous studies have confirmed cardiac expression using the MHCK7 promoter (Salva et al. Mol Ther 15, 320-329 (2007)) and AAVrh74 achieved skeletal, diaphragm, and cardiac muscle expression (Sondergaard et al. Annals of clinical and Transl Neurology 2, 256-270 (2015)), Figure 1 The sequence of the construct was encapsulated into AAVrh.74 virus particles by the capsid. The molecular cloning of the AAVrh.74 serotype was cloned from rhesus monkey lymph nodes and is discussed in Rodino-Klapac et al. Journal of Translational medicine 5, 45 (2007).

[0181] Table 1 shows the molecular characteristics of the plasmid AAVrh74.MHCK7.micro-dystrophin (SEQ ID NO: 3)

[0182]

[0183]

[0184] B) Generation of the AAVrh74.MHCK7. micro - dystrophin construct and plasmid encoding kanamycin (Kan) resistance

[0185] Cloning of MHCK7.μDys.KAN was achieved by the following steps: The MHCK7.μDys fragment and the kanamycin backbone were isolated from the MHCK7.μDys.AMP plasmid and annealed using the NEBuilder cloning workflow. The MHCK7.μDys fragment was isolated via SnaBI restriction enzyme digestion. The digestion was carried out at 37 °C for 1 hour in a 50 μL total reaction volume with 1×CutSmart buffer (NEB) and 1 μL of SnaBI. The resulting fragment was separated by electrophoresis using a 1% agarose gel, which was 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 DNA concentration of the resulting fragment was 10 ng / μL. The Kan backbone fragment was isolated via XbaI restriction enzyme digestion at 37 °C for 1 hour in a 50 μL reaction containing 1×CutSmart buffer (NEB) and 1 μL of XbaI. The resulting fragment was separated by electrophoresis using a 1% agarose gel, which was run at 105 volts for 1.5 hours. The band corresponding to the Kan backbone was excised and purified via a gel purification kit (Macherey - Nagel). The DNA concentration of the resulting fragment was 8.1 ng / μL. These two fragments were annealed using the NEB Builder cloning workflow, which is capable of joining two fragments with overlapping sequences. According to the manufacturer's protocol, in a 1×NEBuilder HiFi DNA Assembly premix, with a total reaction volume of 20 μL, using a 1:1 ratio of MHCK7.μDys to the kanamycin backbone, the NEBuilder cloning reaction was carried out at 50 °C for 15 minutes. The resulting clone was transformed into Stable competent Escherichia coli (E.coli) (C3040) by adding 2.5 μL of the cloning product to the cells, then placing them on ice for 30 minutes, then at 42 °C for 30 seconds, and then on ice for 5 minutes. After transformation, 950 μL of growth medium was added to the cells and grown with shaking at 225 rpm at 30 °C for 1.5 hours. After growth, 450 μL of these cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 30 °C in a dry incubator. Colonies were picked from this plate and grown overnight in LB containing 50 μg / mL kanamycin. Using The DNA was isolated from 3 mL of the culture using a QIAamp DNA Micro Kit (Qiagen). This DNA was used to confirm the cloned product. The cloned product was confirmed via restriction enzyme digestion with PmeI, MscI, and SmaI followed by gel electrophoresis. The cloned product was further confirmed via sequencing. The resulting plasmid is shown as SEQ ID NO: 8 and is shown in Figure 14 and 15 as described above. The sequence of the Figure 13 construct (corresponding to the sequence of SEQ ID NO: 9) and nucleotides 1-4977 of SEQ ID NO: 8 of the capsid were packaged into AAVrh.74 virions.

[0186] Example 2

[0187] Systemic Gene Delivery Clinical Trial for Duchenne Muscular Dystrophy

[0188] This was a single-dose, controlled trial using rAAVrh74.MHCK7.minidystrophin, nucleotides 55-5021 of SEQ ID NO: 3, in DMD subjects. Cohort A will include six subjects aged 3 months to 3 years, and cohort B will include six subjects aged 4 years to 7 years. All subjects will receive an intravenous injection of the minidystrophin vector (2 × 10 14 vg / kg, 10 mL / kg). rAAVrh74.MHCK7.minidystrophin was formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl2), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0189] In this study, rAAVrh74.MHCK7.minidystrophin was infused via the peripheral arm vein so that all muscles in the body could be reached. Six DMD subjects aged 3 months to 3 years were enrolled in cohort A, and six DMD subjects aged 4 years to 7 years were enrolled in cohort B. All subjects received an intravenous injection of the minidystrophin vector (2 × 10 14 vg / kg, 10 mL / kg). The capsid-packaged vector genome titer for the administered dose was determined by quantitative PCR using a Prism 7500 Taqman detector system (PE Applied Biosystems) with primers specific for the MHCK7 promoter, compared to a supercoiled DNA plasmid standard (Pozsgai et al. Mol. Ther. 25(4):855-869, 2017).

[0190] Subjects received an infusion for more than 1 hour in the Pediatric Intensive Care Unit (PICU) of Nationwide Children's Hospital. Muscle biopsies were performed at the screening visit prior to gene therapy. Subjects will undergo a second muscle biopsy to determine whether the gene allows replacement of the missing dystrophin 90 days after birth. After gene transfer, any treatment side effects in the patient should be carefully monitored. This monitoring includes blood and urine tests, as well as 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 no side effects from the gene injection.

[0191] Subjects in cohort A (n = 6) were between 3 months and 3 years of age and received an intravenous injection of the rAAVrh74.MHCK7.minidystrophin vector (2×10 14 vg / kg, 10 mL / kg). One day prior to gene transfer, subjects in cohort A began receiving an injection of 1 mg / kg prednisone or deflazacort and maintained it for 30 days while monitoring the immune response. If it was negative on day 30, the steroid was discontinued within 1 week. If the T cell response to AAV or minidystrophin > 125 SFC / 106 PBMC, the steroid was maintained until the level dropped below this threshold.

[0192] Subjects in cohort B (n = 6) were between 4 years and 7 years of age and received an intravenous injection of the rAAVrh74.MHCK7.minidystrophin vector (2×10 14 vg / kg, 10 mL / kg). These subjects maintained a stable dose of corticosteroids throughout the trial, but if the T cell response to AAV or minidystrophin > 125 SFC / 106 PBMC, the dose could be increased within a short period.

[0193] Acceptance criteria

[0194] The inclusion criteria for this study are as follows:

[0195] ● Inclusion age: Cohort A: 3 months to 7 years of age, Cohort B: 4 years - 7 years (inclusive).

[0196] ● Molecular characterization of the DMD gene with a frameshift (deletion or duplication) or a premature stop codon mutation between exons 18 and 58.

[0197] ● CK elevation > 1000 U / L

[0198] ● Subjects in cohort A: Defined as a scale score ≤ 9 if less than the average in the Bayley-III motor assessment for gross motor movements

[0199] ● Cohort B: Those with a time less than the average in the 100-meter timing test are defined as <predicted 80%

[0200] ● Males of any race.

[0201] ● Able to cooperate with the exercise assessment test.

[0202] ● Subjects in Cohort A: Have not been treated with corticosteroids previously.

[0203] ● Subjects in Cohort B: Have been on a stable dose equivalent of oral corticosteroids for at least 12 weeks before screening and, throughout the study, the expected dose is to remain constant (except for adjustments to accommodate weight changes).

[0204] The exclusion criteria for this study are as follows:

[0205] ● Active viral infection based on clinical observation.

[0206] ● Signs of cardiomyopathy, including echocardiogram with an ejection fraction less than 40%.

[0207] ● Serological evidence of HIV infection, or hepatitis B or C infection.

[0208] ● Diagnosis (or ongoing treatment) of an autoimmune disease.

[0209] ● Abnormal laboratory values considered to be of clinical significance.

[0210] ● The PI believes that concomitant diseases or the need for chronic drug treatment would pose an unnecessary risk to gene transfer.

[0211] ● Subjects with an AAVrh74 or AAV8 antibody titer > 1:400 determined by ELISA immunoassay.

[0212] ● The investigator believes that a medical condition or remission can impair the subject's ability to comply with the tests or procedures required by the protocol, or impair the subject's health, safety, or clinical interpretability.

[0213] ● Severe infection (e.g., pneumonia, pyelonephritis, or meningitis) occurred within 4 weeks before the gene transfer visit (inclusion may be postponed).

[0214] ● Received any investigational drug (other than corticosteroids) or exon skipping drug (including ) or experimental drug within the most recent 6 months before screening for this study.

[0215] ● Have undergone any type of gene therapy, cell-based therapy (e.g., stem cell transplantation), or CRISPR / Cas9 therapy.

[0216] ● Families do not wish to disclose the patient's research participation to primary care physicians and other healthcare providers.

[0217] Outcome measures

[0218] The primary outcome measure is safety based on the number of participants who experienced adverse events (Time frame: 3 years). Adverse reactions are monitored and scored for severity and relevance to the research project.

[0219] The secondary outcome measures are as follows:

[0220] Bayley-III Gross Motor Subtest Scale scores (Time frame: Screening, Day 30 - 3 years): Bayley-III Gross Motor Scale scores measure motor development. For cohort A, Bayley-III Gross Motor Subtest scores are obtained at each follow-up from Day 30 to 3 years. At screening, any subject aged 43 - 47 months (inclusive) has a computed scale score compared to the standard data of children aged 42 months. Bayley-III provides standard data for children aged 1 - 42 months.

[0221] Physical Therapy Assessment 100-meter Timed Test (100m) (Time frame: Screening, Day 30 - 3 years): The 100m is the primary motor outcome for cohort B. The 100-meter Timed Test is an initial exploratory outcome for cohort A at age 3 years.

[0222] Physical Therapy Assessment North Star Ambulatory Assessment (NSAA) (Time frame: Screening, Day 30 - 3 years): The North Star Ambulatory Assessment (NSAA) is an initial exploratory outcome for cohort A and cohort B at age 4 years. The NSAA measures the walking quality of young boys with Duchenne muscular dystrophy.

[0223] Physical Therapy Assessment Timed Up and Go (TUG) modified for children (Time frame: Screening, Day 30 - 3 years): Exploratory outcomes for cohort B include the Timed Up and Go (TUG) modified for children.

[0224] Physical Therapy Assessment Ascending and Descending 4 Steps (Time frame: Screening, Day 30 - 3 years): Exploratory outcomes for cohort B will include ascending and descending 4 steps.

[0225] Physical Therapy Assessment Handheld Dynamometry (HHD) (Time frame: Screening, Day 30 - 3 years): Exploratory outcomes for cohort B include Handheld Dynamometry (HHD) of knee extensors and flexors and elbow flexors and extensors.

[0226] Quantification of micro-dystrophin gene expression by immunofluorescence (time range: screening, day 90): The micro-dystrophin gene expression level was quantified by immunofluorescence and compared before and after muscle biopsy.

[0227] Quantification of micro-dystrophin gene expression by Western blot (time range: screening, day 90): The micro-dystrophin gene expression level was quantified by Western blot analysis and compared before and after muscle biopsy.

[0228] Reduction of CK after gene therapy (time range: 3 years): Reduction of CK level in circulating blood.

[0229] Cardiac magnetic resonance imaging (1 year).

[0230] Minidystrophin gene expression

[0231] Analysis and quantification of the change in micro-dystrophin expression relative to baseline by immunofluorescence (IF) fiber intensity. As Figure 7 shown, after administration of rAAVrh74.MHCK7.micro-dystrophin, Subject 1 (5 years old) showed 78% expression of micro-dystrophin in the muscle fibers of the gastrocnemius biopsy, after administration of rAAVrh74.MHCK7.micro-dystrophin, Subject 2 (4 years old) showed 73.5% expression of micro-dystrophin in the muscle fibers of the gastrocnemius biopsy, after administration of rAAVrh74.MHCK7.micro-dystrophin, Subject 3 (6 years old) showed 77.0% expression of micro-dystrophin in the muscle fibers of the gastrocnemius biopsy. After administration of rAAVrh74.MHCK7.micro-dystrophin, Subject 4 (4 years old) showed 96.2% expression of micro-dystrophin in the muscle fibers of the gastrocnemius biopsy. All patients showed strong expression of the transduced micro-dystrophin, which was appropriately localized in the muscle sarcolemma, as measured by immunohistochemistry. Compared with normal controls, as measured by the percentage of micro-dystrophin-positive fibers, the average gene expression was 76.2% and the average fiber intensity was 74.5%.

[0232] Table 2

[0233]

[0234] The change in micro-dystrophin gene expression from baseline to day 60 was also evaluated by quantifying micro-dystrophin expression, as measured by Western blot of biopsy muscle tissue. As Figure 8A and 8BAs shown, Western blot analysis detected the expression of micro-dystrophin in Subject 1 (5 years old), Subject 2 (4 years old), and Subject 3 (6 years old). Figure 8C Western blot analysis was provided to detect the expression of micro-dystrophin in Subject 4 (4 years old). As measured by Western blot, all post-treatment biopsy samples showed high levels of micro-dystrophin. The average value of Subjects 1-4 was 74.3% compared to the normal value using Method 1 and 95.8% compared to the normal value according to Method 2 (adjusted for adipose and fibrotic tissue).

[0235] For each subject, the vector genome copies of the nuclei of each myofiber were measured. As shown in Table 3, after the administration of rAAVrh74.MHCK7.micro-dystrophin, the vector genome copies of each nuclease were greater than 1 for each subject. One copy of the vector represents approximately 50% of micro-dystrophin gene expression. The average value of the vector copies per nucleus measured in Subjects 1-3 was 1.6, which was consistent with the observed high levels of micro-dystrophin expression. When the value of Subject 4 was included, the average vector copy / μg DNA > 10 5 , and the average value of the vector copies per nucleus was 3.3.

[0236] Table 3

[0237] Subjects Vector copies / μg DNA Copies per nucleus 1 <![CDATA[>10 5 > 1.7 2 <![CDATA[>10 5 > 1.3 3 <![CDATA[>10 5 > 1.9

[0238] Before and after the administration of rAAVrh74.MHCK7.micro-dystrophin, the protein levels of α-sarcoglycan and β-sarcoglycan in muscle biopsy tissues were measured by immunohistochemistry. The administration of rAAVrh74.MHCK7 also caused an upregulation of DAPC protein in the subjects. As shown in Figure 9, in Subject 1 ( Figure 9A ), Subject 2 ( Figure 9B ), and Subject 3 ( Figure 9C ), before the administration of rAAVrh74.MHCK7, the expression of α-sarcoglycan and β-sarcoglycan in muscle biopsy tissues increased compared to the levels of these proteins in muscle biopsy samples.

[0239] Circulating serum CK levels

[0240] In the rAAVrh74.MHCK7.micro-dystrophin vector (2×10 14After intravenous infusion at 10 vg / kg, 10 mL / kg, blood samples were taken every 30 days. CK levels were measured at each visit and compared to the baseline levels obtained prior to administration of rAAVrh74.MHCK7.minidystrophin (visit on day 0). Baseline serum CK levels (units / liter) are provided in Table 4 below. As Figure 10 shown, about 87% reduction in the level of circulating serum CK was observed 2 months after administration of rAAVrh74.MHCK7.minidystrophin. All subjects showed a significant reduction in serum creatine kinase (CK) levels, with an average reduction of greater than 87% in CK 2 months after treatment (n = 3). CK is an enzyme associated with muscle damage, and patients with DMD always exhibit high levels of CK. In fact, significantly elevated CK is usually used as a preliminary diagnostic tool for DMD, followed by confirmatory genetic tests.

[0241] Table 5 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 decreased significantly over time after administration of rAAVrh74.MHCK7.minidystrophin. The average baseline CK level of 27,064 U / L (average of Table 4) decreased by about 63% to an average of 9,982 U / L (average, day 270, Table 5).

[0242] Table 4

[0243]

[0244] Table 5: Changes in CK levels from baseline to day 270

[0245]

[0246] Efficacy assessment

[0247] In addition to minidystrophin and CK levels, efficacy was also measured by the following functional tests: time to stand from the floor, ascend 4 steps, North Star Ambulatory Assessment (NSAA), time to stand test, ascend 4 steps test, 10-meter timed test (10m), and 100-meter timed test (100m). The data are provided in Tables 6 and 7 below, and the data show sustained and durable improvement 9 months after administration of rAAVrh74.MHCK7.minidystrophin. Figure 12 Improvement in NSAA over time is also provided.

[0248] Table 6: Changes in NSAA from baseline to day 270

[0249]

[0250] Table 7: Changes from Baseline to Day 270

[0251]

[0252]

[0253] Safety assessment

[0254] No serious adverse events (SAEs) were observed in the study. Three subjects had elevated gamma-glutamyl transferase (GGT), which resolved with an increase in steroids within one week and returned to baseline levels. No other clinically significant laboratory findings were observed. Patients generally had transient nausea during the first week of treatment, coinciding with an increase in steroid dose. This was not associated with elevated liver enzymes or any other abnormalities.

[0255] Example 3

[0256] Randomized, Double-Blind, Placebo-Controlled Phase I / IIa Clinical Trial of Systemic Gene Delivery

[0257] This was a randomized, double-blind, single-dose trial using rAAVrh74.MHCK7. microdystrophin in DMD subjects. The study included twenty-four subjects aged 4 to 7 years. Subjects were randomly assigned to the treatment group or the placebo group at the time of enrollment. Twelve subjects received intravenous administration of the rAAVrh74.MHCK7. microdystrophin vector (2×10 14 vg / kg, approximately 10 mL / kg), while another twelve subjects received 10 mL / kg of placebo (lactated Ringer's solution). Placebo subjects will continue treatment in the same manner as the 12 previously treated subjects one year after the last treated subject is dosed. Subjects received an infusion of rAAV carrying microdystrophin or lactated Ringer's solution over approximately 1 hour. Needle muscle biopsies of the gastrocnemius muscle were performed before and after treatment (90 days).

[0258] The primary objective of this study was to evaluate the safety of intravenous administration of rAAVrh74.MHCK7.minidystrophin via the peripheral limb veins in DMD subjects. Safety endpoints were evaluated by hematology, serum chemistry, urine analysis, immune responses to rAAVrh74 and minidystrophin, and changes in reported medical history and symptom observations. Dystrophin gene expression was included as a primary outcome measure together with safety. Quantitative measurements were performed using validated immunofluorescence and immunoblot assays. Reduction in CK after gene therapy was a secondary outcome. Efficacy was measured by the following functional tests: time to stand, four-step ascent, North Star Ambulatory Assessment (NSAA), 10-meter timed test (10m), 100-meter timed test (100m). Exploratory measures included hand-held dynamometry (HHD) of knee extensors and flexors and elbow flexors and extensors.

[0259] The inclusion criteria for this study were as follows:

[0260] ● Inclusion age: between 4 and 7 years of age (inclusive).

[0261] ● Molecular characterization of the DMD gene with a frameshift (deletion or duplication) or premature stop codon mutation between exons 18 and 58.

[0262] ● Symptomatic muscular dystrophy indication: CK elevation > 1000 U / L and percentage less than the mean predicted time in the 100-meter walk test

[0263] ● Males of any race were eligible for inclusion.

[0264] ● Ability to cooperate with the motor assessment tests.

[0265] ● Stable dose equivalent of oral corticosteroids for at least 12 weeks prior to screening and, throughout the study, the expected dose was to remain constant (except for potential adjustments to accommodate weight changes).

[0266] The exclusion criteria for this study were as follows:

[0267] ● Active viral infection based on clinical observation.

[0268] ● Signs of cardiomyopathy, including echocardiogram with ejection fraction less than 40%.

[0269] ● Serological evidence of HIV infection, or hepatitis B or C infection.

[0270] ● Diagnosis of an autoimmune disease (or ongoing treatment).

[0271] ● Abnormal laboratory values are considered clinically significant (GGT > 3X ULN, bilirubin ≥ 3.0 mg / dL, creatinine ≥ 1.8 mg / dL, Hgb < 8 or > 18 g / Dl; WBC > 18,500 / cmm), platelets ≤ 50,000.

[0272] ● The PI believes that concomitant diseases or the need for chronic drug therapy pose unnecessary risks to gene transfer.

[0273] ● Subjects with AAVrh74 or AAV8 antibody titers > 1:400 determined by ELISA immunoassay. If the endpoint titer is positive at screening, the test can be repeated before exclusion.

[0274] ● The investigator believes that there are medical conditions or remissions that can impair the subject's ability to comply with the tests or procedures required by the protocol, or that can impair the subject's health, safety, or clinical interpretability.

[0275] ● Severe infections (e.g., pneumonia, pyelonephritis, or meningitis) occurred within 4 weeks before the gene transfer visit (inclusion may be postponed).

[0276] ● Within the most recent 6 months before screening for this study, any investigational drug (other than corticosteroids) or exon skipping drug (including ) or experimental drug has been received.

[0277] ● Any type of gene therapy, cell-based therapy (e.g., stem cell transplantation), or CRISPR / Cas9 therapy has been performed.

[0278] ● The family does not wish to disclose the patient's study participation to the primary care physician and other healthcare providers.

[0279] Efficacy assessment

[0280] Dystrophin gene expression, together with safety, is used as the primary outcome measure. Quantitative analysis is performed using validated immunofluorescence and immunoblot assays. Reduction of CK after gene therapy is used as a secondary outcome. In addition, efficacy is measured by the following functional tests: time to stand from the floor, ascending 4 steps, North Star Ambulatory Assessment Scale (NSAA), 10-meter timed test (10m), 100-meter timed test (100m). Exploratory measures include handheld dynamometry (HHD) of the knee extensors and flexors, and the elbow flexors and extensors.

[0281] Ultrasound-guided muscle biopsies were used to perform quantitative comparisons of transgene expression at baseline and day 90. Muscle biopsies were performed on the muscle in the other leg that was the same as the original biopsy. All subjects were dosed after one year, and 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 received a muscle biopsy at day 90 (total of 3 muscle biopsies). Cryosections were stained for dystrophin using indirect immunofluorescence (IF). Whole-slide scanning was performed using a validated image scanning and MuscleMapTM analysis algorithm, and quantification of micro-dystrophin intensity and percentage of positive fibers was carried out. Muscle morphometry, including fiber size histograms, was performed in a blinded manner. A validated Western blot method was used, and blinded frozen muscle biopsy scrapings were used for quantitative protein analysis of micro-dystrophin.

[0282] Muscle needle biopsies of the gastrocnemius muscle (unless the PI deems it contraindicated in a particular subject, in which case the PI will select an alternative muscle for biopsy) were used to quantify micro-dystrophin expression.

[0283] Efficacy analysis

[0284] The primary efficacy endpoint was the change in the amount of micro-dystrophin expression from baseline to day 90, as measured by Western blot of biopsy muscle tissue. Treatment group differences for the primary efficacy endpoint were evaluated using an analysis of covariance (ANCOVA) model, where treatment was a fixed factor and the baseline value was a covariate. The Wilcoxon rank sum test was performed as a supportive analysis. Similarly, changes in micro-dystrophin expression relative to baseline were analyzed via immunofluorescence (IF) fiber intensity.

[0285] Supportive efficacy endpoints included changes from baseline to each scheduled assessment in time to stand from the floor, step up 4 stairs, NSAA, 10-meter timed test (10m), 100-meter timed test (100m), and CK. Exploratory measures included HHD of the knee extensors and flexors and elbow flexors and extensors. Treatment group differences were evaluated using an ANCOVA model, where treatment was a fixed factor and the baseline value was a covariate. The Wilcoxon rank sum test was performed as a supportive analysis.

[0286] Example 4

[0287] The experiments and studies described in Examples 2 and 3 above can alternatively be carried out using the rAAVrh74.MHCK7.minidystrophin construct shown in SEQ ID NO:9; nucleotides 1-4977 shown in SEQ ID NO:8; or nucleotides 56-5022 shown in SEQ ID NO:6.

[0288] Example 5

[0289] Generation of the pAAV.MCK.minidystrophin construct

[0290] The pAAV.MCK.minidystrophin plasmid was constructed by inserting the MCK expression cassette driving the codon-optimized human minidystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski et al., J. Virol. 61(10):3096-3101). Muscle-specific regulatory elements were included in the construct to drive muscle-specific gene expression. The regulatory element contains the murine MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). After the core promoter, the construct contains the 53 bp endogenous murine MCK exon 1 (untranslated) for efficient transcriptional initiation, followed by the SV40 late 16S / 19S splice signal (97 bp) and a small 5'UTR (61 bp). The intron and 5'UTR are derived from the plasmid pCMVβ (Clontech). The minidystrophin cassette has a consensus Kozak sequence before the ATG start codon and a small 53 bp synthetic polyA signal for mRNA termination. The human minidystrophin cassette contains the (R4-R23 / Δ71-78) domain as previously described by Harper et al., Nat. Med. 8(3):253-61, 2002.

[0291] The pAAV.MCK.minidystrophin plasmid contains the human minidystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 5 ). This sequence is packaged into AAVrh.74 viral particles by the capsid. The molecular clone of the AAVrh.74 serotype was cloned from rhesus monkey lymph nodes and is described in Rodino-Klapac et al., Journal of Tran. Med. 45 (2007).

[0292] References

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[0305] 13. Cullen, M.J. & Jaros, E. Ultrastructure of the skeletal muscle in the X chromosome-linked dystrophic (mdx) mouse. Comparison with Duchenne muscular dystrophy. Acta Neuropathol 77, 69 - 81 (1988).

[0306] 14. Dupont-Versteegden, E.E. & McCarter, R.J. Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of mdx mice. Muscle Nerve 15, 1105 - 1110 (1992).

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[0308] 16. Deconinck, A.E., et al. Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 90, 717 - 727 (1997).

[0309] 17. Grady, R.M., et al. Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 90, 729 - 738 (1997).

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[0312] 20. Tinsley, J., et al. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat Med 4, 1441 - 1444 (1998).

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Claims

1. Use of recombinant adeno-associated virus (rAAV) in the preparation of a medicament for the treatment of Duchenne muscular dystrophy in human subjects aged from 3 months to 7 years in need thereof, wherein the rAAV is serotype rh.74 and comprises a polynucleotide containing the nucleotide sequence of SEQ ID NO:9, wherein the medicament is formulated for the systemic administration route, and the dose of the rAAV is 1×10 14 vg / kg to 4×10 14 vg / kg.

2. The use according to claim 1, wherein the drug is formulated for intravenous administration and the dose of the rAAV is 2×10 14 vg / kg.

3. The use according to claim 2, wherein the dose of the rAAV is determined by using a supercoiled DNA standard.

4. The use according to claim 1 or 2, wherein the dose of the rAAV is formulated for administration at 10 mL / kg.

5. The use according to claim 1, wherein the medicament is formulated for administration by an infusion over one hour.

6. Use of recombinant adeno-associated virus (rAAV) in the preparation of a medicament for treating Duchenne muscular dystrophy in a human subject in need thereof, wherein the rAAV is serotype rh74 and comprises a polynucleotide containing the nucleotide sequence of SEQ ID NO:9, wherein the medicament is formulated for administration by intravenous infusion over one hour, and the dose of rAAV is 2×10 14 vg / kg, and wherein the age of the subject is from 3 months to 7 years.

7. The use according to claim 6, wherein the Duchenne muscular dystrophy gene of the subject has a frameshift or premature termination codon mutation between exons 18 and 58.

8. The use according to claim 6, wherein before administration of the rAAV to the subject, the serum creatine kinase (CK) level of the subject > 1000 U / L.

9. The use according to claim 6, wherein before administration of the rAAV to the subject, the subject is below the average in the Bayley-III motor assessment.

10. The use according to claim 6, wherein before administration of the rAAV to the subject, the subject is below the average in the 100-meter timed test.

11. The use according to claim 6, wherein before administration of the rAAV to the subject, the AAVrh74 or AAV8 antibody titer determined by ELISA immunoassay of the subject < 1:

400.

12. The use according to claim 6, wherein the dose of the rAAV is determined by using a supercoiled DNA standard.

13. The use according to claim 1 or 6, wherein compared with the serum CK level before administration, after administration of the rAAV to the subject, the serum CK level in the subject decreases.

14. The use according to claim 7, wherein the dose of the rAAV is determined by using a supercoiled DNA standard.

15. The use according to claim 1 or 6, wherein compared with the level of micro-dystrophin gene expression before administration of the rAAV, after administration of the rAAV, the level of micro-dystrophin gene expression in the cells of the subject increases.

16. The use according to claim 1 or 6, wherein compared with the number of micro-dystrophin positive fibers before administration of the rAAV, after administration of the rAAV, the number of micro-dystrophin positive fibers in the muscle tissue of the subject increases.

17. The use according to claim 1 or 6, wherein compared with the level of α-sarcoglycan and / or β-sarcoglycan before administration of the rAAV, after administration of the rAAV, the level of α-sarcoglycan and / or β-sarcoglycan in the subject increases.

18. The use according to claim 1 or 6, wherein after administration of the rAAV, the disease progression of the subject is delayed, as measured by any one of the following tests: six-minute walk test, time to stand up, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment Scale (NSAA), 10-meter timed test, 100-meter timed test, hand-held dynamometry (HHD), timed up and go, and / or gross motor subtest scale (Bayley-III) score.

19. Use of muscle cells in the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, wherein the muscle cells have been transduced with recombinant adeno-associated (rAAV) serotype rh74, the recombinant adeno-associated (rAAV) serotype rh74 comprising the micro-dystrophin nucleotide sequence of SEQ ID NO:9 and 5' inverted terminal repeat sequences (ITRs), a promoter sequence, a chimeric intron sequence, a polyA sequence, and the 3' ITR of SEQ ID NO:

9.

20. The use according to claim 19, wherein the medicament is formulated for intramuscular injection, intravenous injection, subcutaneous injection, or intraperitoneal injection.

21. A composition comprising muscle cells transduced with recombinant adeno-associated (rAAV) serotype rh74, the recombinant adeno-associated (rAAV) serotype rh74 comprising the micro-dystrophin nucleotide sequence of SEQ ID NO:9 and 5' inverted terminal repeat (ITR), a promoter sequence, a chimeric intron sequence, a polyA sequence, and the 3' ITR of SEQ ID NO:9.

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