Adeno-associated viral vector delivery of muscle-specific micro-dystrophin for the treatment of muscular dystrophy

Abstract

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

Description

[0001] This application claims the benefit of priority to 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, each of which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials

[0003] As a separate part of the present disclosure, this application contains a sequence listing in computer readable form, which is incorporated herein by reference in its entirety and identified as follows: File name: 53169_Seqlisting.txt; Size: 60,056 bytes, Creation Date: June 17, 2019. Technical Field

[0004] The present invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, that express a miniaturized human micro-dystrophin gene, and methods of using these vectors to express micro-dystrophin in skeletal muscle, including diaphragm and cardiac muscle, and to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in subjects with muscular dystrophy. Background Art

[0005] The importance of muscle mass and strength for daily activities (such as movement and breathing) and systemic metabolism is obvious. Insufficient muscle function can cause muscular dystrophy (MD) (characterized by muscle weakness and emaciation) and can seriously affect the quality of life. The most well-characterized MD is the result of mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs are the result of membrane fragility associated with the loss of sarcolemmal cytoskeleton tethering caused by DAPC. Duchenne muscular dystrophy (DMD) is one of the most lethal muscle diseases, affecting 1 in 5000 newborn boys.

[0006] DMD is caused by mutations in the DMD gene, which lead to a reduction in mRNA and a lack of dystrophin, a 427kD sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51 (6): 919-28, 1987). DAPC is composed of a variety of proteins at the muscle sarcolemma, which form structural connections between the extracellular matrix (ECM) and the cytoskeleton via dystrophin (an actin-binding protein) and α-dystrophy protein glycan (a laminin-binding protein). These structural connections are used to stabilize the muscle cell membrane during contraction and prevent damage caused by contraction. With the loss of dystrophin, membrane fragility causes sarcolemmal tearing and calcium influx, thereby triggering 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 eventually depletes the muscle stem cell population (Sacco et al., Cell, 2010. 143(7): 1059-71; Wallace et al., Annu Rev Physiol, 2009. 71: 37-57), leading to progressive muscle weakness, endomysial inflammation, and fibrotic scarring.

[0007] In the absence of membrane stability from dystrophin or micro-dystrophin, DMD will show an uncontrolled cycle of tissue damage, and the final repair of the lost muscle fiber is replaced by fibrotic scar tissue through connective tissue proliferation. Fibrosis is characterized by 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 main pathological feature of DMD is muscle fiber degeneration and necrosis, fibrosis as a pathological result has the same consequences. The excessive production of fibrotic tissue limits muscle regeneration and leads to progressive muscle weakness in DMD patients. In a study, the presence of fibrosis in initial DMD muscle biopsy is highly associated with the adverse motor outcomes of 10 years of follow-up (Desguerre et al., J Neuropathol Exp Neurol, 2009.68 (7): 762-7 pages). These results show that fibrosis is the main factor leading to DMD muscle dysfunction, and emphasizes the necessity of early intervention before obvious fibrosis.

[0008] For patients with DMD, treatments are needed to increase muscle strength and prevent muscle loss. Summary of the invention

[0009] The present invention relates to a gene therapy vector, such as AAV, for expressing a micro-dystrophin gene into skeletal muscle (including diaphragm muscle and cardiac muscle) to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis.

[0010] The present invention provides therapies and methods for delivering micro-dystrophin using gene therapy vectors to address the genetic defects observed in DMD, thereby increasing muscle strength and / or increasing muscle mass. Example 2 describes a systemic gene delivery clinical trial for Duchenne muscular dystrophy in which subjects received 2×1014 vg / kg AAVrh74.MHCK7. micro-dystrophin. The clinical study described in Example 3 provides a novel pivotal clinical protocol that includes a randomized double-blind placebo-controlled design. At the beginning of the study, subjects were randomized and received 2×10 14 vg / kg AAVrh74.MHCK7.micro-dystrophin or lactated Ringer's solution.

[0011] The present invention provides a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 3, 8 or 9. The present invention also provides a rAAV comprising a 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 a rAAV particle comprising a 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.

[0012] Another aspect of the present invention provides a composition comprising a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 3, 8 or 9, a rAAV comprising a 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 a rAAV particle comprising a 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 of the methods disclosed herein can be performed using these compositions.

[0013] The present invention provides a method for treating muscular dystrophy in a human subject in need thereof, the method comprising the step of administering a recombinant adenovirus (rAAV)-associated rAAV.MHCK7.micro-dystrophin, wherein the rAAV is expressed at 5.0×10 12 vg / kg to about 1.0×10 15The dose of vg / kg is administered by a systemic route of administration. The muscular dystrophy may be Duchenne muscular dystrophy or Becker muscular dystrophy.

[0014] For example, the dose of rAAV administered is about 5.0 x 10 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 about 1.0x10 14 vg / kg, or about 5.0x10 13 vg / kg to 1.0x10 14 vg / kg, or about 5.0x10 13 vg / kg to about 2.0x10 14 vg / kg, or about 5.0x10 13 vg / kg to about 1.0x10 14 vg / kg, or about 5.0x10 13 vg / kg to about 3.0x10 14 vg / kg, or about 5.0x10 13 vg / kg to about 5.0x10 14 vg / kg, or about 5.0x10 13 vg / kg to about 6.0x10 14 vg / kg, or 5.0x10 13 vg / kg to about 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg to about 6.0x10 14 vg / kg, or 1.0x10 14 vg / kg to about 5.0x10 14 vg / kg, or 1.0x10 14 vg / kg to about 4.0x10 14 vg / kg, or 1.0x10 14 vg / kg to about 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg to about 3.0x10 14 vg / kg, or about 1.0x10 14 vg / kg to about 2.5x10 14 vg / kg, or 1.0x10 14 vg / kg to about 2.0x10 14 vg / kg, or about 1.25x10 14 vg / kg to about 3.75x10 14 vg / kg, or about 1.25x10 14 vg / kg to 6.0x10 14 , or about 1.25x10 14 vg / kg to 5.0x10 14 , or about 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.

[0015] In one embodiment, the method of the present invention comprises systemic administration of rAAV, wherein the systemic administration route is intravenous and the dose of rAAV administered is about 2.0×10 14 In another embodiment, the method of the present invention comprises systemic administration of rAAV, wherein the systemic administration route is intravenous, 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, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin of SEQ ID NO:5.

[0016] In any of the methods of the 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 / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In a specific embodiment, the dose or rAAV is administered at about 10 mL / kg. In one embodiment, rAAV is AAVrh74.MHCK7. micro-dystrophin or AAVrh74.MCK. micro-dystrophin. In one embodiment, rAAV is 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 of nucleotides 56-4820 of SEQ ID NO:5.

[0017] In any one 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 about one hour. In addition, the dose of rAAV is administered by intravenous route of peripheral limb vein (such as, peripheral arm vein or 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. In one embodiment, rAAV is AAVrh74.MHCK7. micro-dystrophin. In one embodiment, AAVrh74.MHCK7. micro-dystrophin is SEQ ID NO: 9 AAVrh74.MHCK7. micro-dystrophin, SEQ ID NO: 3 nucleotides 55-5021, SEQ ID NO: 8 nucleotides 1-4977 or SEQ ID NO: 6 nucleotides 56-5022. In one embodiment, rAAV is AAVrh74.MCK. micro-dystrophin. In one embodiment, the AAVrh74.MCK.micro-dystrophin is AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0018] The rAAV administered by any of the methods of the invention may comprise the human micro-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 invention comprises the human micro-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 may comprise the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, the AAVrh74.MHCK7.micro-dystrophin is 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.

[0019] In one embodiment, the rAAV is AAVrh74.MCK.micro-dystrophin. In one embodiment, the AAVrh74.MCK.micro-dystrophin is AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0020] In any of the methods of the invention, the rAAV administered is of serotype AAVrh7.4.

[0021] In some embodiments, the methods of the invention treat Duchenne muscular dystrophy or Becker muscular dystrophy. An exemplary embodiment is a method of treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, the method comprising the step of administering a dose of recombinant adenovirus (rAAV)-associated rAAV.MHCK7.micro-dystrophin, wherein the route of administration is intravenous infusion, and the dose of rAAV administered is about 2×10 14 vg / kg, and wherein the rAAV vector comprises the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, or nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, AAVrh74.MHCK7.micro-dystrophin is 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 AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0022] In one embodiment, the invention provides 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% sequence identity with SEQ ID NO: 1, more usually at least 90%, 91%, 92%, 93% or 94%, even more usually at least 95%, 96%, 97%, 98%, 99% or 100%, 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, rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, AAVrh74.MHCK7.Microdystrophin is AAVrh74.MHCK7.Microdystrophin of SEQ ID NO:9, or nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, rAAV is AAVrh74.MCK.Microdystrophin. In one embodiment, AAVrh74.MCK.Microdystrophin is AAVrh74.MCK.Microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0023] The present invention also provides rAAV, wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes with the nucleic acid sequence of SEQ ID NO: 1 or its complementary sequence under stringent conditions and encodes a functional micro-dystrophin protein.

[0024] In one embodiment, rAAV is a non-replicating recombinant adeno-associated virus (AAV), i.e., 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 minimum elements required for gene expression, including AAV2 inverted terminal repeats (ITRs), micro-dystrophin, SV40 introns (SD / SA), and synthetic polyadenylation (poly A) signals, all of which are under the control of the MHCK7 promoter / enhancer. A schematic diagram of the vector genome and expression cassette is shown in FIG. Figure 1Following intravenous administration, AAVrh74 serotype can be used to achieve efficient gene transfer in skeletal and cardiac muscle.

[0025] The term "stringent" is used to refer to conditions that are generally understood in the art to be stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate 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 LaboratoryManual , 2nd edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide or other denaturants) may also be used, however, the rate of hybridization may be affected. In the case of deoxyoligonucleotide hybridization, 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).

[0026] To reduce nonspecific and / or background hybridization, other reagents may be included in the hybridization and wash 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 may be used. The concentration and type of these additives may be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed 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 one skilled in the art to accommodate these variables and allow DNA of different sequence relatedness to form hybrids.

[0027] 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 a construct comprising the muscle-specific control elements MCKH7 promoter, MCK promoter and MCK enhancer.

[0028] The term "operably linked" refers to the positioning of a regulatory element nucleotide sequence (eg, a promoter nucleotide sequence) so as to confer expression of the nucleotide sequence by the regulatory element.

[0029] In one aspect, the present invention provides rAAV, wherein the muscle-specific control element is a human skeletal muscle actin gene element, a cardiac actin gene element, a myocyte-specific enhancer binding factor (MEF), muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), a hybrid α-myosin heavy chain enhancer / MCK enhancer-promoter (MHCK7), C5-12, a murine creatine kinase enhancer element, a skeletal muscle fast-twitch troponin c gene element, a slow-twitch cardiac troponin c gene element, a slow-twitch troponin i gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0030] 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. In addition, in any of the rAAV vectors of the present invention, the muscle-specific control element nucleotide sequence (eg, MHCK7 or MCK nucleotide sequence) is operably linked to a nucleotide sequence encoding micro-dystrophin. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO: 2 or SEQ ID NO: 7) is operably linked to a human micro-dystrophin coding sequence (SEQ ID NO: 1), such as Figure 1 or Figure 2 (SEQ ID NO:3) or Fig.13 In another example, the MCK promoter (SEQ ID NO: 4) is operably linked to the human microdystrophin coding sequence (SEQ ID NO: 1), such as Figure 5 or Figure 6(SEQ ID NO:5) is shown in the construct provided. In another aspect, the present invention provides a 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 a rAAV vector, the rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:4.

[0031] In another aspect, the present invention provides an rAAV construct contained in a plasmid, the plasmid comprising the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8. For example, the AAVrh74.MHCK7.microdystrophin vector comprises a nucleotide sequence within the ITR of SEQ ID NO: 3 and including the termini, such as Figure 2 The rAAV vector comprises a 5'ITR, a MHCK7 promoter, a chimeric intron sequence, a coding sequence of a human microdystrophin gene, poly A, and a 3'ITR. In one embodiment, the vector comprises nucleotides 55-5021 of SEQ ID NO: 3. The plasmid shown in SEQ ID NO: 3 also comprises ampicillin resistance and a pGEX plasmid backbone with a pBR322 replication origin.

[0032] In another aspect, the present invention provides an rAAV comprising the nucleotide sequence of SEQ ID NO: 9. For example, the AAVrh74.MHCK7.micro-dystrophin vector construct comprises the nucleotide sequence of SEQ ID NO: 9, such as Fig.13 As shown. The rAAV vector construct comprises the MHCK7 promoter, a chimeric intron sequence, a coding sequence of the human microdystrophin gene, and poly A. In one embodiment, the rAAV vector construct further comprises the 5' ITR of the promoter and the 3' ITR of the poly A. In one embodiment, the rAAV is AAVrh74.

[0033] In another aspect, the AAVrh74.MHCK7.micro-dystrophin vector comprises a nucleotide sequence within the ITR of SEQ ID NO: 8 and including the termini, such as Fig.15The rAAV vector comprises a 5'ITR, a MHCK7 promoter, a chimeric intron sequence, a coding sequence of a human microdystrophin gene, poly A, and a 3'ITR. In one embodiment, the vector comprises nucleotides 1-4977 of SEQ ID NO: 9. The plasmid shown in SEQ ID NO: 3 also comprises a pGEX plasmid backbone with kanamycin resistance and a pBR322 replication origin.

[0034] In another aspect, the present invention provides a plasmid comprising an AAVrh74.MHCK7.micro-dystrophin vector construct. In one embodiment, the plasmid comprises a 5'ITR, a MHCK7 promoter, a chimeric intron sequence, a coding sequence of a human micro-dystrophin gene, poly A, and a 3'ITR. In one embodiment, the plasmid comprises kanamycin resistance and optionally comprises a pGEX plasmid backbone with a pBR322 replication origin. In a specific embodiment, the plasmid is as shown in SEQ ID NO: 8, and in Fig.14 and 15 Displayed in.

[0035] The present invention provides a recombinant AAV vector, which comprises a human micro-dystrophin nucleotide sequence of SEQ ID NO: 1 and a MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7. The rAAV vector is AAV serotype AAVrh.74.

[0036] The present invention also provides rAAV comprising the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence within the ITRs and including the ends in SEQ ID NO: 3, the nucleotide sequence within the ITRs and including the ends in SEQ ID NO: 8, or the nucleotide sequence as shown in SEQ ID NO: 9. The rAAV vector is AAV serotype AAVrh.74.

[0037] The rAAV vector of the present invention may be any AAV serotype, such as serotype AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13.

[0038] The present invention also provides pharmaceutical compositions (or sometimes simply referred to herein as "compositions") comprising any of the rAAV vectors of the present invention.

[0039] In another embodiment, the present invention provides a method for producing rAAV vector particles, the method comprising culturing cells transfected with any rAAV vector of the present invention, and recovering rAAV particles from the supernatant of the transfected cells. The present invention also provides a viral particle comprising any of the recombinant AAV vectors of the present invention.

[0040] In any of the methods for treating muscular dystrophy, the level of micro-dystrophin gene expression in cells of the subject is increased after administration of rAAV. Micro-dystrophin levels are measured by Western blot in biopsied muscle before and after administration of rAAV to detect expression of the micro-dystrophin gene in cells. Specifically, 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%, compared to the level of micro-dystrophin before administration of rAAV. For example, after administration of rAAV, 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% compared to the level of micro-dystrophin before administration of rAAV.

[0041] In addition, the expression of the micro-dystrophin gene in the cell is detected by measuring the level of micro-dystrophin by immunohistochemistry in muscle biopsies before and after the administration of rAAV. Compared with the level of micro-dystrophin before the administration of rAAV, after the 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, after the administration of rAAV, 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%, compared with the level of micro-dystrophin before the administration of rAAV.

[0042] In any of the methods for treating muscular dystrophy, serum CK levels in the subject are reduced after administration of rAAV compared to serum CK levels before administration of rAAV. For example, serum CK levels in the subject are reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% 60 days after administration of rAAV compared to serum CK levels before administration of rAAV. Specifically, in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 87% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 72% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 73% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 78% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 95% 60 days after rAAV administration compared to the serum CK level before rAAV administration. In any of the methods of treating muscular dystrophy, the number of micro-dystrophin-positive fibers in the subject's muscle tissue increases after administration of rAAV compared to the number of micro-dystrophin-positive fibers before administration of rAAV. For example, micro-dystrophin levels are measured by Western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV to detect the number of micro-dystrophin-positive fibers.

[0043] In any one of the methods for treating muscular dystrophy, the administration of rAAV upregulates the expression of DAPC proteins (such as α-sarcoglycan or β-sarcoglycan). For example, compared with the level of α-sarcoglycan before the administration of rAAV, after the administration of rAAV, the level of α-sarcoglycan in the subject increases. In addition, compared with the level of β-sarcoglycan before the administration of rAAV, after the administration of rAAV, the level of β-sarcoglycan in the subject increases. Before and after the administration of rAAV, α-sarcoglycan or β-sarcoglycan protein levels are measured by Western blotting or immunohistochemistry for muscle biopsy to detect the level of α-sarcoglycan or β-sarcoglycan.

[0044] In any of the methods of treating muscular dystrophy, following administration of the rAAV, the subject has a delay in disease progression as measured by any of the following tests: six minute walk test, time to stand, ascending 4 stairs, ascending and descending 4 stairs, North Star Ambulatory Assessment Scale (NSAA), 10 meter timed test, 100 meter timed test, handheld dynamometer (HHD), timed up-and-go, and / or gross motor subtest scale (Bayley-III) scores.

[0045] For example, in any method, the subject's NSAA score has an improvement of at least 6 points at least 270 days after rAAV administration, compared to the NSAA score before rAAV administration. In addition, in any method, the subject's stand-up time has an improvement of at least about 0.8 seconds at least 270 days after rAAV administration, compared to the stand-up time before rAAV administration. In addition, in any method, the subject's time to rise 4 stairs test has an improvement of at least about 1.2 seconds at least 270 days after rAAV administration, compared to the time to rise 4 stairs test before rAAV administration. In addition, in any method, the subject's 100m time test has an improvement of at least about 7 seconds at least 270 days after rAAV administration, compared to the 100m time test before rAAV administration.

[0046] In another embodiment, the present invention provides a method for expressing a micro-dystrophin gene in a patient's cells, the method comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, micro-dystrophin levels are measured by protein blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct, thereby detecting the expression of the micro-dystrophin gene in the patient's cells. In addition, the expression of the micro-dystrophin gene in the patient is measured by detecting the number of vector genomes per nucleus more, wherein 1 vector genome per nucleus is about 50% micro-dystrophin expression, and greater than 1 copy per nucleus is consistent with micro-dystrophin expression levels. 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.

[0047] 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 an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. For example, serum CK levels in the patient are 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% at 60 days after administration of rAAV, compared to serum CK levels before administration of rAAV. Specifically, 60 days after rAAV administration, the subject's serum CK level is reduced by about 87% compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 72% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 73% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 78% 60 days after rAAV administration compared to the serum CK level before rAAV administration, or in any of the methods for treating muscular dystrophy of the present invention, the subject's serum CK level is reduced by about 95% 60 days after rAAV administration compared to the serum CK level before rAAV administration.

[0048] The present invention also provides a method for increasing micro-dystrophin positive fibers in the muscle tissue of a patient, the method comprising administering 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 the patient. For example, before and after administration of rAAV, the level of dystrophin is measured by protein blotting or immunohistochemistry of muscle biopsies 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 more vector genomes per cell nucleus, wherein 1 vector genome per cell nucleus is about 50% micro-dystrophin expression, and greater than 1 copy per cell nucleus is consistent with the expression level of micro-dystrophin. 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.

[0049] In another embodiment, the present invention provides a method of increasing the expression of alpha-sarcoglycan in a patient in need thereof, 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, the level of alpha-sarcoglycan is detected by measuring the level of alpha-sarcoglycan protein by Western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

[0050] In addition, the present invention provides a method of increasing the expression of β-sarcoglycan in a patient in need thereof, 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, the level of β-sarcoglycan is detected by measuring the level of β-sarcoglycan protein by Western blot or immunohistochemistry on muscle biopsies before and after administration of rAAV.

[0051] The present invention also provides a method of treating a patient suffering from Duchenne muscular dystrophy or Becker muscular dystrophy, the method comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, so that the patient's disease progression is delayed as measured by any of the following tests: six-minute walk test, stand time, ascent of 4 stairs, ascent and descent of 4 stairs, North Star Ambulatory Assessment Scale (NSAA), 10-meter timed test, 100-meter timed test, hand-held dynamometer (HHD), timed up-and-go and / or gross motor subtest scale (Bayley-III) score.

[0052] For example, in any method, the subject's NSAA score has an improvement of at least 6 points at least 270 days after rAAV administration, compared to the NSAA score before rAAV administration. In addition, in any method, the subject's stand-up time has an improvement of at least about 0.8 seconds at least 270 days after rAAV administration, compared to the stand-up time before rAAV administration. In addition, in any method, the subject's time to rise 4 stairs test has an improvement of at least about 1.2 seconds at least 270 days after rAAV administration, compared to the time to rise 4 stairs test before rAAV administration. In addition, in any method, the subject's 100m time test has an improvement of at least about 7 seconds at least 270 days after rAAV administration, compared to the 100m time test before rAAV administration.

[0053] "Fibrosis" refers to the excessive or uncontrolled deposition of extracellular matrix (ECM) components and the abnormal repair process of tissues (including skeletal muscle, myocardium, liver, lung, kidney and pancreas) after injury. The deposited ECM components include fibronectin and collagen (e.g., collagen 1, collagen 2 or collagen 3).

[0054] The present invention also provides a method of reducing or preventing fibrosis in a subject with muscular dystrophy, the method comprising administering a therapeutically effective amount of rAAV or 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 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. 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, 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.

[0055] 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 a human micro-dystrophin nucleotide sequence of SEQ ID NO: 1 and a MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7; or a rAAV vector comprising an AAV74.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, any of the rAAVs of the present invention can be administered to a subject with muscular dystrophy to prevent fibrosis, for example, before fibrosis is observed in the subject, the rAAV of the present invention expresses the administered human micro-dystrophin. In addition, the rAAV of the present invention expressing the human micro-dystrophin gene can be administered to a subject at risk of developing fibrosis, such as those subjects suffering from or diagnosed with muscular dystrophy (e.g., DMD). The rAAV of the invention can be administered to subjects suffering from muscular dystrophy to prevent new fibrosis in these subjects.

[0056] The present invention contemplates administering rAAV before fibrosis is observed in a subject. In addition, rAAV can be administered to subjects at risk of developing fibrosis, such as those suffering from or diagnosed with muscular dystrophy (e.g., DMD). rAAV can be administered to subjects with muscular dystrophy who have developed fibrosis to prevent new fibrosis in these subjects.

[0057] The present invention also provides a method for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, the method comprising administering a therapeutically effective amount of a human micro-dystrophin nucleotide sequence of SEQ ID NO: 1 and a MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7; or rAAV comprising an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6.

[0058] The present invention contemplates administering a rAAV vector to a subject diagnosed with DMD before fibrosis is observed in the subject, or before a decrease in muscle strength, or before a decrease in muscle mass.

[0059] 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 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 to subjects with muscular dystrophy who have developed fibrosis to prevent new fibrosis in these subjects or reduce fibrosis in these subjects. The present invention also provides a method of 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 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 to a subject with muscular dystrophy whose muscle strength has been reduced or whose muscle mass has been reduced to protect the muscle from further damage.

[0060] In any of the methods of the invention, the subject may be suffering from a muscular dystrophy such as DMD or any dystrophin-related muscular dystrophy thereof.

[0061] In other embodiments of any of the methods of the invention described herein, the subject's serum CK level after administration of rAAV is reduced to a percentage level selected from the group consisting of:

[0062] a) at least 78% at 90, 180 or 270 days after administration;

[0063] 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;

[0064] c) at least 72%, 73%, 74% or 95% at 180 days after administration;

[0065] d) at least 87%, 88%, 93% or 95% at 90 days after administration;

[0066] e) at least 70% at 270 days after administration;

[0067] f) 70% to 95% at 90, 180 or 270 days after administration;

[0068] 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

[0069] 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.

[0070] 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 adenovirus (rAAV)-associated rAAV.MHCK7.micro-dystrophin, 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.micro-dystrophin. In one embodiment, AAVrh74.MHCK7.micro-dystrophin is 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, AAVrh74.MCK.micro-dystrophin is AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0071] 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 vg / kg, or about 1.75×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.75×10 14 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 14vg / kg dose of rAAV. In one embodiment, the rAAV is AAVrh74.MHCK7. micro-dystrophin. In one embodiment, AAVrh74.MHCK7. micro-dystrophin is 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, AAVrh74.MCK. micro-dystrophin is AAVrh74.MCK. micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0072] In one embodiment, the composition of the invention is formulated for intravenous administration and comprises about 2.0×10 14 In another embodiment, the composition of the invention is formulated for intravenous administration and comprises 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 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 In one embodiment, the rAAV is AAVrh74.MHCK7. micro-dystrophin. In one embodiment, AAVrh74.MHCK7. micro-dystrophin is 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 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.

[0073] In any of the compositions of the invention, the dose of rAAV is delivered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In a specific embodiment, the composition comprises a dose of rAAV delivered at about 10 mL / kg. In one embodiment, rAAV is AAVrh74.MHCK7. micro-dystrophin. In one embodiment, AAVrh74.MHCK7. micro-dystrophin is 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 another embodiment, the rAAV is AAVrh74.MCK.micro-dystrophin.In one embodiment, the AAVrh74.MCK.micro-dystrophin is AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0074] 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. In addition, the compositions of the present invention are 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 about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0075] Any of the compositions of the present invention comprises rAAV or an rAAV vector, wherein the rAAV comprises the human micro-dystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7, and 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.

[0076] Specifically, the composition of the present invention is used to treat Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present invention provides a composition for treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of recombinant adenovirus (rAAV)-related rAAV.MHCK7.micro-dystrophin, wherein the composition is formulated for administration by intravenous infusion over about one hour, and the dose of rAAV administered is about 2×10 14 vg / kg, and 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.

[0077] 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 rAAV vector for preventing fibrosis in a subject suffering from muscular dystrophy.

[0078] The present invention also provides a composition comprising rAAV for use in increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy. In another embodiment, the present invention provides a composition comprising any of the rAAVs of the present invention for use in treating muscular dystrophy.

[0079] In other embodiments of any of the compositions of the invention, after administration of the composition to a human subject in need of treatment for muscular dystrophy, the subject's serum CK level is reduced to a percentage level selected from the group consisting of:

[0080] a) at least 78% at 90, 180 or 270 days after administration;

[0081] b) at least 46%, 55%, 70% or 85% at 270 days after administration;

[0082] c) at least 72%, 73%, 74% or 95% at 180 days after administration;

[0083] d) at least 87%, 99%, 93% or 95% at 90 days after administration;

[0084] e) at least 70% at 270 days after administration;

[0085] f) 70% to 95% at 90, 180 or 270 days after administration;

[0086] 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

[0087] 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.

[0088] In another embodiment, the present invention provides a use of a dose of a recombinant adenovirus (rAAV)-associated rAAV.MHCK7.micro-dystrophin for the preparation of a medicament for treating muscular 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 is about 4×101 4 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, AAVrh74.MHCK7.micro-dystrophin is 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, AAVrh74.MCK.micro-dystrophin is AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0089] For example, a drug contains approximately 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 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 vg / kg, or about 1.75×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 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 dose of rAAV. In one embodiment, the rAAV is AAVrh74.MHCK7. micro-dystrophin. In one embodiment, AAVrh74.MHCK7. micro-dystrophin is 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, AAVrh74.MCK. micro-dystrophin is AAVrh74.MCK. micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0090] 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 an intravenous route and the dose of rAAV administered is about 2.0×10 14 In another embodiment, the medicament of the present invention is formulated for systemic administration of a dose of rAAV, wherein the systemic administration route is an 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.micro-dystrophin. In one embodiment, AAVrh74.MHCK7.micro-dystrophin is 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, AAVrh74.MCK.micro-dystrophin is AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0091] In any of the uses of the invention, the medicament comprises a dose of rAAV of about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In a specific embodiment, the dose or rAAV is about 10 mL / kg. In one embodiment, rAAV is AAVrh74.MHCK7. micro-dystrophin. In one embodiment, AAVrh74.MHCK7. micro-dystrophin is 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 AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0092] In any of the uses of the present invention, the drug is formulated for administration by injection, injection or transplantation. For example, the drug is formulated for administration by infusion over about one hour. In addition, 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 about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0093] In any of the uses of the present invention, the drug comprises rAAV, wherein the rAAV comprises the human micro-dystrophin 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 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.

[0094] A particular use of the present invention is the preparation of a medicament for treating Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present invention provides a use of a dose of a recombinant adenovirus (rAAV)-associated rAAV.MHCK7.micro-dystrophin for the preparation of a medicament for treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the medicament is formulated for administration by intravenous infusion over about one hour, and the dose of the rAAV administered is about 2×10 14 vg / kg, and 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.

[0095] In another embodiment, the invention provides the use of rAAV for the preparation of a medicament for reducing fibrosis in a subject in need thereof. For example, the subject in need thereof may suffer from muscular dystrophy (such as DMD) or any other dystrophin-related muscular dystrophy.

[0096] In another embodiment, the invention provides the use of rAAV for the preparation of a medicament for preventing fibrosis in a subject suffering from muscular dystrophy.

[0097] Furthermore, the present invention provides the use of rAAV for the preparation of a medicament for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy.

[0098] The present invention also provides the use of rAAV for preparing a drug for treating muscular dystrophy.

[0099] The present invention provides the use of rAAV vectors for preparing drugs for treating muscular dystrophy: the rAAV vectors contain 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 rAAV vectors for treating muscular dystrophy: the rAAV vectors contain 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.

[0100] In other embodiments of any of the uses of the invention, after rAAV is administered to the subject, the serum CK level of the subject is reduced to a percentage level selected from the group consisting of:

[0101] a) at least 78% at 90, 180 or 270 days after administration;

[0102] b) at least 46%, 55%, 70% or 95% at 270 days after administration;

[0103] c) at least 72%, 73%, 74% or 95% at 180 days after administration;

[0104] d) at least 87%, 88%, 93% or 95% at 90 days after administration;

[0105] e) at least 70% at 270 days after administration;

[0106] f) 70% to 95% at 90, 180 or 270 days after administration;

[0107] 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

[0108] 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.

[0109] In any of the uses of a composition for treating muscular dystrophy or a drug for treating muscular dystrophy, after administration of the composition or drug, the level of micro-dystrophin gene expression in cells of the subject increases. Before and after administration of the composition or drug, the level of micro-dystrophin is measured by Western blot in biopsied muscle to detect the expression of the micro-dystrophin gene in cells. Specifically, after administration of the composition or drug, 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%, compared to the level of micro-dystrophin before administration of the composition or drug. For example, 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%, compared to the level of micro-dystrophin before administration of the composition or medicament.

[0110] In addition, the expression of the micro-dystrophin gene in cells is detected by measuring the level of micro-dystrophin by immunohistochemistry in muscle biopsies before and after administration of the composition or medicament. Compared to the level of micro-dystrophin before administration of the composition or medicament, the level of micro-dystrophin after administration of rAAV 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, 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% compared to the level of micro-dystrophin before administration of the composition or medicament.

[0111] In any of the compositions for treating muscular dystrophy, the subject's serum CK level is reduced after administration of the rAAV compared to the serum CK level before administration of the composition or medicament. For example, the subject's serum CK level is reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament. Specifically, in any of the compositions for treating muscular dystrophy of the present invention, the serum CK level of the subject is reduced by about 87% 60 days after administration of the composition or drug, compared with the serum CK level before administration of the composition or 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, the serum CK level of the subject is reduced by about 72% 60 days after administration of the composition or drug, or in any of the compositions for treating muscular dystrophy of the present invention, the serum CK level of the subject is reduced by about 10% compared with the serum CK level before administration of the composition or drug. In any of the uses of the composition for treating muscular dystrophy or the medicament for treating muscular dystrophy of the present invention, the serum CK level of the subject is reduced by about 73% compared to the serum CK level before administration of the composition, or in any of the uses of the composition for treating muscular dystrophy or the medicament for treating muscular dystrophy of the present invention, the serum CK level of the subject is reduced by about 78% 60 days after administration of the composition or the medicament compared to the serum CK level before administration of the composition, or in any of the uses of the composition for treating muscular dystrophy or the medicament for treating muscular dystrophy of the present invention, the serum CK level of the subject is reduced by about 95% 60 days after administration of the composition or the medicament compared to the serum CK level before administration of the composition or the medicament. In any of the uses of the composition for treating muscular dystrophy or the medicament for treating muscular dystrophy, the number of micro-dystrophin-positive fibers in the muscle tissue of the subject increases after administration of the composition or the medicament compared to the number of micro-dystrophin-positive fibers before administration of the composition or the medicament. For example, micro-dystrophin levels are measured by Western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or medicament to detect the number of micro-dystrophin-positive fibers.

[0112] In any of the uses of a composition for treating muscular dystrophy or a drug for treating muscular dystrophy, the administration of the composition or drug upregulates the expression of a DAPC protein, such as α-sarcoglycan or β-sarcoglycan. For example, the level of α-sarcoglycan in the subject is increased after the administration of the composition or drug compared to the level of α-sarcoglycan before the administration of the composition or drug. In addition, the level of β-sarcoglycan in the subject is increased after the administration of the composition or drug compared to the level of β-sarcoglycan before the administration of the composition or drug. Before and after the administration of the composition or drug, the level of α-sarcoglycan or β-sarcoglycan protein is measured by Western blotting or immunohistochemistry for muscle biopsy to detect the level of α-sarcoglycan or β-sarcoglycan.

[0113] 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 subject's disease progression is delayed as measured by any of the following tests: six-minute walk test, stand time, ascending 4 stairs, ascending and descending 4 stairs, 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.

[0114] For example, after administration of any of the compositions for treating muscular dystrophy or the uses of the drug for treating muscular dystrophy, the NSAA score of the subject has an improvement of at least 6 points compared to the NSAA score before the administration of rAAV, at least 270 days after the administration of the composition or drug. In addition, in any method, the stand-up time of the subject has an improvement of at least about 0.8 seconds compared to the stand-up time before the administration of the composition or drug, at least 270 days after the administration of the composition or drug. In addition, in any of the methods or uses of the present invention, the test of the time to rise 4 stairs of the subject has an improvement of at least about 1.2 seconds compared to the time test to rise 4 stairs before the administration of the composition or drug, at least 270 days after the administration of the composition or drug. In addition, in any of the methods or uses of the present invention, the 100m timing test of the subject has an improvement of at least about 7 seconds compared to the 100m timing test before the administration of the composition or drug, at least 270 days after the administration of the composition or drug.

[0115] In another embodiment, the present invention provides a composition for expressing a micro-dystrophin gene in a patient's cells, the composition comprising the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In another embodiment, the present invention 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 to prepare a medicament for expressing a 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, thereby detecting the expression of the micro-dystrophin gene in the patient's cells. In addition, the expression of the micro-dystrophin gene in the patient is measured by detecting more vector genomes per nucleus, wherein 1 vector genome per nucleus is about 50% micro-dystrophin expression, and greater than 1 copy per nucleus is consistent with the expression level of micro-dystrophin. For example, the cell has 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0116] In another embodiment, the present invention provides a composition for reducing serum CK levels 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. In addition, the present invention 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 to prepare a medicament for reducing serum CK levels in a patient in need thereof. For example, serum CK levels in the patient are 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% 60 days after administration of the composition or medicament, compared to serum CK levels before administration of the composition or medicament. Specifically, the serum CK level of the subject is reduced by about 87% 60 days after administration of the composition or drug, compared with the serum CK level before administration of the composition or drug, or reduced by about 72% 60 days after administration of the composition or drug, compared with the serum CK level before administration of the composition or drug, or reduced by about 73% 60 days after administration of the composition or drug, compared with the serum CK level before administration of the composition or drug, or reduced by about 78% 60 days after administration of the composition or drug, compared with the serum CK level before administration of the composition or drug, or reduced by about 95% 60 days after administration of the composition or drug, compared with the serum CK level before administration of the composition or drug.

[0117] The present invention also provides a composition for increasing micro-dystrophin-positive fibers in the patient's muscle tissue, the composition comprising the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In addition, the present invention 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 to prepare a drug for increasing micro-dystrophin-positive fibers in the patient's muscle tissue. For example, before and after administration of the composition or drug, the level of dystrophin is measured by Western blot or immunohistochemistry on 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 a greater number of vector genomes per nucleus, wherein 1 vector genome per nucleus is approximately 50% expression of micro-dystrophin, and greater than 1 copy per nucleus is consistent with micro-dystrophin expression levels. For example, 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.

[0118] 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 to prepare 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 level of α-sarcoglycan protein by Western blot or immunohistochemistry on muscle biopsy before and after administration of the composition or medicament.

[0119] 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 to prepare 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 level of β-sarcoglycan protein by Western blot or immunohistochemistry on muscle biopsy before and after administration of the composition or medicament.

[0120] The present invention also provides a dose of the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, such that administration of the medicament causes a delay in disease progression in the patient, as measured by any of the following tests: six-minute walk test, stand time, ascending 4 stairs, ascending and descending 4 stairs, North Star Ambulatory Assessment Scale (NSAA), 10-meter timed test, 100-meter timed test, hand-held dynamometer (HHD), timed up-and-go and / or gross motor subtest scale (Bayley-III) score.

[0121] For example, compared to the NSAA score before the application of the composition or medicine, at least 270 days after the application of the composition or medicine, the NSAA score of the subject has an improvement of at least 6 points. In addition, compared to the stand-up time before the application of the composition or medicine, at least 270 days after the application of the composition or medicine, the stand-up time of the subject has an improvement of at least about 0.8 seconds. In addition, compared to the time test of rising 4 ladders before the application of the composition or medicine, at least 270 days after the application of the composition or medicine, the test of the time of rising 4 ladders of the subject has an improvement of at least about 1.2 seconds. In addition, compared to the 100m timing test before the application of the composition or medicine, at least 270 days after the application of the composition or medicine, the 100m timing test of the subject has an improvement of at least about 7 seconds. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1The rAAV.MHCK7.micro-dystrophin construct is presented. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). The construct is characterized by the deletion of the in-frame rod domain (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 micro-dystrophin (3579 bp) is directed by the MHCK7 promoter (795 bp). Introns and 5'UTR are derived from the plasmid pCMVβ (Clontech). The micro-dystrophin cassette has a consensus sequence Kozak before the ATG start codon and a small 53 bp synthetic poly A 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)).

[0123] Figure 2 The nucleic acid sequence of AAVrh74.MHCK7.micro-dystrophin is provided (SEQ ID NO: 3).

[0124] Figure 3 A map of the pNLREP2-Caprh74 AAV helper plasmid is provided.

[0125] Figure 4 The Ad helper plasmid pHELP is provided.

[0126] Figure 5 rAAV.MCK.micro-dystrophin plasmid constructs are shown.

[0127] Figure 6 The nucleic acid sequence of rAAVrh74.MCK.micro-dystrophin is provided (SEQ ID NO: 5).

[0128] Figure 7 Shown is micro-dystrophin gene expression in myofiber biopsies of gastrocnemius muscle measured by immunocytochemistry.

[0129] Figures 8A-8C A Western blot showing expression of micro-dystrophin at the correct molecular weight is provided. Figure 8C In the analysis, the sample from subject 4 (*) was diluted 1:4 (to the linear range) because the ULDQ (>80%) was exceeded in the initial analysis, and the mean value was multiplied by the dilution correction factor of the final value relative to the normal value. The mean micro-dystrophin expression relative to the normal value was 182.7% in method 1 and 222.0% in method 2.

[0130] Figures 9A-9C It was demonstrated that administration of rAAVrh74.MHCK7.micro-dystrophin upregulated the expression of DAPC protein, α-sarcoglycan, and β-sarcoglycan.

[0131] Fig.10 A sustained and significant reduction in creatine kinase (CK) was demonstrated with administration of rAAVrh74.MHCK7.micro-dystrophin.

[0132] Figure 11 provides the mean CK change from baseline to day 270. The data demonstrate that following administration of rAAVrh74.MHCK7.micro-dystrophin, CK was significantly reduced over time.

[0133] Fig.12 Mean NSAA change and mean CK change from baseline to day 270 are presented. The data demonstrate that NSAA increased significantly over time following administration of rAAVrh74.MHCK7.micro-dystrophin.

[0134] Fig.13 The nucleic acid sequence of AAVrh74.MHCK7.micro-dystrophin is provided (SEQ ID NO:9).

[0135] Fig.14 AAVrh74.MHCK7.micro-dystrophin plasmid constructs are shown.

[0136] Fig.15 The nucleic acid sequence of an AAVrh74.MHCK7.microdystrophin plasmid construct comprising a kanamycin resistance gene is provided (SEQ ID NO: 8). DETAILED DESCRIPTION

[0137] The present invention provides gene therapy vectors (e.g., rAAV vectors) that overexpress human microdystrophin and methods for reducing and preventing fibrosis in patients with muscular dystrophy. Muscle biopsy samples collected early in the diagnosis of DMD show significant connective tissue hyperplasia. Muscle fibrosis has harmful effects in many ways. It reduces the normal transport of endomysial nutrients through connective tissue barriers, reduces blood flow, deprives muscles of vascular-derived nutrients, and functionally causes early loss of walking ability due to limb contracture. Over time, due to obvious muscle fibrosis, treatment challenges are increasing. This can be observed in muscle biopsies comparing connective tissue hyperplasia at consecutive time points. The process continues to intensify, leading to loss of walking ability and accelerated loss of control, especially in wheelchair-dependent patients.

[0138] Without early treatment (including parallel approaches to reduce fibrosis), it is impossible to fully achieve the beneficial effects of exon skipping, stop codon read-through, or gene replacement therapy. Even small molecule or protein replacement strategies may fail if methods to reduce muscle fibrosis are not performed. Previous studies on elderly mdx mice that have been fibrotic and treated with AAV. microdystrophin showed that we could not achieve complete 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 scarring and fibrosis of the ventricular wall.

[0139] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells where certain functions are provided by co-infection helper viruses. Thirteen AAV serotypes have been identified. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is entirely expected that these same principles will also apply to other AAV serotypes, because the following fact is well known: even at the genetic level, various serotypes are very closely related in structure and function. (See, for example, Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165-174, JRPattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further indicated by heteroduplex analysis, which shows extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that replication functions in each serotype are under similar regulatory control.

[0140] As used herein, "AAV vector" refers to a vector comprising one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). When such AAV vectors are present in host cells that have been transfected with vectors encoding and expressing rep and cap gene products, they can be replicated and packaged into infectious viral particles.

[0141] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle composed of at least one AAV capsid protein and a polynucleotide AAV vector encapsidated 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 a mammalian cell), the particle is generally referred to as an "AAV vector particle" or simply "AAV vector". Therefore, the production of an AAV vector particle necessarily includes the production of an AAV vector, because such a vector is contained within the AAV vector particle.

[0142] AAV

[0143] Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of about 4.7 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). AAV has multiple serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is provided in Srivastava et al., J Virol, 45:555-564 (1983), and 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 No. NC_002077; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the complete genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least a portion of the genomes of AAV-7 and AAV-8 are provided in GenBank Accession No. NC_001862, respectively. 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 chromosomal integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), in conjunction with differential splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), produce four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The Rep proteins have multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3.Alternative splicing and non-consensus translation initiation sites are responsible for the production of three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0144] AAV has unique characteristics that make it an attractive vector for delivering exogenous 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, which may target many different tissues in vivo. In addition, AAV transduces slowly dividing and non-dividing cells, and can persist in the life cycle of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, which makes the construction of a recombinant genome possible. In addition, due to the signal that directs AAV replication, the genome capsid is encapsulated and integrated and is contained in the ITR of the AAV genome, so part or all of the internal approximately 4.3kb genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with exogenous DNA, such as a gene cassette containing a promoter, DNA of interest, and polyadenylation signals. Rep and cap proteins can be provided in trans. Another notable feature of AAV is that it is an extremely stable and strong virus. It can easily withstand the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV no longer a critical factor. AAV can even be lyophilized. Finally, AAV-infected cells are not tolerant to superinfection.

[0145] Several studies have shown that recombinant AAV-mediated proteins can be expressed in muscle for long periods of 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). Furthermore, since muscle is highly vascularized, recombinant AAV transduction results in the appearance of transgene products in the systemic circulation following intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers have cytokines necessary for correct antibody glycosylation, folding, and secretion, suggesting that muscle can stably express secreted protein therapeutics.

[0146] 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 rAAVs is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22 (11): 1900-1909 (2014). As described in the background technology 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.

[0147] The DNA plasmid of the present invention comprises the rAAV genome of the present invention. The DNA plasmid is transferred to cells that allow the helper virus (e.g., adenovirus, adenovirus or herpes virus deleted by AAV) to be infected, so that the rAAV genome is assembled into infectious viral particles. The technology for producing rAAV particles is a standard technology in the art, in which the AAV genome, rep and cap genes and helper virus functional elements to be packaged are provided to cells. The production of rAAV requires providing the following components in a single cell (referred to herein as packaging cell): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome) and the helper virus functional elements. The AAV rep and cap genes can be from any AAV serotype from which recombinant virus can be derived, and can be from an AAV serotype different from the rAAV genome ITRs, 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. The generation of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0148] The method for producing packaging cells is to produce a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, an AAV rep and cap gene separated from the rAAV genome, and a selective marker (such as a 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 GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79: 2077-2081), adding a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23: 65-73), or by direct blunt end connection (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 cell is selectable and suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or the rep and cap genes into packaging cells.

[0149] The general principles of rAAV production are reviewed in, for example, 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., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al., J. Virol., 63:3822-3828 (1989); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. 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. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the documents relating to rAAV production.

[0150] Therefore, 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 (homologous 293 cell lines). In another embodiment, the packaging cells are untransformed cancer cells, such as low-passage 293 cells (human embryonic kidney cells transformed with E1 of adenovirus), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus embryonic lung cells).

[0151] The recombinant AAV of the present invention (i.e., the rAAV particles wrapped by the infectious capsid) comprises the rAAV genome. In an exemplary embodiment, the genomes of the two rAAVs are both lacking AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAVs that can be constructed to comprise nucleic acid molecules 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.

[0152] 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 AAV vector plasmids rAAV.MHCK7. micro-dystrophin, pNLRep2-Caprh74 and pHelp, rAAV containing a micro-dystrophin gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs). This sequence is encapsidated into AAVrh74 virus particles. The plasmid contains the micro-dystrophin sequence and the MHCK7 enhancer and core promoter elements of the muscle-specific promoter that drives 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 is used for efficient transcription termination.

[0153] pNLREP2-Caprh74 is an AAV helper plasmid that encodes four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from serotype rh74. A schematic diagram of the pNLREP2-Caprh74 plasmid is shown in Figure 3 shown.

[0154] The pHELP adenoviral helper plasmid is 11,635 bp and was obtained from Applied Viromics. The plasmid contains regions of the adenoviral genome that are important for AAV replication, namely E2A, E4ORF6, and VA RNA (the adenoviral E1 functional elements are provided by 293 cells). The adenoviral sequences present in this plasmid represent only ~40% of the adenoviral genome and do not contain replication-critical cis elements, such as adenoviral terminal repeats. Therefore, infectious adenovirus is not expected to be produced from this production system. A schematic diagram of the pHELP plasmid is shown in Figure 2. Figure 4 shown.

[0155] rAAV can be purified by methods that are standard in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Pat. No. 6,566,118 and methods disclosed in WO 98 / 09657.

[0156] 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).

[0157] The titer of the rAAV to be administered in the methods of the invention will vary depending on, for example, the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type or cells being targeted, and can be determined by methods that are standard in the art. The titer of the rAAV can range from about 1×10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 About 1×10 14or more DNase-resistant particles (DRPs). The dose can also be expressed in units of viral genomes (vg). An exemplary method for determining the titer of capsid-encapsulated vector genomes uses quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25(4): 855-869, 2017).

[0158] The present invention contemplates methods for transducing target cells with rAAV in vivo or in vitro. The in vivo method includes the step of administering an effective dose or multiple effective doses of a composition comprising rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the development of the disorder / disease, the administration is preventive. If the dose is administered after the development of the disorder / disease, the administration is therapeutic. In an embodiment 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 a disorder / disease state, slows or prevents the progression of a disorder / disease state, reduces the extent of the disease, causes alleviation of the disease (partial or complete), and / or prolongs survival. An example of a disease that is contemplated for prevention or treatment using the methods of the present invention is DMD.

[0159] The present invention also contemplates combination therapy. Combination as used herein includes both simultaneous and sequential treatment. Combinations of the methods of the present invention with standard drug treatments (e.g., corticosteroids) are specifically contemplated, as are combinations with novel therapies.

[0160] Administration of an effective dose of the composition can be performed by routes that are standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal or vaginal routes. One skilled in the art can select and / or match one or more routes of administration and one or more serotypes of the AAV components (especially, 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 / tissue(s) to be expressed in the micro-dystrophin protein.

[0161] The present invention provides local and systemic administration of effective doses of rAAV and compositions of the present invention. For example, systemic administration is administration to the circulatory system, thereby affecting the body as a whole. Systemic administration includes enteral administration (such as absorption through the gastrointestinal tract) and parenteral administration (by injection, infusion or transplantation).

[0162] Specifically, the actual application of the rAAV of the present invention can be accomplished by using any physical method for transporting the rAAV recombinant vector to the target tissue of the animal. The application according to the present invention includes, but is not limited to, injection into the muscle and injection into the bloodstream. The rAAV is simply resuspended in phosphate-buffered saline to show a medium sufficient to provide expression in muscle tissue, and there are no known restrictions on the carrier or other components that can be co-administered with the rAAV (although the composition that degrades 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 preparation or a topical preparation delivered to the muscle by transdermal transport. Many preparations for intramuscular injection and transdermal transport have been developed previously, and these preparations can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and treatment.

[0163] In one embodiment of the present invention, the AAVrh74.MHCK7.micro-dystrophin 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.

[0164] 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 therapeutic goal, the individual, and the cell type or cells being targeted, and can be determined by methods that are standard in the art. The titer of each rAAV administered can range from about 1×10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 , about 2×10 14 or about 1×10 15 or more DNase-resistant particles (DRP). Doses can also be expressed in units 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). Doses can also be expressed in units 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).

[0165] Specifically, the actual application of the rAAV of the present invention can be accomplished by using any physical method for transporting the rAAV recombinant vector to the target tissue of the animal. The application according to the present invention includes, but is not limited to, injection into the muscle and injection into the bloodstream. The rAAV is simply resuspended in phosphate-buffered saline to show a medium sufficient to provide expression in muscle tissue, and there are no known restrictions on the carrier or other components that can be co-administered with the rAAV (although the composition that degrades 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 preparation or a topical preparation delivered to the muscle by transdermal transport. Many preparations for intramuscular injection and transdermal transport have been developed previously, and these preparations can be used in the practice of the present invention. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and treatment.

[0166] For intramuscular injection, a solution dissolved in an adjuvant (such as sesame oil or peanut oil) or in a propylene glycol aqueous solution, and a sterile aqueous solution can be used. If necessary, such an aqueous solution can be a buffer solution, and the liquid diluent is first isotonic treated with saline or glucose. A solution of rAAV as a free acid (DNA contains an acidic phosphate group) or a pharmacologically acceptable salt can be prepared by appropriately mixing with a surfactant (such as hydroxypropyl cellulose) in water. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, the sterile aqueous medium used can be easily obtained by standard techniques well known to those skilled in the art.

[0167] The pharmaceutical carrier, diluent or excipient suitable for injection purposes includes sterile aqueous solution or dispersion and sterile powder for the temporary preparation of sterile injection solution or dispersion. In all cases, the form must be sterile and must have fluidity that is easy to inject. It must be stable under manufacturing and storage conditions and must be able to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), their suitable mixtures and vegetable oils. For example, by using a coating (such as lecithin), by maintaining the required particle size for dispersions, and by using a surfactant to maintain appropriate fluidity. The prevention of microbial action can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferred to include isotonic agents (such as sugar or sodium chloride). The extended absorption of the composition for injection can be achieved by using agents (such as aluminum monostearate and gelatin) that delay absorption.

[0168] Sterile injectable solutions are prepared by incorporating the desired amount of rAAV into an appropriate solvent with the various other ingredients listed above, followed by filter sterilization as required. Dispersions are generally prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other components listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which produce powders of the active ingredient and any additional desired ingredients from previously sterile filtered solutions.

[0169] Transduction using rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, allogeneic or xenogeneic muscle cells can be used that do not produce an inappropriate immune response in the subject.

[0170] Suitable methods for transducing and re-transducing transduced cells into a subject are known in the art. In one embodiment, the cells can be transduced in vitro by combining rAAV with muscle cells, for example in an appropriate culture medium, and screening for those cells with the DNA of interest using conventional techniques (such as Southern blotting and / or PCR) or by using a selective marker. The transduced cells can then be formulated into a pharmaceutical composition and introduced into a subject by various techniques (such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection) or by using, for example, catheter injection into smooth muscle and myocardium.

[0171] The result of transduction of cells using the rAAV of the present invention is the sustained expression of micro-dystrophin. Therefore, the present invention provides a method of administering / delivering rAAV expressing micro-dystrophin to an animal (preferably a human). These methods include using one or more rAAVs of the present invention to transduce tissues (including but not limited to tissues (such as muscles), organs (such as liver and brain) and glands (such as salivary glands)). Transduction can be performed 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 tissues directed by muscle-specific control elements, 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)), derived from the human skeletal muscle actin gene (Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)), the cardiac actin gene, the muscle creatine kinase sequence element (see Johnson et al., Mol Cell Biol, 11:4090-4091 (1987)), 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)), derived from the human skeletal muscle actin gene (Muscat et al., Mol Cell Biol, 11:4090-4091 (1987)), the cardiac actin gene, the muscle creatine kinase sequence element (see Johnson et al., Mol Cell Biol, 11:4090-4091 (1987)), 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, 11:4090-4091 (1987)), the muscle cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson Biol, 9:3393-3399 (1989)) and the murine creatine kinase enhancer (mCK) element, the control elements derived from the skeletal muscle fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc Natl Acad Sci USA, 88:5680-5684 (1991)), steroid-inducible elements and promoters (including the glucocorticoid response element (GRE)) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)) and other control elements.

[0172] 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 sustained expression of micro-dystrophin from transduced myofibers.

[0173] By "muscle cell" or "muscle tissue" is meant a cell or cell population derived from any type of muscle (e.g., skeletal muscle and smooth muscle, such as from the digestive tract, bladder, blood vessels, or heart tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyocytes.

[0174] The term "transduction" is used to refer to the in vivo or in vitro administration / delivery of the coding region of micro-dystrophin to recipient cells, which results in the expression of micro-dystrophin by the recipient cells via the replication-defective rAAV of the present invention.

[0175] Thus, the present invention provides methods for administering an effective dose (or doses administered substantially simultaneously, or doses administered at intervals) of a rAAV encoding micro-dystrophin to a subject in need thereof.

[0176] The following examples are provided by way of illustration and not limitation.The numerical ranges described include each integer value within each range, including the minimum and maximum stated integer values.

[0177] Example

[0178] Example 1

[0179] A) Generation of AAVrh74.MHCK7.micro-dystrophin constructs

[0180] The AAVrh74.MHCK7.microdystrophin plasmid contains the human microdystrophin 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 hinges 1, 2 and 4 and the cysteine-rich domain can still produce a 138kDa protein. The expression of micro-dystrophin (3579bp) is directed by the MHCK7 promoter (792bp). The plasmid is constructed by removing the MCK promoter from the rAAV.MCK.micro-dystrophin plasmid and inserting the MHCK7 promoter. After the core promoter, a 53bp endogenous mouse MCK exon 1 (untranslated) is provided for efficient transcription initiation, followed by the SV40 late 16S / 19S splicing signal (150bp) and a small 5'UTR (61bp). The intron and 5'UTR are derived from the plasmid pCMVβ (Clontech). The micro-dystrophin cassette has a consensus sequence Kozak before the ATG start codon and a small 53bp synthetic poly A signal for mRNA termination. The human micro-dystrophin cassette contains the (R4-R23 / Δ71-78) domains 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 repeats of AAV2, which are required for viral DNA replication and packaging. The micro-dystrophin cassette has a small 53 bp synthetic poly A signal for mRNA termination.

[0181] Previous studies have demonstrated cardiac expression using the MHCK7 promoter (Salva et al. Mol Ther 15, 320-329 (2007)) and AAVrh74 achieved skeletal muscle, diaphragm muscle, and cardiac muscle expression (Sondergaard et al. Annals of Clinical and Transl Neurology 2, 256-270 (2015)). Figure 1 The sequences of the constructs are encapsidated into AAVrh.74 virions. The molecular clone of the AAVrh.74 serotype was cloned from rhesus monkey lymph nodes and discussed in Rodino-Klapac et al. Journal of Translational medicine 5, 45 (2007).

[0182] Table 1 shows the molecular characteristics of plasmid AAVrh74.MHCK7.microdystrophin (SEQ ID NO: 3)

[0183]

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

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

[0186] Example 2

[0187] A clinical trial of systemic gene delivery in Duchenne muscular dystrophy

[0188] This is a single-dose controlled trial of rAAVrh74.MHCK7. micro-dystrophin, 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 intravenous micro-dystrophin vector (2×10 14 rAAVrh74.MHCK7.micro-dystrophin was prepared 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.micro-dystrophin was infused via a peripheral arm vein, which can reach all muscles in the body. Six DMD subjects aged 3 months to 3 years were included in Cohort A, and six DMD subjects aged 4 to 7 years were included in Cohort B. All subjects received intravenous micro-dystrophin vector (2×10 14 vg / kg, 10 mL / kg) injection. By quantitative PCR, using the Prism 7500 Taqman detector system (PE Applied Biosystems), primers for the MHCK7 promoter were used to determine the titer of the vector genome encapsidated for the administered dose compared to supercoiled DNA plasmid standards (Pozsgai et al. Mol. Ther. 25 (4): 855-869, 2017).

[0190] Subjects receive the infusion over 1 hour in the Pediatric Intensive Care Unit (PICU) at Nationwide Children's Hospital. A muscle biopsy is performed at the screening visit before gene therapy. Subjects will have a second muscle biopsy to determine if the gene allows for replacement of the missing dystrophin protein 90 days after delivery. After the gene transfer, patients will be carefully monitored for any side effects of treatment. This monitoring includes blood and urine tests, as well as physical exams during the screening visit and at days 0, 1, 7, 14, 30, 60, 90, and 180, and at months 9, 12, 18, 24, 30, and 36 to ensure there are no side effects from the gene injection.

[0191] Cohort A subjects (n=6) were aged between 3 months and 3 years and received intravenous rAAVrh74.MHCK7.micro-dystrophin vector (2×10 14 vg / kg, 10mL / kg) injection. One day before gene transfer, subjects in cohort A started injections of 1mg / kg prednisone or deflazacort and maintained for 30 days while monitoring immune responses. If negative on day 30, steroids were discontinued within 1 week. If the T cell response to AAV or micro-dystrophin was >125SFC / 106PBMC, steroids were maintained until the level dropped below this threshold.

[0192] Cohort B subjects (n = 6) were aged 4 to 7 years and received intravenous rAAVrh74.MHCK7.micro-dystrophin vector (2 × 10 14 These subjects were maintained on a stable dose of corticosteroids throughout the trial, but the dose could be increased on a short-term basis if the T cell response to AAV or micro-dystrophin was >125 SFC / 106 PBMC.

[0193] Eligibility criteria

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

[0195] Age of inclusion: Cohort A: 3 months to 7 years old, Cohort B: 4 years to 7 years old (inclusive).

[0196] Molecular characterization of DMD genes with frameshift (deletion or duplication) or premature stop codon mutations between exons 18 to 58.

[0197] CK increased > 1000 U / L

[0198] Cohort A subjects: less than average on the Bayley-III Motor Assessment for gross motor activity defined as a scale score ≤ 9

[0199] Cohort B: Below average in the 100m timed test is defined as < 80% of predicted

[0200] Males of any race.

[0201] ·Ability to collaborate with movement assessment testing.

[0202] Cohort A subjects: no prior corticosteroid treatment.

[0203] Cohort B subjects: Stable dose equivalent of oral corticosteroids for at least 12 weeks prior to screening, and the dose is expected to remain constant throughout the study (except for adjustments to accommodate changes in body weight).

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

[0205] Active viral infection based on clinical observation.

[0206] Signs of cardiomyopathy, including an ejection fraction less than 40% on echocardiography.

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

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

[0209] Abnormal laboratory values ​​were considered clinically significant.

[0210] The PI believes that concomitant illness or the need for chronic medication would present an unnecessary risk to gene transfer.

[0211] Subjects with AAVrh74 or AAV8 antibody titers >1:400 as determined by ELISA immunoassay.

[0212] The medical condition or mitigating circumstances, in the opinion of the investigator, could impair the subject's ability to comply with tests or procedures required by the protocol or could otherwise compromise the subject's health, safety, or clinical interpretability.

[0213] Serious infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (enrollment may be delayed).

[0214] Receipt of any study medication (except corticosteroids) or exon skipping drugs (including ) or experimental drugs.

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

[0216] The family does not want the patient's research participation disclosed to primary care physicians and other medical providers.

[0217] Outcome Measures

[0218] The primary outcome measure was safety based on the number of participants experiencing adverse events (time frame: 3 years). Adverse events were monitored and scored for severity and relevance to the study items.

[0219] The secondary outcome measures are as follows:

[0220] Gross Motor Subtest Scale (Bayley-III) Scores (Time Frame: Screening, Day 30-3 Years): Gross Motor Scale Scores measure motor development. Cohort A was scored on the Bayley-III Gross Motor Subtest at each visit from Day 30 to 3 years. At Screening, any subject aged 43-47 months (inclusive) had a calculated scale score compared to normative data for children aged 42 months. The Bayley-III provides normative data for children aged 1-42 months.

[0221] Physical Therapy Assessment 100-meter timed test (100m) (Time Frame: Screening, Day 30-3 years): 100m was the primary motor outcome for Cohort B. The 100-meter timed test was the initial exploratory outcome for Cohort A when children were 3 years old.

[0222] Physical Therapy Assessments North Star Ambulatory Assessment Scale (NSAA) (Time Frame: Screening, Day 30-3 Years): The North Star Ambulatory Assessment Scale (NSAA) was an initial exploratory outcome at age four for Cohort A and at age four for Cohort B. The NSAA measures the quality of walking in 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 included the Timed Up and Go (TUG) Modified for Children.

[0224] Physical Therapy Assessment Up and Down 4 Tiers (Time Frame: Screening, Day 30-3 Years): Exploratory outcomes for Cohort B will include up and down 4 tiers.

[0225] Physical Therapy Assessment Handheld Dynamometer (HHD) (Time Frame: Screening, Day 30-3 Years): Exploratory outcomes for Cohort B included handheld dynamometer (HHD) of the knee extensors and knee flexors and elbow flexors and elbow extensors.

[0226] Quantification of micro-dystrophin gene expression by immunofluorescence (time frame: screening, day 90): Micro-dystrophin gene expression levels were quantified by immunofluorescence and compared before and after muscle biopsy.

[0227] Quantification of micro-dystrophin gene expression by Western blotting (Time frame: screening, day 90): Micro-dystrophin gene expression levels were quantified by Western blot analysis and compared before and after muscle biopsy.

[0228] Reduction in CK after gene therapy (time frame: 3 years): Reduction in CK levels in circulating blood.

[0229] Cardiac Magnetic Resonance Imaging (1 year).

[0230] Micro-dystrophin gene expression

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

[0232] Table 2

[0233]

[0234] Changes in micro-dystrophin gene expression from baseline to day 60 were also assessed by quantification of micro-dystrophin expression as measured by Western blot of biopsied muscle tissue. Fig. 8A and 8BAs shown, Western blot analysis detected micro-dystrophin expression in subject 1 (5 years old), subject 2 (4 years old), and subject 3 (6 years old). Figure 8C Western blot analysis is provided to detect micro-dystrophin expression in Subject 4 (4 years old). All post-treatment biopsy samples showed elevated levels of micro-dystrophin as measured by Western blot, with a mean for Subjects 1-4 of 74.3% compared to normal values ​​using Method 1 and 95.8% compared to normal values ​​according to Method 2 (adjusted for fat and fibrotic tissue).

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

[0236] Table 3

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

[0238] The protein levels of α-sarcoglycan and β-sarcoglycan in muscle biopsies were measured by immunohistochemistry before and after administration of rAAVrh74.MHCK7.micro-dystrophin. Administration of rAAVrh74.MHCK7 also caused upregulation of DAPC protein in the subjects. As shown in FIG9 , in subject 1 ( Fig. 9A ), Subject 2( Fig. 9B ) and subject 3 ( Fig. 9C ), the expression of α-sarcoglycan and β-sarcoglycan in muscle biopsies was increased compared to the levels of these proteins in muscle biopsy samples before administration of rAAVrh74.MHCK7.

[0239] Circulating serum CK levels

[0240] In rAAVrh74.MHCK7.micro-dystrophin vector (2×10 14Blood samples were obtained every 30 days after administration of an intravenous infusion of 10 mL / kg (vg / kg, 10 mL / kg). CK levels were measured at each visit and compared to baseline levels obtained before administration of rAAVrh74.MHCK7.micro-dystrophin (Day 0 visit). Baseline serum CK levels (units / liter) are provided in Table 4 below. Fig.10 As shown, 2 months after the administration of rAAVrh74.MHCK7. micro-dystrophin, the level of circulating serum CK decreased by about 87%. All subjects showed a significant reduction in serum creatine kinase (CK) levels, with a mean CK reduction of more than 87% (n=3) 2 months after treatment. CK is an enzyme associated with muscle damage, and patients with DMD consistently show high levels of CK. In fact, significantly elevated CK is often used as a preliminary diagnostic tool for DMD, followed by confirmatory genetic testing.

[0241] Table 5 and Fig.10 CK levels for each subject are provided. Figure 11 provides mean CK levels over time and demonstrates that after administration of rAAVrh74.MHCK7.micro-dystrophin, mean CK levels were significantly reduced over time. The mean baseline CK level of 27,064 U / L (mean of Table 4) was reduced by approximately 63% to a mean of 9,982 U / L (mean, day 270, Table 5).

[0242] Table 4

[0243]

[0244] Table 5: Changes in CK Levels from Baseline to Day 270

[0245]

[0246] Efficacy evaluation

[0247] In addition to micro-dystrophin and CK levels, efficacy was measured by the following functional tests: floor stand time, 4-stair rise, North Star Ambulatory Assessment Scale (NSAA), stand time test, 4-stair rise test, 10-meter timed test (10m), and 100-meter timed test (100m). The data are provided in Tables 6 and 7 below and demonstrate sustained, lasting improvements 9 months after administration of rAAVrh74.MHCK7.micro-dystrophin. Fig.12 NSAA improvements over time are 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-glutamyltransferase (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 typically experienced transient nausea during the first week of treatment, concurrent with steroid dose increases. This was not associated with elevated liver enzymes or any other abnormalities.

[0255] Example 3

[0256] A randomized, double-blind, placebo-controlled phase I / IIa clinical trial of systemic gene delivery

[0257] This was a randomized, double-blind, single-dose trial of rAAVrh74.MHCK7.micro-dystrophin in DMD subjects. Twenty-four subjects aged 4 to 7 years were included in the study. Subjects were randomized to treatment or placebo at inclusion. Twelve subjects received intravenous rAAVrh74.MHCK7.micro-dystrophin vector (2×10 14 vg / kg, approximately 10 mL / kg), while the other twelve subjects will receive 10 mL / kg 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 was dosed. Subjects receive an infusion of rAAV carrying micro-dystrophin or lactated Ringer's solution over approximately 1 hour. Needle muscle biopsies are performed on the gastrocnemius muscle before and after treatment (90 days).

[0258] The primary objective of the study is to evaluate the safety of intravenous administration of rAAVrh74.MHCK7. micro-dystrophin via a peripheral limb vein in DMD subjects. Safety endpoints were assessed by hematology, serum chemistry, urinalysis, immune responses to rAAVrh74 and micro-dystrophin, and changes in reported medical history and symptom observations. Dystrophin gene expression was used as the primary outcome measure along with safety. Quantification was performed using validated immunofluorescence and immunoblotting assays. CK reduction after gene therapy was a secondary outcome. Efficacy was measured by the following functional tests: stand-up time, 4-stair rise, North Star Mobile Assessment Scale (NSAA), 10-meter timed test (10m), 100-meter timed test (100m). Exploratory indicators included handheld muscle strength measurement (HHD) of the knee extensors and knee flexors and elbow flexors and elbow extensors.

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

[0260] Age of inclusion: between 4 and 7 years old (inclusive).

[0261] Molecular characterization of DMD genes with frameshift (deletion or duplication) or premature stop codon mutations between exons 18 to 58.

[0262] Indications for symptomatic muscular dystrophy: CK elevation > 1000 U / L and less than the mean percent predicted time in the 100-meter walk test

[0263] Men of any race were eligible.

[0264] ·Ability to collaborate with movement assessment testing.

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

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

[0267] Active viral infection based on clinical observation.

[0268] Signs of cardiomyopathy, including an ejection fraction less than 40% on echocardiography.

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

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

[0271] Abnormal laboratory values ​​were 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 illness or the need for chronic medication would present an unnecessary risk to gene transfer.

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

[0274] Have a medical condition or mitigating condition that, in the opinion of the investigator, could impair the subject's ability to comply with tests or procedures required by the protocol or otherwise compromise the subject's health, safety, or clinical interpretability.

[0275] Serious infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (enrollment may be delayed).

[0276] Have received any study medication (except corticosteroids) or exon skipping drugs (including ) or experimental drugs.

[0277] Have had any type of gene therapy, cell-based therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy.

[0278] The family does not want the patient's research participation disclosed to primary care physicians and other medical providers.

[0279] Efficacy evaluation

[0280] Dystrophin gene expression served as the primary outcome measure along with safety. Quantification was performed using validated immunofluorescence and immunoblotting assays. CK reduction after gene therapy served as a secondary outcome. In addition, efficacy was measured by the following functional tests: floor stand time, 4-stair ascent, North Star Ambulatory Assessment Scale (NSAA), 10-meter timed test (10m), 100-meter timed test (100m). Exploratory measures included handheld dynamometer (HHD) of the knee extensors and knee flexors and elbow flexors and elbow extensors.

[0281] Ultrasound-guided muscle biopsy was used to compare baseline and day 90 transgene expression quantification. The same muscle as the original biopsy, but on the other leg, was biopsied. All subjects were dosed one year later, and placebo crossover subjects will restart the study schedule at visit 1. Placebo subjects will not undergo the following operations at the second baseline screening: cardiac MRI and muscle biopsy. Placebo subjects received a muscle biopsy on day 90 (3 muscle biopsies in total). Frozen sections were stained for dystrophin using indirect immunofluorescence (IF). Whole slide scans were performed using validated image scanning and MuscleMapTM analysis algorithms, and micro-dystrophin intensity and percentage of positive fibers were quantified. Muscle morphology measurements, including fiber size histograms, were performed by blinding. Blinded frozen muscle biopsy scrapings were used for quantitative protein analysis for micro-dystrophin using validated protein blotting methods.

[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) are used to quantify micro-dystrophin expression.

[0283] Efficacy analysis

[0284] The primary efficacy endpoint was the change in the number of micro-dystrophin proteins expressed from baseline to day 90, as measured by Western blot of biopsied muscle tissue. The treatment group differences for the primary efficacy endpoint were assessed by an analysis of covariance (ANCOVA) model, where treatment was a fixed factor and baseline values ​​were covariates. 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 include changes from baseline to each scheduled assessment in ground stand time, 4-stair rise, NSAA, 10-meter timed test (10m), 100-meter timed test (100m), and CK changes. Exploratory indicators include HHD of knee extensors and knee flexors and elbow flexors and elbow extensors. Treatment group differences were assessed by ANCOVA models, in which treatment was a fixed factor and baseline values ​​were covariates. Wilcoxon rank sum tests were performed as supportive analyses.

[0286] Example 4

[0287] The experiments and studies described above in Examples 2 and 3 may alternatively be performed using the rAAVrh74.MHCK7.micro-dystrophin construct set forth in SEQ ID NO:9; nucleotides 1-4977 set forth in SEQ ID NO:8; or nucleotides 56-5022 set forth in SEQ ID NO:6.

[0288] Example 5

[0289] Generation of pAAV.MCK.micro-dystrophin constructs

[0290] pAAV.MCK. micro-dystrophin plasmid (Samulski et al., J.Virol.61(10):3096-3101) was constructed by inserting the MCK expression cassette of the human micro-dystrophin cDNA sequence driven by codon optimization into the AAV cloning vector psub201. Muscle-specific regulatory elements are included in the construct to drive muscle-specific gene expression. The regulatory element comprises a mouse MCK core enhancer (206bp) fused to a 351bp MCK core promoter (proximal end). After the core promoter, the construct comprises a 53bp endogenous mouse MCK exon 1 (non-translated) for efficient transcription initiation, followed by a SV40 late 16S / 19S splicing signal (97bp) and a small 5'UTR (61bp). Introns and 5'UTR are derived from plasmid pCMVβ (Clontech). The micro-dystrophin cassette has a consensus sequence Kozak before the ATG start codon and a small 53 bp synthetic poly A signal for mRNA termination. The human micro-dystrophin 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.microdystrophin plasmid contains the human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 5 ). This sequence is encapsidated into AAVrh.74 virions. The molecular clone of the AAVrh.74 serotype was cloned from rhesus monkey lymph nodes and described in Rodino-Klapac et al. Journal of Tran. Med. 45 (2007).

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Claims

1. Use of a recombinant adeno-associated virus (rAAV) for the preparation of a medicament for treating Duchenne muscular dystrophy in a human subject in need thereof, wherein the rAAV is of serotype rh 74 and comprises a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, wherein the medicament is formulated for systemic administration 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 medicament is formulated for intravenous administration and the dose of rAAV is 2×10 14 vg / kg.

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

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

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

6. Use of a recombinant adeno-associated virus (rAAV) for the preparation of a medicament for treating Duchenne muscular dystrophy in a human subject in need thereof, wherein the rAAV is serotype rh 74 and comprises a polynucleotide comprising 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 the rAAV is 2×10 14 vg / kg.

7. The use according to claim 1 or 6, wherein the serum CK level of the subject is reduced after rAAV is administered to the subject compared to the serum CK level before administration.

8. The use according to claim 6, wherein the dose of rAAV is determined by using supercoiled DNA standards.

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