Adeno-associated virus vector delivery for muscular dystrophy

Abstract

To provide adeno-associated virus vector delivery for muscular dystrophy.SOLUTION: The present disclosure provides a method of treating muscular dystrophy in a subject in need thereof comprising administering a gene therapy vector, such as an adeno-associated virus (AAV) vector, expressing a miniaturized human micro-dystrophin gene in combination with suppressing the immune system of the subject. The importance of muscle mass and strength for daily activities such as exercise and respiration, as well as systemic metabolism, is clear. The loss of muscle function results in muscular dystrophy (MD), which is characterized by loss of muscle strength and wasting and has a profound impact on quality of life.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 039,252, filed June 15, 2020, U.S. Provisional Application No. 63 / 083,953, filed September 27, 2020, U.S. Provisional Application No. 63 / 160,376, filed March 12, 2021, and U.S. Provisional Application No. 63 / 188,266, filed May 13, 2021, each of which is incorporated by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials This application contains a sequence listing in computer readable form, which is incorporated by reference in its entirety as a separate part of this disclosure and identified as Filename: 55714_Seqlisting.txt, Size: 83,380 bytes, Created: June 7, 2021.

[0003] The present disclosure provides methods for treating diseases, such as muscular dystrophy, in a subject in need thereof, comprising administering a gene therapy vector, such as an adeno-associated virus (AAV) vector, and expressing a transgene of interest, such as a truncated human microdystrophin gene or a β-sarcoglycan gene, in combination with suppressing the subject's immune system. [Background technology]

[0004] The importance of muscle mass and strength for daily activities such as locomotion and breathing, as well as for whole-body metabolism, is clear. Deficits in muscle function result in muscular dystrophies (MDs), characterized by muscle weakness and wasting, severely impacting quality of life. The most well-characterized MDs result from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs result from membrane fragility associated with loss of sarcolemmal-cytoskeleton anchoring by DAPCs. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in 5,000 newborn males.

[0005] DMD is caused by mutations in the DMD gene, which result in reduced mRNA and the absence of dystrophin, a 427-kD sarcolemmal protein that binds to the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51(6):919-28, 1987). The DAPC is composed of multiple proteins in the sarcolemma, forming structural links with the extracellular matrix (ECM) and the cytoskeleton via the actin-binding protein dystrophin and the laminin-binding protein alpha-dystroglycan. These structural links act to stabilize the muscle membrane during contraction and protect it from contraction-induced damage. Loss of dystrophin leads to membrane fragility, resulting in sarcolemmal rupture and calcium influx, 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):p.1059-71; Wallace et al., Annu Rev Physiol, 2009.71:p.37-57), resulting in progressive muscle weakness, endomysitis, and fibrous scarring.

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

[0007] Another group of MDs is limb-girdle muscle myopathy (LGMD). LGMD is a rare condition, and symptoms vary from person to person in terms of age of onset, areas of muscle weakness, cardiac and respiratory involvement, rate of progression, and severity. LGMD can begin in childhood, adolescence, young adulthood, or later. Both genders are affected equally. LGMD causes weakness in the shoulders and pelvic girdle, and nearby muscles in the upper limbs and arms may also weaken over time. Leg weakness often precedes arm weakness. Facial muscles are usually unaffected. As the condition progresses, people may have trouble walking and may need to use a wheelchair over time. Involvement of shoulder and arm muscles can make it difficult to lift the arms overhead or lift objects. Depending on the type of LGMD, cardiac and respiratory muscles may also be involved.

[0008] [Table 1]

[0009] Specialist tests for LGMD are now available through the National Commissioning Group (NCG), the national scheme for diagnosis. As gene therapies are developed to treat muscular dystrophies such as DMD or LGMD, there is a need to optimize these therapies and evaluate the effect of immunosuppression on the expression of microdystrophin transgenes by optimized gene therapy vectors. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Hoffman et al., Cell 51(6):919-28,1987 [Non-patent document 2] Straub et al.,Curr Opin.Neurol.10(2):168-75,1997 [Non-patent document 3] Sacco et al.,Cell,2010.143(7):p.1059-71 [Non-patent document 4] Wallace et al.,Annu Rev Physiol,2009.71:p.37-57 [Non-Patent Document 5] Desguerre et al.,J Neuropathol Exp Neurol,2009.68(7):p.762-7 Summary of the Invention [Means for solving the problem]

[0011] The present disclosure relates to a gene therapy vector, e.g., AAV, that expresses a transgene of interest in skeletal muscle to treat muscular dystrophy in combination with the administration of an immunosuppressant. Additionally, the present disclosure includes a method for re-administering AAV gene therapy to a subject, in which the subject's plasma is subjected to therapeutic plasma exchange (TPE) to remove AAV antibodies produced in response to the initial administration of AAV gene therapy.

[0012] The present disclosure relates to gene therapy vectors, e.g., AAV, that express the microdystrophin gene in skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in combination with the administration of immunosuppressants. Additionally, the present disclosure includes a method for re-administering AAV gene therapy to a subject, in which the subject's plasma is subjected to therapeutic plasma exchange (TPE) to remove AAV antibodies produced in response to the initial administration of AAV gene therapy.

[0013] Additionally, the present disclosure relates to a gene therapy vector, e.g., AAV, that expresses a β-sarcoglycan gene in skeletal muscle, including the diaphragm and cardiac muscle, in combination with the administration of an immunosuppressant. Additionally, the present disclosure includes a method of re-administering AAV gene therapy to a subject, in which the subject's plasma is subjected to therapeutic plasma exchange (TPE) to remove AAV antibodies produced in response to the initial administration of AAV gene therapy.

[0014] The present disclosure provides therapies and approaches for increasing muscle strength and / or muscle mass using gene therapy vectors to deliver micro-dystrophin to address the genetic defect observed in DMD. In particular, the present disclosure provides studies demonstrating gene expression following systemic delivery of rAAVrh74.MHCK7.micro-dystrophin in non-human primate models using different immunosuppressive regimens that vary in duration, dose, and type of immunosuppression. The present disclosure also provides studies demonstrating micro-dystrophin transgene expression after using TPE to remove AAV viral antibodies from previously administered non-human primates and systemically re-administering rAAVrh74.MHCK7.micro-dystrophin to the primates.

[0015] The present disclosure also provides an approach for treating a human subject suffering from DMD with an anti-AAVrh.74 antibody prior to administration of rAAVrh74.MHCK7.microdystrophin, wherein the subject is subjected to multiple sessions of TPE prior to administration of rAAVrh74.MHCK7.microdystrophin.

[0016] The present disclosure provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, 8, or 9. The present disclosure also provides an rAAV comprising the nucleic acid sequence of SEQ ID NO: 9, or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3, and an rAAV particle comprising the nucleic acid sequence of SEQ ID NO: 9, nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3.

[0017] Another aspect of the disclosure provides compositions comprising the nucleic acid sequence of SEQ ID NO: 3, 8, or 9, an rAAV comprising the nucleic acid sequence of SEQ ID NO: 9, or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3, and rAAV particles comprising the nucleic acid sequence of SEQ ID NO: 9, or nucleotides 1 to 4977 of SEQ ID NO: 8, or nucleotides 55 to 5021 of SEQ ID NO: 3. Any of the methods disclosed herein can be performed using these compositions.

[0018] Additionally, the present disclosure provides compositions comprising a nucleic acid comprising an rAAV genome of one of AAVrh.74.tMCK.CAP N3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05.

[0019] The present disclosure provides a method for treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an anti-inflammatory steroid. Additionally, the present disclosure provides the use of a combination therapy comprising a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an anti-inflammatory steroid for the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, such that the rAAV and the anti-inflammatory steroid are administered simultaneously or separately. The present disclosure also provides a combination therapy for treating muscular dystrophy, comprising a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an anti-inflammatory steroid, and the rAAV and the anti-inflammatory steroid are administered simultaneously or separately. The present disclosure also provides a combination therapy for treating muscular dystrophy, comprising a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an anti-inflammatory steroid, and the rAAV and the anti-inflammatory steroid are administered simultaneously or separately. For example, the muscular dystrophy is DMD or Becker muscular dystrophy. For example, the anti-inflammatory steroid is a glucocorticoid. In some embodiments, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort. In some embodiments, the anti-inflammatory steroid is administered orally. The anti-inflammatory steroid may be administered both before and after administration of rAAV. Alternatively, the anti-inflammatory steroid is administered only before or only after administration of rAAV.

[0020] Additionally, the present disclosure provides a method of treating limb-girdle muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated virus (rAAV) and an anti-inflammatory steroid, wherein the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05. Additionally, the present disclosure provides the use of a combination therapy comprising a recombinant adenovirus-associated virus (rAAV) and an anti-inflammatory steroid for the preparation of a medicament for treating limb-girdle muscular dystrophy in a human subject in need thereof, wherein the rAAV and the anti-inflammatory steroid are co-administered separately, e.g., the rAAV and the anti-inflammatory steroid are co-administered simultaneously or sequentially, wherein the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05. The present disclosure also provides a combination therapy for treating limb-girdle muscular dystrophy, the combination therapy comprising a recombinant adenovirus-associated virus (rAAV) and an anti-inflammatory steroid, wherein the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and the rAAV and the anti-inflammatory steroid are administered separately in combination, e.g., the rAAV and the anti-inflammatory steroid are administered simultaneously or sequentially. For example, the anti-inflammatory steroid is a glucocorticoid. In some embodiments, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort.In some embodiments, the anti-inflammatory steroid is administered orally. The anti-inflammatory steroid may be administered both before and after administration of rAAV. Alternatively, the anti-inflammatory steroid is administered only before or only after administration of rAAV.

[0021] For example, in any of the methods, uses or combination therapies disclosed herein, the anti-inflammatory steroid is administered about 12 hours prior to administration of rAAV, or about 24 hours prior to administration of rAAV, or about 36 hours prior to administration of rAAV, or about 48 hours prior to administration of rAAV, or about 60 hours prior to administration of rAAV, or about 72 hours prior to administration of rAAV, or about 96 hours prior to administration of rAAV. In some embodiments, the inflammatory steroid is administered about 5 days prior to administration of the rAAV, about 6 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, or about 8 days prior to administration of the rAAV, or about 9 days prior to administration of the rAAV, or about 10 days prior to administration of the rAAV, or about 11 days prior to administration of the rAAV, or about 12 days prior to administration of the rAAV, or about 13 days prior to administration of the rAAV, or about 14 days prior to administration of the rAAV, or about 30 days prior to administration of the rAAV.

[0022] Additionally, in any of the disclosed methods, uses, or combination therapies, the anti-inflammatory steroid is administered at least once daily about 7 days prior to administration of the rAAV, or at least once daily about 14 days prior to administration of the rAAV, or at least once daily about 21 days prior to administration of the rAAV, or at least once daily about 28 days prior to administration of the rAAV, or at least once daily about 30 days prior to administration of the rAAV, or at least once daily about 45 days prior to administration of the rAAV, or at least once daily about 60 days prior to administration of the rAAV. In some embodiments, the anti-inflammatory steroid is administered 30-60 days prior to administration of the rAAV.

[0023] For example, in any of the disclosed methods, uses, or combination therapies, the anti-inflammatory steroid is administered prior to administration of rAAV, and the anti-inflammatory steroid is administered at least once daily from day 1 to day 30 after administration of rAAV, at least once daily from day 1 to day 60 after administration of rAAV, or at least once daily from day 1 to day 7 after administration of rAAV, or at least once daily from day 1 to day 14 after administration of rAAV, or at least once daily from day 1 to day 21 after administration of rAAV, or at least once daily from day 1 to day 24 after administration of rAAV, or at least once daily from day 1 to day 28 after administration of rAAV, or at least from day 1 to day 30 after administration of rAAV, or at least from day 30 to day 60 after administration of rAAV.

[0024] In any of the methods, uses, or combination therapies disclosed herein, the anti-CD20 specific antibody is administered prior to administration of rAAV. In some embodiments, the anti-CD20 specific antibody is administered at least 7 days prior to administration of rAAV. The term anti-CD20 specific antibody refers to an antibody that specifically binds to CD20 or inhibits or reduces the expression or activity of CD20. Exemplary anti-CD20 antibodies include rituximab, ocrelizumab, or ofatumumab.

[0025] In any of the disclosed methods, uses, or combination therapies, the anti-CD20-specific antibody is administered about 60 days prior to administration of the rAAV, or about 45 days prior to administration of the rAAV, or about 30 days prior to administration of the rAAV, about 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and within about 24 hours of administration of the rAAV. In some embodiments, the anti-CD20-specific antibody is administered 30 to 60 days prior to administration of the rAAV. In some embodiments, the anti-CD20-specific antibody is administered after administration of the rAAV. For example, the anti-CD20-specific antibody is administered both before and after administration of the rAAV. Alternatively, the anti-CD20-specific antibody is administered before administration of the rAAV, or the anti-CD20-specific antibody is administered after administration of the rAAV.

[0026] Additionally, any of the disclosed methods, uses, or combination therapies may include administering an immunosuppressant macrolide. The term immunosuppressant macrolide refers to a macrolide drug that suppresses or modulates a subject's immune system. Macrolides are a class of drugs that contain a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desoamine, are attached. The lactone ring is typically 14, 15, or 16 members. Macrolides belong to the polyketide class of drugs and can be natural products. Examples of immunosuppressant macrolides include tacrolimus, pimecrolimus, and sirolimus. In some embodiments, the immunosuppressant macrolide is orally administered to the subject. In some embodiments, the immunosuppressant macrolide may be administered both before and after administration of rAAV. Alternatively, the immunosuppressant macrolide is administered before administration, or the rAAV or the immunosuppressant macrolide is administered after administration of rAAV.

[0027] In some embodiments, the immunosuppressant macrolide is administered at least once daily for at least 3 days prior to administration of the rAAV, or at least 4 days prior to administration of the rAAV, or at least 5 days prior to administration of the rAAV, or at least 6 days prior to administration of the rAAV, or at least 7 days prior to administration of the rAAV, or at least 10 days prior to administration of the rAAV, or at least 14 days prior to administration of the rAAV, or at least 30 days prior to administration of the rAAV, or at least 45 days prior to administration of the rAAV, or at least 60 days prior to administration of the rAAV. In some embodiments, the immunosuppressant macrolide is administered 30-60 days prior to administration of the rAAV.

[0028] The present disclosure also provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an immunosuppressive regimen, wherein the immunosuppressive regimen comprises administering one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide. The present disclosure also provides use of a combination therapy comprising recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an immunosuppressive regimen for the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, wherein the immunosuppressive regimen comprises administering one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide, for example, wherein in the disclosed medicament, the rAAV and one or more components of the immunosuppressive regimen are co-administered separately, for example, the rAAV and one or more components of the immunosuppressive regimen are co-administered simultaneously or sequentially. The present disclosure also provides a combination therapy for treating muscular dystrophy in a human subject in need thereof, the combination therapy comprising a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an immunosuppressive regimen, and the rAAV and the immunosuppressive regimen are administered separately in combination, e.g., the rAAV and one or more components of the immunosuppressive regimen are administered simultaneously or sequentially in combination. The term immunosuppressive regimen refers to a method of treatment or therapy that suppresses or modulates a subject's immune system. The regimen includes the administration of one or more immunosuppressive agents. In some embodiments, the immunosuppressive regimen includes administering an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide.

[0029] The present disclosure also provides a method of treating limb-girdle muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and an immunosuppressive regimen, wherein the immunosuppressive regimen comprises administering one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide. The present disclosure also provides use of a combination therapy comprising an rAAV and an immunosuppressive regimen for the preparation of a medicament for treating limb-girdle muscular dystrophy in a human subject in need thereof, wherein the rAAV comprises an rAAV recombinant adeno-associated virus (rAAV) selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and the immunosuppressive regimen comprises administering one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide, e.g., in the disclosed medicament, the rAAV and one or more components of the immunosuppressive regimen are co-administered separately, e.g., the rAAV and one or more components of the immunosuppressive regimen are co-administered simultaneously or sequentially.The present disclosure also provides a combination therapy for treating limb-girdle muscular dystrophy in a human subject in need thereof, the combination therapy comprising a recombinant adenovirus-associated virus (rAAV) selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05; and the immunosuppressive regimen comprising administering one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide; wherein the rAAV and one or more components of the immunosuppressive regimen are co-administered separately, e.g., the rAAV and one or more components of the immunosuppressive regimen are co-administered simultaneously or sequentially. In some embodiments, the immunosuppressive regimen comprises administering an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressant macrolide.

[0030] In one exemplary immunosuppressive regimen, an anti-inflammatory steroid is administered about 24 hours prior to administration of the rAAV. In another exemplary immunosuppressive regimen, an anti-inflammatory steroid is administered prior to administration of the rAAV and the anti-inflammatory steroid is administered at least once daily from day 1 to day 30 after administration of the rAAV, or the anti-inflammatory steroid is administered at least once daily from day 1 to day 60 after administration of the rAAV.

[0031] In any of the disclosed immunosuppressive regimens, the anti-inflammatory steroid is a glucocorticoid such as prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort. In some embodiments, the anti-inflammatory steroid is administered orally.

[0032] A further exemplary immunosuppressive regimen involves administering an anti-CD20 specific antibody prior to administration of the rAAV. For example, the anti-CD20 antibody is administered by intravenous infusion. Exemplary anti-CD20 specific antibodies include rituximab, ocrelizumab, or ofatumumab.

[0033] In one embodiment, the anti-CD20-specific antibody is administered at least 14 days prior to administration of the rAAV. In another embodiment, the anti-CD20-specific antibody is administered about 60 days prior to administration of the rAAV, about 45 days prior to administration of the rAAV, about 30 days prior to administration of the rAAV, 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and within about 24 hours of administration of the rAAV. Additionally, the anti-CD20-specific antibody is administered 30 to 60 days prior to administration of the rAAV. The disclosed immunosuppressive regimens also include administering the anti-CD20-specific antibody after administration of the rAAV.

[0034] Additionally, the disclosed immunosuppressive regimens include administering an immunosuppressant macrolide at least once daily for at least three days prior to administration of the rAAV. The immunosuppressive regimen may also include administering an immunosuppressant macrolide after administration of the rAAV. In any of the disclosed immunosuppressive regimens, the immunosuppressant macrolide is administered orally. Exemplary immunosuppressant macrolides include tacrolimus, pinecrolimus, or sirolimus.

[0035] In some embodiments, the disclosed immunosuppressive regimens are administered 30 to 60 days prior to administration of rAAV. Additionally, the immunosuppressive regimen is administered about 60 days prior to administration of rAAV, about 45 days prior to administration of rAAV, about 30 days prior to administration of rAAV, about 14 days prior to administration of rAAV, about 7 days prior to administration of rAAV, or about 24 hours prior to administration of rAAV.

[0036] In certain embodiments, the present disclosure provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin and an immunosuppressive regimen, the immunosuppressive regimen comprising: i) orally administering an anti-inflammatory steroid about 24 hours prior to administration of the rAAV and at least once daily from day 1 to day 30 after administration of the rAAV; or ii) administering an anti-inflammatory steroid at least once daily for at least 1 to 60 days after administration of the rAAV; ii) administering an anti-CD20 antibody intravenously about 14 days before administration of the rAAV, about 7 days before administration of the rAAV, and about 24 hours after administration of the rAAV, and optionally administering an anti-CD20 antibody after administration of the rAAV; and iii) administering an immunosuppressant macrolide orally at least once daily for at least 3 days before administration of the rAAV, and optionally administering an immunosuppressant macrolide after administration of the rAAV. For example, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort, the anti-CD20 specific antibody is rituximab, ocrelizumab, or ofatumumab, or more of these anti-inflammatory steroids, anti-CD20 antibodies, or immunosuppressive macrolides, and the immunosuppressive macrolide is tacrolimus, pinecrolimus, or sirolimus. In an exemplary embodiment, the immunosuppressive regimen includes the anti-inflammatory steroid prednisone or prednisolone, the anti-CD20 antibody rituximab, and the immunosuppressive macrolide sirolimus.

[0037] Also provided is the use of a combination therapy comprising rAAV and an immunosuppressive regimen for the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, wherein the rAAV is rAAV.MHCK7.microdystrophin, and the immunosuppressive regimen includes: i) orally administering an anti-inflammatory steroid about 24 hours before administration of the rAAV and at least once daily from day 1 to day 30 after administration of the rAAV; or ii) orally administering an anti-inflammatory steroid at least once daily from day 1 to day 30 after administration of the rAAV. ii) administering an anti-inflammatory steroid at least once daily for at least 60 days prior to administration of the rAAV; ii) administering an anti-CD20 antibody intravenously about 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and about 24 hours after administration of the rAAV, and optionally administering an anti-CD20 antibody after administration of the rAAV; and iii) administering an immunosuppressant macrolide orally at least once daily for at least 3 days prior to administration of the rAAV, and optionally administering an immunosuppressant macrolide after administration of the rAAV. For example, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort, the anti-CD20 specific antibody is rituximab, ocrelizumab, or ofatumumab, or more of these anti-inflammatory steroids, anti-CD20 antibodies, or immunosuppressive macrolides, and the immunosuppressive macrolide is tacrolimus, pinecrolimus, or sirolimus. In an exemplary embodiment, the immunosuppressive regimen includes the anti-inflammatory steroid prednisone or prednisolone, the anti-CD20 antibody rituximab, and the immunosuppressive macrolide sirolimus.

[0038] The present disclosure provides a combination therapy comprising an rAAV and an immunosuppressive regimen for treating muscular dystrophy in a human subject in need thereof, wherein the rAAV is rAAV.MHCK7.microdystrophin, and the immunosuppressive regimen comprises: i) orally administering an anti-inflammatory steroid about 24 hours before administration of the rAAV and orally administering an anti-inflammatory steroid at least once daily from day 1 to day 30 after administration of the rAAV, or administering an anti-inflammatory steroid at least once daily from day 1 to day 60 after administration of the rAAV; ii) intravenously administering an anti-CD20 antibody about 14 days before administration of the rAAV, about 7 days before administration of the rAAV, and about 24 hours after administration of the rAAV, and optionally administering an anti-CD20 antibody after administration of the rAAV; and iii) orally administering an immunosuppressive macrolide at least once daily for at least 3 days before administration of the rAAV, and optionally administering an immunosuppressive macrolide after administration of the rAAV. For example, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort, the anti-CD20 specific antibody is rituximab, ocrelizumab, or ofatumumab, or more of these anti-inflammatory steroids, anti-CD20 antibodies, or immunosuppressive macrolides, and the immunosuppressive macrolide is tacrolimus, pinecrolimus, or sirolimus. In an exemplary embodiment, the immunosuppressive regimen includes the anti-inflammatory steroid prednisone or prednisolone, the anti-CD20 antibody rituximab, and the immunosuppressive macrolide sirolimus.

[0039] In another specific embodiment, the present disclosure provides a method of treating limb-girdle muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and an immunosuppressive regimen, wherein the immunosuppressive regimen comprises: i) administration of an anti-inflammatory steroid about 24 hours prior to administration of the rAAV. and administering an anti-inflammatory steroid at least once daily from day 1 to day 30 after administration of the rAAV, or an anti-inflammatory steroid is administered at least once daily from day 1 to day 60 after administration of the rAAV; ii) administering an anti-CD20 antibody intravenously about 14 days prior to administration of the rAAV, 7 days prior to administration of the rAAV, and within about 24 hours of administration of the rAAV, and optionally administering an anti-CD20 antibody after administration of the rAAV; and iii) administering an immunosuppressant macrolide orally at least once daily for at least 3 days prior to administration of the rAAV, and optionally administering an immunosuppressant macrolide after administration of the rAAV.

[0040] In another specific embodiment, the disclosure provides use of a combination therapy comprising an rAAV and an immunosuppressive regimen for the preparation of a medicament for treating limb-girdle muscular dystrophy in a human subject in need thereof, wherein the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and the immunosuppressive regimen comprises administering an anti-inflammatory drug (e.g., an anti-inflammatory drug) to a subject in need thereof about 24 hours prior to administration of the rAAV. The method includes: orally administering a steroid and administering an anti-inflammatory steroid at least once daily from day 1 to day 30 after administration of the rAAV, or administering an anti-inflammatory steroid at least once daily from day 1 to day 60 after administration of the rAAV; ii) intravenously administering an anti-CD20 antibody about 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and about 24 hours after administration of the rAAV, and optionally administering an anti-CD20 antibody after administration of the rAAV; and iii) orally administering an immunosuppressive macrolide at least once daily for at least 3 days prior to administration of the rAAV, and optionally administering an immunosuppressive macrolide after administration of the rAAV.

[0041] In another specific embodiment, the disclosure provides a combination therapy comprising an rAAV and an immunosuppressive regimen for treating limb-girdle muscular dystrophy in a human subject in need thereof, wherein the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and the immunosuppressive regimen comprises: i) administering an anti-inflammatory steroid about 24 hours prior to administration of the rAAV; orally administering an anti-inflammatory steroid at least once daily from day 1 to day 30 after administration of the rAAV, or an anti-inflammatory steroid is administered at least once daily from day 1 to day 60 after administration of the rAAV; ii) intravenously administering an anti-CD20 antibody about 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and about 24 hours after administration of the rAAV, and optionally administering an anti-CD20 antibody after administration of the rAAV; and iii) orally administering an immunosuppressive macrolide at least once daily for at least 3 days prior to administration of the rAAV, and optionally administering an immunosuppressive macrolide after administration of the rAAV.

[0042] In another embodiment, the disclosure provides a method of treating muscular dystrophy in a human subject in need thereof, comprising subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administration of a second dose of adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, wherein the subject received a first dose of rAAV prior to being subjected to TPE.

[0043] In another embodiment, the present disclosure provides for the use of a combination therapy for treating muscular dystrophy in a human subject in need thereof, the combination therapy comprising subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administration of a second dose of adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, wherein the subject received a first dose of rAAV prior to being subjected to TPE.

[0044] In another embodiment, the present disclosure provides a combination therapy for treating muscular dystrophy in a human subject in need thereof, the combination therapy comprising subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administration of a second dose of adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, wherein the subject received a first dose of rAAV prior to being subjected to TPE.

[0045] The present disclosure provides a method of treating muscular dystrophy in a human subject in need thereof, comprising subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administration of a second dose of a recombinant adenovirus-associated (rAAV) selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, wherein the subject received a first dose of the rAAV prior to being subjected to TPE.

[0046] In further embodiments, the present disclosure provides methods of treating muscular dystrophy in a human subject in need thereof, comprising: a) administering a first dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin; b) administering the subject's plasma to at least one therapeutic plasma exchange (TPE); and c) administering a second dose of rAAV. In any of the disclosed methods, the subject's plasma is subjected to at least two TPEs or at least three TPEs prior to administration of the second dose or rAAV. In some embodiments, the subject's plasma is subjected to at least four TPEs prior to administration of the second dose of rAAV, or the subject's plasma is subjected to five TPEs prior to administration of the second dose of rAAV, or the subject's plasma is subjected to six TPEs prior to administration of the second dose of rAAV, or the subject's plasma is subjected to seven TPEs prior to administration of the second dose of rAAV.

[0047] In another embodiment, the present disclosure provides a method of treating limb-girdle muscular dystrophy in a human subject in need thereof, comprising: Methods are provided that include the steps of: a) administering a first dose of a recombinant adenovirus-related virus selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05; b) subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE); and c) administering a second dose or rAAV. In any of the disclosed methods, the subject's plasma is subjected to at least two TPEs or at least three TPEs prior to administration of the second dose or rAAV. In some embodiments, the subject's plasma is subjected to at least four TPEs before administration of the second dose of rAAV, or the subject's plasma is subjected to five TPEs before administration of the second dose of rAAV, or the subject's plasma is subjected to six TPEs before administration of the second dose of rAAV, or the subject's plasma is subjected to seven TPEs before administration of the second dose of rAAV.

[0048] In further embodiments, the present disclosure provides methods for treating muscular dystrophy in a human subject in need thereof, comprising: a) subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administering a recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin; and b) administering the rAAV. In any of the disclosed methods, the subject's plasma is subjected to at least two TPEs prior to administering the rAAV, at least three TPEs prior to administering the rAAV, at least four TPEs prior to administering the rAAV, at least five TPEs prior to administering the rAAV, at least six TPEs prior to administering the rAAV, or at least seven TPEs prior to administering the rAAV. In these disclosed methods, the subject is administered an anti-inflammatory steroid about 24 hours prior to administering the rAAV. Additionally, in some embodiments, the subject is administered an anti-inflammatory steroid at least once daily from day 1 to day 60 after administration of the rAAV. For example, the anti-inflammatory steroid is administered orally. Additionally, the anti-inflammatory steroid is a glucocorticoid, such as prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort.

[0049] Additionally, the present disclosure provides a method of treating limb-girdle muscular dystrophy in a human subject in need thereof, comprising the steps of: a) subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administering a recombinant adenovirus (rAAV) selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05; and b) administering the rAAV. In any of the disclosed methods, the subject's plasma is subjected to at least two TPEs before administering rAAV, at least three TPEs before administering rAAV, at least four TPEs before administering rAAV, at least five TPEs before administering rAAV, at least six TPEs before administering rAAV, or at least seven TPEs before administering rAAV. In these disclosed methods, the subject is administered an anti-inflammatory steroid about 24 hours before administering rAAV. Additionally, in some embodiments, the subject is administered an anti-inflammatory steroid at least once daily from day 1 to day 60 after administration of rAAV. For example, the anti-inflammatory steroid is administered orally. Additionally, the anti-inflammatory steroid is a glucocorticoid such as prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort.

[0050] In any of the disclosed methods, the subject's plasma is subjected to TPE for at least 9 days, at least 7 days, 5 days, or 2 days prior to administration of the rAAV. Additionally, there is about 24 to about 48 hours between sessions of TPE performed on the subject's plasma prior to administration of the rAAV. In certain embodiments, the subject's plasma is subjected to at least two TPE sessions prior to administration of the rAAV, with about 48 hours between TPE sessions.

[0051] In any of the methods described herein, the subject has an anti-AAVrh.74 antibody level of about 1:400 or less upon administration of rAAV. For example, the subject has an anti-AAVrh.74 antibody level of about 1:100 to about 1:400, or about 1:100 to 1:300, or about 1:100 to 1:200, or about 1:250 to 1:500, or about 1:200 to 1:400 upon administration of rAAV. The antibody titer is determined as a total antibody binding titer. In any of the disclosed methods of treating muscular dystrophy, these methods further include determining the presence of anti-AAVrh.74 antibodies in the subject's serum or plasma. The step of determining the presence of anti-AAVrh.74 antibodies can be performed before administration of rAAV, after administration of rAAV, before an immune response or adverse event is observed, or after an immune response or adverse event is observed. In addition, this determining step can be performed before administering an immunosuppressive regimen or TPE. For example, this determining step is performed before administration of any AAV to the subject, or this determining step is performed before administration of any AAVrh.74 to the subject.

[0052] The disclosure also provides methods that further include comparing the level of anti-AAVrh.74 antibodies in the subject's serum or plasma to a positive control, for example, the positive control utilizes an anti-AAVrh.74 monoclonal antibody.

[0053] In any of the disclosed methods, determining the presence of anti-AAVrh.74 may be determined using an immunofluorescence assay, an immunohistochemical assay, a Western blot, a direct enzyme-linked immunosorbent assay (ELISA), an indirect ELISA, a sandwich ELISA, a competitive ELISA, a reverse ELISA, a chemiluminescence assay, a radioimmunoassay, or an immunoprecipitation assay.

[0054] In any of the disclosed methods, the step of determining the presence of anti-AAVrh.74 antibodies may include determining the presence of anti-AAVrh.74 antibodies using a monoclonal antibody comprising a VH CDR1 amino acid sequence selected from the group consisting of NYGMN (SEQ ID NO:20), DYGMN (SEQ ID NO:22), YTFTNYGMN (SEQ ID NO:21), and YTFTKYGMN (SEQ ID NO:23), or a monoclonal antibody comprising a VH CDR2 amino acid sequence selected from the group consisting of WINTYTGEPTYADDFKG (SEQ ID NO:24), WINTNTGEPTYGDDFKG (SEQ ID NO:25), and WMGWINTYTGEPTY (SEQ ID NO:26), or a VH CDR2 amino acid sequence selected from the group consisting of GVAHYSDSRFAFDY (SEQ ID NO:27), GNAHPGGSAFVY (SEQ ID NO:28), RGSYYYDSSPAWFAY (SEQ ID NO:29), RGVDSSGYGAFAY (SEQ ID NO:30), and TRGTSTMISTFAFVY (SEQ ID NO:31). The present invention also includes utilizing a monoclonal antibody comprising a VL CDR1 amino acid sequence selected from the group consisting of SVSSSVSYMH (SEQ ID NO: 32), SASSGVTYMH (SEQ ID NO: 33), SSVSYMH (SEQ ID NO: 34), and SSVRYMH (SEQ ID NO: 35), or a monoclonal antibody comprising a VL CDR2 amino acid sequence selected from the group consisting of YTSNLAS (SEQ ID NO: 36), RTSNLAS (SEQ ID NO: 37), LWIYSTSNLAS (SEQ ID NO: 38), and VWIYSTSNLAS (SEQ ID NO: 39), or a monoclonal antibody comprising a VH CDR3 amino acid sequence selected from the group consisting of QQRSSYPFT (SEQ ID NO: 40), QQRSTYPF (SEQ ID NO: 41), QQRSFYPF (SEQ ID NO: 42), and QQRTYYPF (SEQ ID NO: 43).

[0055] In exemplary embodiments, the disclosed methods include determining the presence of anti-AAVrh.74 antibodies using an anti-AAVrh.74 monoclonal antibody, such as a monoclonal antibody comprising a variable heavy chain (VH) sequence set forth in SEQ ID NO: 10, 12, 14, 16, or 18, or a monoclonal antibody comprising a variable light chain (VL) sequence set forth in SEQ ID NO: 11, 13, 15, 17, or 19.

[0056] In a further embodiment, the disclosed method includes determining the presence of an anti-AAVrh.74 antibody using an anti-AAVrh.74 monoclonal antibody comprising a variable heavy chain (VH) sequence set forth in SEQ ID NO: 10, 12, 14, 16, or 18, and a variable light chain (VL) sequence set forth in SEQ ID NO: 11, 13, 15, 17, or 19.

[0057] In any of the disclosed methods, the step of determining the presence of anti-AAVrh.74 antibodies is quantitative, the subject is identified as seropositive for anti-AAVrh.74 antibodies based on the quantification, and the immunosuppressive regimen or TPE is selectively administered to seropositive subjects. In any of the methods disclosed herein, the rAAV is administered at a concentration of about 5.0 x 10 12 vg / kg ~ approx. 1.0×10 15 The muscular dystrophy may be Duchenne muscular dystrophy or Becker muscular dystrophy.

[0058] For example, the dose of rAAV administered is about 5.0x10 12 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg~1.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg~approx.2.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg~approx.5.0x10 13 vg / kg, or approximately 5.0 x 10 12 vg / kg~approx.2.0x10 13 vg / kg, or approximately 5.0 x 10 12 vg / kg ~ approx. 1.0x10 13 vg / kg, or 1.0x10 14 vg / kg ~ approx. 1.0x10 15 vg / kg, or 1.0x10 13 vg / kg ~ approx. 1.0x10 14vg / kg, or approximately 1.0 x 10 13 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.2.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.5.0x10 13 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.5.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.6.0x10 14 vg / kg, or 1.0x10 13 vg / kg ~ approx. 1.0x10 15 vg / kg, or 5.0x10 13 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~1.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.2.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.3.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.5.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.6.0x10 14 vg / kg, or 5.0x10 13 vg / kg ~ approx. 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg~approx.6.0x10 14 vg / kg, or 1.0x10 14 vg / kg~approx.5.0x10 14 vg / kg, or 1.0x10 14vg / kg ~ approx. 4.0x10 14 vg / kg, or 1.0x10 14 vg / kg ~ approx. 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.0 x 10 14 vg / kg ~ approx. 2.5x10 14 vg / kg, or 1.0x10 14 vg / kg~approx.2.0x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~6.0x10 14 , or approximately 1.25x10 14 vg / kg~5.0x10 14 , or approximately 1.25x10 14 vg / kg~4.0x10 14 , or approximately 1.25x10 14 vg / kg~1.0x10 15 , or approximately 1.25x10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.2.75x10 14 vg / kg, or approximately 1.25x10 14 vg / kg ~ approx. 2.5x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.2.0x10 14 vg / kg, or 1.25x10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.3.5x10 14 vg / kg, or 1.5x10 14 vg / kg~1.0x10 15 vg / kg, or approximately 1.5x10 14 vg / kg~6.0x10 14 , or about 1.5x1014 vg / kg~5.0x10 14 , or about 1.5x10 14 vg / kg~4.0x10 14 , or about 1.5x10 14 vg / kg~approx.3.75x10 14 , or about 1.5x10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.3.25x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.2.75x10 14 vg / kg, or approximately 1.5x10 14 vg / kg ~ approx. 2.5x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.2.0x10 14 vg / kg, or 1.75x10 14 vg / kg ~ approx. 1.0x10 15 vg / kg, or approximately 1.75x10 14 vg / kg~6.0x10 14 , or approximately 1.75x10 14 vg / kg~5.0x10 14 , or approximately 1.75x10 14 vg / kg~4.0x10 14 , or approximately 1.75x10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.3.25x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.2.75x10 14 vg / kg, or approximately 1.75x10 14 vg / kg ~ approx. 2.5x10 14vg / kg, or approximately 1.75x10 14 vg / kg~approx.2.25x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.2.0x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg ~ approx. 1.0x10 15 , or approximately 2.0x10 14 vg / kg~6.0x10 14 , or approximately 2.0x10 14 vg / kg~5.0x10 14 , or approximately 2.0x10 14 vg / kg ~ approx. 4.0x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg~approx.3.25x10 14 vg / kg.

[0059] In one embodiment, the method of the disclosure includes systemically administering rAAV, wherein the systemic route of administration is intravenous, and the dose of rAAV administered is about 2.0 x 10 14 In another embodiment, the methods of the disclosure include systemically administering rAAV, wherein the systemic route of administration is intravenous, and the dose of rAAV administered is about 5.0 x 10 12 vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 10 12 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13 vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 1013 vg / kg, or approximately 3.0 × 10 13 vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14 vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 10 14 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14 vg / kg, or approximately 1 × 10 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, nucleotides 56-4820.

[0060] In any of the methods, combination therapies, or uses disclosed herein, the dose of rAAV may be administered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the dose or rAAV is administered at about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin or AAVrh74.MCK.microdystrophin. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin, nucleotides 56-4820 of SEQ ID NO:5.

[0061] In any of the methods, combination therapies, or medicaments disclosed herein, the dose of rAAV may be administered by injection, infusion, or implantation. For example, the dose of rAAV may be administered by infusion over about 1 hour. Additionally, the dose of rAAV may be administered intravenously via a peripheral limb vein, such as a peripheral arm vein or a peripheral leg vein. Alternatively, the infusion may be administered over about 30 minutes, about 1.5 hours, about 2 hours, about 2.5 hours, or about 3 hours. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin 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. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0062] The rAAV administered by any of the methods, combination therapies, or uses of the disclosure may comprise the human microdystrophin nucleotide sequence of SEQ ID NO: 1, and the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. Additionally, the rAAV administered by any of the methods of the disclosure may comprise the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. For example, the rAAV may comprise the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is the AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6.

[0063] In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0064] In any of the methods, combination therapies or uses of the disclosure, the rAAV administered is of the serotype for AAVrh.74.

[0065] In some embodiments, the disclosed methods, combination therapies, or uses treat Duchenne muscular dystrophy or Becker muscular dystrophy. An exemplary embodiment is a method, combination therapy, or medicament for treating Duchenne muscular dystrophy or Becker muscular dystrophy in need thereof, comprising administering a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, wherein the route of administration is intravenous infusion, and the dose of rAAV administered is about 2 x 10 over about 1 hour. 14 vg / kg, and the rAAV vector is the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, or nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, or nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0066] In one embodiment, the present disclosure provides an rAAV comprising a nucleotide sequence of a muscle-specific regulatory element and a nucleotide sequence encoding a micro-dystrophin protein. For example, the nucleotide sequence encodes a functional micro-dystrophin protein, where the nucleotide sequence has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, and the protein retains micro-dystrophin activity. The micro-dystrophin protein provides stability to muscle membranes during muscle contraction; for example, micro-dystrophin functions as a shock absorber during muscle contraction. In one embodiment, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, or nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0067] The present invention also provides an rAAV, wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 1, or its complement, and encodes a functional micro-dystrophin protein.

[0068] In one embodiment, the rAAV is a non-replicating recombinant adeno-associated virus (AAV) designated 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. This vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeats (ITRs) under the control of the MHCK7 promoter / enhancer, microdystrophin, an SV40 intron (SD / SA), and a synthetic polyadenylation (polyA) signal. A schematic diagram of the genome and expression cassette is shown in Figure 1. The AAVrh74 serotype can be used to achieve efficient gene transfer in skeletal and cardiac muscle following IV administration.

[0069] In another embodiment, the disclosure provides a method of treating limb-girdle muscular dystrophy in a human subject in need thereof, comprising administering an rAAV comprising the nucleotide sequence of SEQ ID NO:44.

[0070] In another aspect, the present disclosure provides a method, combination therapy, or use for treating limb-girdle muscular dystrophy in a subject in need thereof, comprising administering to a subject a dose of about 5.0 x 10 mAb of the compound of formula (I) or (II) based on supercoiled plasmid as a quantitative standard. 13 vg / kg or approximately 2.0 × 10 14 vg / kg, or approximately 1.85 x 10 based on linearized plasmid as a quantification standard. 13 vg / kg or approximately 7.41 x 10 13The present disclosure provides a method, combination therapy, or use comprising administering to a subject an rAAV intravenous infusion at a dose of 1000 mg / kg over about 1-2 hours, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO: 44. In another aspect, the disclosure describes a method of expressing a beta-sarcoglycan gene in cells of a subject, comprising administering to the subject an scAAVrh74.MHCK7.hSGCB construct comprising a nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO: 19. In one aspect, the disclosure provides a method of increasing beta-sarcoglycan-positive fibers and / or decreasing CK levels in muscle tissue of a subject, comprising administering to the subject an scAAVrh74.MHCK7.hSGCB construct nucleotide sequence at least 90%, 95%, or 99% identical to SEQ ID NO: 44.

[0071] In another aspect, described herein is a recombinant AAV vector comprising a polynucleotide sequence encoding β-sarcoglycan. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan comprises a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to, for example, the nucleotide sequence set forth in SEQ ID NO: 45, and encodes a protein that retains β-sarcoglycan activity. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan comprises the nucleotide sequence set forth in SEQ ID NO: 45. In some embodiments, the polynucleotide sequence encoding β-sarcoglycan consists of the nucleotide sequence set forth in SEQ ID NO: 45.

[0072] In another aspect, the recombinant AAV vector described herein comprises a polynucleotide sequence encoding a β-sarcoglycan that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, or 99% sequence identical to the amino acid sequence of SEQ ID NO: 46, and the protein retains β-sarcoglycan activity.

[0073] In another aspect, the recombinant AAV vector described herein comprises a polynucleotide sequence encoding a β-sarcoglycan having the amino acid sequence of SEQ ID NO:46.

[0074] In another aspect, described herein is a recombinant AAV vector comprising a polynucleotide sequence encoding a functional β-sarcoglycan comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 45, or its complement.

[0075] In certain embodiments, the disclosure provides methods, combination therapies, or uses for treating LGMD2E in a human subject in need thereof, comprising administering rAAV scAAVrh74.MHCK7.HSGCB. For example, in any of the methods for treating LGMD2E, scAAVrh74.MHCK7.HSGCB is administered at a concentration of about 0.5×10 14 vg / kg or approximately 2 × 10 14 The doses described herein for scAAVrh74.MHCK7.HSGCB, including those immediately above, are based on the use of supercoiled qPCR standards. 14 vg / kg and 2x10 14 vg / kg is 1.85 x 10 when measured using a linear qPCR standard 13 and 7.41 x 10 13 Corresponds to vg / kg.

[0076] In certain embodiments, in any of the methods, combination therapies or uses for treating limb-girdle muscular dystrophy in a human subject, the human subject is afflicted with LGMD2E and the rAAV is administered in an amount of about 2 x 10 14 The rAAV is administered by intravenous infusion at a dose of 1000 mg / kg, and the rAAV is scAAVrh74.MHCK7.HSGCB, which comprises the nucleotide sequence of SEQ ID NO:44.

[0077] In a related embodiment, the human subject has LGMD2E muscular dystrophy and the rAAV is administered in an amount of about 2x10 14 and scAAVrh74.MHCK7.HSGCB administered by intravenous infusion at a dose of 100 mg / kg / day of scAAVrh74.MHCK7.HSGCB. The method further comprises administering to the subject 1 mg / kg / day of prednisone starting 1 day (12-24 hours) prior to administration of the rAAV and continuing for 60 days with or without tapering.

[0078] In another related embodiment, the human subject has LGMD2E muscular dystrophy and the rAAV is administered at a dose of about 0.5 x 10 14 and scAAVrh74.MHCK7.HSGCB administered by intravenous infusion at a dose of 100 mg / kg / day of scAAVrh74.MHCK7.HSGCB. The method further comprises administering to the subject 1 mg / kg / day of prednisone starting 1 day (12-24 hours) prior to administration of the rAAV and continuing for 30 days with or without tapering.

[0079] It will be appreciated that the 30-day and 60-day administration of prednisone can be tapered by one of skill in the art according to the subject's particular clinical condition, and as otherwise further described herein.

[0080] The term "stringent" refers to conditions generally understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agents) can also be used, although the rate of hybridization will be affected. Where deoxyoligonucleotide hybridization is involved, examples of additional stringent hybridization conditions include 6×SSC at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos). A wash with 0.05% sodium pyrophosphate is included.

[0081] Other agents can be included in the hybridization and wash buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4 (SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents may also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8-7.4, although the rate of hybridization is largely independent of pH under typical ionic strength conditions. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art to take these variables into account and allow DNAs of different sequence similarities to form hybrids.

[0082] The term "muscle-specific control element" refers to a nucleotide sequence that regulates expression of a coding sequence specific for expression in muscle tissue. These control elements include enhancers and promoters. The present disclosure provides constructs that include the muscle-specific control elements MCKH7 promoter, MCK promoter, and MCK enhancer.

[0083] In one aspect, the disclosure provides an rAAV, wherein the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF), a muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), a hybrid alpha-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7), C5-12, a mouse creatine kinase enhancer factor, a fast-twitch skeletal 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).

[0084] For example, the muscle-specific control element is the MHCK7 promoter nucleotide sequence SEQ ID NO:2, or the muscle-specific control element is the MCK nucleotide sequence SEQ ID NO:4. Additionally, in any of the rAAV vectors of the disclosure, the muscle-specific control element nucleotide sequence, e.g., the MHCK7 or MCK nucleotide sequence, is operably linked to a nucleotide sequence encoding a microdystrophin protein. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO:2 or SEQ ID NO:7) is operably linked to a human microdystrophin coding sequence (SEQ ID NO:1) as depicted in the construct presented in Figure 1 or Figure 2 (SEQ ID NO:3) or Figure 13 (SEQ ID NO:9). In another example, the MCK promoter (SEQ ID NO:4) is operably linked to a human microdystrophin coding sequence (SEQ ID NO:1) as depicted in the construct presented in Figure 5 or Figure 6 (SEQ ID NO:5). In another aspect, the disclosure provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, or SEQ ID NO:1 and SEQ ID NO:7. The present invention also provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:4.

[0085] In a further aspect, the disclosure provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:8. For example, the AAVrh74.MHCK7.microdystrophin vector is within and includes the ITR of SEQ ID NO:3 and comprises the nucleotide sequence shown in Figure 2. The rAAV vector comprises the 5' ITR, the MHCK7 promoter, a chimeric intron sequence, the human microdystrophin gene, polyA, and the coding sequence for the 3' ITR. In one embodiment, the vector comprises nucleotides 55-5021 of SEQ ID NO:3. The plasmid set forth in SEQ ID NO:3 further comprises a pGEX plasmid backbone with ampicillin resistance and a pBR322 origin of replication.

[0086] In another aspect, the disclosure provides an rAAV comprising the nucleotide sequence of SEQ ID NO:9, for example, the AAVrh74.MHCK7.microdystrophin vector construct comprises the nucleotide sequence of SEQ ID NO:9 and is shown in FIG. 7. This rAAV vector construct comprises an MHCK7 promoter, a chimeric intron sequence, a coding sequence for the human microdystrophin gene, and a polyA. In one embodiment, the rAAV vector construct further comprises an ITR 5' to the promoter and an ITR 3' to the polyA. In one embodiment, the rAAV is AAVrh74.

[0087] In another aspect, for example, the AAVrh74.MHCK7.microdystrophin vector comprises the nucleotide sequence shown in Figure 9, within and including the ITRs of SEQ ID NO:8. The rAAV vector comprises the 5' ITR, the MHCK7 promoter, a chimeric intron sequence, the human I microdystrophin gene, polyA, and the coding sequence for the 3' ITR. In one embodiment, this vector comprises nucleotides 1-4977 of SEQ ID NO:9. The plasmid shown in SEQ ID NO:3 further comprises a pGEX plasmid backbone with kanamycin resistance and a pBR322 origin of replication.

[0088] In another aspect, the present disclosure provides a plasmid comprising the AAVrh74.MHCK7.microdystrophin vector construct. In one embodiment, the plasmid comprises a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, a coding sequence for the human microdystrophin gene, polyA, and a 3' ITR. In one embodiment, the plasmid comprises a pGEX plasmid backbone containing kanamycin resistance and, optionally, a pBR322 origin of replication. In a specific embodiment, the plasmid is set forth in SEQ ID NO: 8 and depicted in Figures 8 and 9.

[0089] The present disclosure provides a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7. The rAAV vector is of the AAV serotype AAVrh.74.

[0090] The present disclosure also provides an rAAV comprising the AAVrh74.MHCK7.microdystrophin vector construct nucleotide sequence within and including SEQ ID NO:3, the nucleotide sequence within and including SEQ ID NO:8, or the nucleotide sequence set forth in SEQ ID NO:9. This rAAV vector is of the AAV serotype AAVrh.74.

[0091] The rAAV vectors of the present disclosure may be of any AAV serotype, such as serotypes AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0092] The present disclosure also provides pharmaceutical compositions (or sometimes referred to herein simply as "compositions") comprising any of the rAAV vectors of the present disclosure.

[0093] In another embodiment, the present disclosure provides a method for producing rAAV vector particles, comprising culturing cells transfected with any of the rAAV vectors of the present disclosure and recovering the rAAV particles from the supernatant of the transfected cells. The present disclosure also provides viral particles comprising any of the recombinant AAV vectors of the present disclosure.

[0094] In any of the methods for treating muscular dystrophy, the level of a transgene of interest, such as beta-sarcoglycan or microdystrophin gene expression, is increased in cells of the subject after administration of rAAV. Transgene expression of the gene of interest in cells is detected by measuring the level of the protein of interest by Western blot in muscle biopsied before and after administration of rAAV. Specifically, the level of the protein of interest increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of rAAV compared to the level of the protein of interest before administration of rAAV. For example, the level of the protein of interest is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the rAAV compared to the level of the protein of interest before administration of the rAAV.

[0095] Additionally, intracellular microdystrophin gene expression is detected by measuring the level of a protein of interest by immunohistochemistry in muscle biopsies before and after administration of rAAV. The level of the protein of interest is increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of rAAV compared to the level of the protein of interest before administration of rAAV. For example, the level of the protein of interest is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of rAAV compared to the level of the protein of interest before administration of rAAV.

[0096] In any of the methods, combination therapies, or uses for treating muscular dystrophy, the serum CK level in the subject is reduced after administration of rAAV compared to the serum CK level before administration of rAAV. For example, the serum CK level in the subject is reduced by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90% by 60 days after administration of rAAV compared to the serum CK level before administration of rAAV. In particular, in any of the methods, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 87% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV; or in any of the methods, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 72 .... or in any of the methods, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 73% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV; or in any of the methods, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 78% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV; or in any of the methods, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 95% by 60 days after administration of the rAAV compared to serum CK levels before administration of the rAAV. In any of the methods, combination therapies, or uses for treating muscular dystrophy, the number of muscle fibers positive for the protein of interest in the muscle tissue of the subject is increased after administration of the rAAV compared to the number of muscle fibers positive for the protein of interest before administration of the rAAV. For example, the number of muscle fibers positive for a protein of interest is detected by measuring the level of the protein of interest in positive muscle fibers by Western blot or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0097] In any of the methods, combination therapies, or uses for treating muscular dystrophy, administration of rAAV upregulates the expression of DAPC proteins, such as alpha-sarcoglycan or beta-sarcoglycan. For example, the level of alpha-sarcoglycan in a subject increases after administration of rAAV compared to the level of alpha-sarcoglycan before administration of rAAV. In addition, the level of beta-sarcoglycan in a subject increases after administration of rAAV compared to the level of beta-sarcoglycan before administration of rAAV. The level of alpha-sarcoglycan is detected by measuring the level of alpha-sarcoglycan protein or beta-sarcoglycan protein by Western blot or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0098] In any of the methods, combination therapies or uses for treating muscular dystrophy, disease progression in the subject is slowed following administration of the rAAV as measured by any of the 6-minute walk test, stand time, 4-step climb, 4-step climb, North Star Ambulatory Assessment (NSAA), timed 10 meter test, timed 100 meter test, handheld dynamometry (HHD), Timed Up and Go, and / or Gross Motor Subtest Scaled (Bayley-III) score.

[0099] For example, in any of the methods, combination therapies, or uses, the subject has an improvement of at least 6 points in NSAA score at least 270 days after administration of rAAV, compared to the NSAA score before administration of rAAV. Further, in any of the methods, combination therapies, or uses, the subject has an improvement of at least about 0.8 seconds in rise time at least 270 days after administration of rAAV, compared to the rise time before administration of rAAV. In addition, in any of the methods, combination therapies, or uses, the subject has an improvement of at least about 1.2 seconds in the 4-step rise time test at least 270 days after administration of rAAV, compared to the 4-step rise time test before administration of rAAV. In addition, in any of the methods, combination therapies, or uses, the subject has an improvement of at least about 7 seconds in the 100m timed test at least 270 days after administration of rAAV, compared to the 100m timed test before administration of rAAV.

[0100] In another embodiment, the disclosure provides a method, composition, combination therapy, or use for expressing a micro-dystrophin gene in a patient's cells, comprising administering to the patient an AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, expression of the micro-dystrophin gene in a patient's cells is detected by measuring micro-dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. Additionally, micro-dystrophin gene expression is measured in a patient by detecting a greater number of vector genomes per nucleus, with one vector genome per nucleus being approximately 50% micro-dystrophin expression and greater than one copy per nucleus being 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.

[0101] In further embodiments, the disclosure provides methods, compositions, combination therapies, or uses for reducing serum CK levels in a patient in need thereof, the methods comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. For example, the patient's serum CK level is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, by 60 days after administration of the rAAV, compared to the serum CK level before administration of the rAAV. In particular, the serum CK level in the subject is reduced by about 87% by 60 days after administration of the rAAV compared to the serum CK level before administration of the rAAV; or in any of the methods, compositions, combination therapies, or uses for treating muscular dystrophy disclosed herein, the serum CK level in the subject is reduced by about 72% by 60 days after administration of the rAAV compared to the serum CK level before administration of the rAAV; or in any of the methods, compositions, combination therapies, or uses for treating muscular dystrophy disclosed herein, the serum CK level in the subject is reduced by about 72% by 60 days after administration of the rAAV compared to the serum CK level before administration of the rAAV. or in any of the methods, compositions, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 73% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods, compositions, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 78% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or in any of the methods, compositions, combination therapies, or uses for treating muscular dystrophy disclosed herein, serum CK levels in the subject are reduced by about 95% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV.

[0102] The present disclosure also provides methods, compositions, combination therapies, or uses for increasing microdystrophin-positive fibers in muscle cells of a patient, comprising administering to the patient the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, the number of beta-sarcoglycan-positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after rAAV administration. Additionally, microdystrophin gene expression is measured in the patient by detecting a higher number of vector genomes per nucleus, with one vector genome per nucleus representing approximately 50% microdystrophin expression and greater than one copy per nucleus corresponding to a microdystrophin expression level. For example, the cells have 1.2 vector copies per nucleus, or 1.3 vector copies per nucleus, or 1.4 vector copies per nucleus, or 1.5 vector copies per nucleus, or 1.6 vector copies per nucleus, or 1.7 vector copies per nucleus, or 1.8 vector copies per nucleus, or 1.9 vector copies per nucleus.

[0103] In another embodiment, the disclosure provides a method, composition, combination therapy, or use for increasing alpha-sarcoglycan expression in a patient in need thereof, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. For example, alpha-sarcoglycan levels are detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.

[0104] Additionally, the disclosure provides methods, compositions, combination therapies, or uses for increasing beta-sarcoglycan expression in a patient in need thereof, comprising administering to the patient the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. For example, beta-sarcoglycan levels are detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after rAAV administration.

[0105] The disclosure also provides methods, compositions, combination therapies, or uses for treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, comprising administering to the patient an AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, so as to slow disease progression in the subject as measured by any of the 6-minute walk test, stand time, 4-step climb, 4-step climb, North Star Ambulatory Assessment (NSAA), timed 10 meter test, timed 100 meter test, handheld dynamometry (HHD), Timed Up and Go, and / or Gross Motor Subtest Scaled (Bayley-III) score.

[0106] For example, in any of the methods, compositions, combination therapies, or uses, the subject has an improvement of at least 6 points in NSAA score at least 270 days after administration of rAAV, compared to the NSAA score before administration of rAAV. Further, in any of the methods, the subject has an improvement of at least about 0.8 seconds in rise time at least 270 days after administration of rAAV, compared to the rise time before administration of rAAV. Additionally, in any of the methods, the subject has an improvement of at least about 1.2 seconds in the 4-step rise time test at least 270 days after administration of rAAV, compared to the 4-step rise time test before administration of rAAV. Additionally, in any of the methods, the subject has an improvement of at least about 7 seconds in the 100m timed test at least 270 days after administration of rAAV, compared to the 100m timed test before administration of rAAV.

[0107] "Fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and the abnormal repair process of tissues upon injury, including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas. Deposited ECM components include fibronectin and collagen, such as collagen 1, collagen 2, or collagen 3.

[0108] The present disclosure also provides a method of reducing or preventing fibrosis in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of nucleotides 55-5021 of SEQ ID NO: 3. In another embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO: 9. In another embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin from nucleotides 1 to 4977 of SEQ ID NO: 8 or from nucleotides 56 to 5066 of SEQ ID NO: 6. In a further embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO: 5.

[0109] In another embodiment, the present disclosure provides a method for preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAV74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, any of the rAAVs of the present disclosure can be administered to a subject suffering from muscular dystrophy to prevent fibrosis, e.g., an rAAV of the present disclosure expressing a human microdystrophin protein is administered before fibrosis is observed in the subject. In addition, an rAAV of the present disclosure expressing a human microdystrophin gene can be administered to a subject at risk of developing fibrosis, such as a subject suffering from or diagnosed with a muscular dystrophy, e.g., DMD. The rAAV of the present disclosure can be administered to subjects suffering from muscular dystrophy to prevent de novo fibrosis in these subjects.

[0110] The present disclosure 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 subjects suffering from or diagnosed with muscular dystrophy, e.g., DMD. rAAV can be administered to subjects suffering from muscular dystrophy who have already developed fibrosis to prevent new fibrosis in these subjects.

[0111] The present disclosure also provides a method of increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6.

[0112] The present invention contemplates administering the rAAV vectors of the invention to patients diagnosed with DMD before fibrosis is observed in the subject, or before muscle strength is reduced, or before muscle mass is reduced.

[0113] The present disclosure also includes administering to subjects suffering from muscular dystrophy who have already developed fibrosis an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, to prevent new fibrosis in these subjects or reduce fibrosis in these subjects. The disclosure also includes administering to a subject suffering from muscular dystrophy who already has reduced muscle strength or reduced muscle mass an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 to protect the muscle from further damage.

[0114] In any of the methods of the present disclosure, the subject may be suffering from a muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0115] In other embodiments of any of the disclosed methods described herein, the serum CK level in the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the rAAV by a percent level selected from the group consisting of: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 85% by 270 days post-dose; c) at least 72, 73, 74, or 95% by 180 days post-dose; d) at least 87, 88, 93, or 95% by 90 days post-dose; e) at least 70% by 270 days post-dose; f) 70–95% by 90, 180, or 270 days after administration; g) at least about 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration; and h) at least about 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration.

[0116] In another embodiment, the present disclosure provides a composition for treating muscular dystrophy in a human subject in need thereof, the composition comprising a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, wherein the composition is formulated for a systemic route of administration, and the dose of the rAAV is about 1 x 10 14 vg / kg ~ approx. 4×10 14 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0117] For example, the composition of the present disclosure may be about 5.0x10 12 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg~1.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg~approx.2.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 12 vg / kg~approx.5.0x10 13 vg / kg, or approximately 5.0 x 10 12 vg / kg~approx.2.0x10 13 vg / kg, or approximately 5.0 x 10 12 vg / kg ~ approx. 1.0x10 13 vg / kg, or 1.0x10 14 vg / kg ~ approx. 1.0x10 15 vg / kg, or 1.0x10 13 vg / kg ~ approx. 1.0x1014 vg / kg, or approximately 1.0 x 10 13 vg / kg~1.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.2.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.5.0x10 13 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.5.0x10 14 vg / kg, or approximately 1.0 x 10 13 vg / kg~approx.6.0x10 14 vg / kg, or 1.0x10 13 vg / kg ~ approx. 1.0x10 15 vg / kg, or 5.0x10 13 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~1.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.2.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg ~ approx. 1.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.3.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.5.0x10 14 vg / kg, or approximately 5.0 x 10 13 vg / kg~approx.6.0x10 14 vg / kg, or 5.0x10 13 vg / kg ~ approx. 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg~approx.6.0x10 14 vg / kg, or 1.0x10 14 vg / kg~approx.5.0x10 14 vg / kg, or 1.0x10 14vg / kg ~ approx. 4.0x10 14 vg / kg, or 1.0x10 14 vg / kg ~ approx. 1.0x10 15 vg / kg, or 1.0x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.0 x 10 14 vg / kg ~ approx. 2.5x10 14 vg / kg, or 1.0x10 14 vg / kg~approx.2.0x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~6.0x10 14 , or approximately 1.25x10 14 vg / kg~5.0x10 14 , or approximately 1.25x10 14 vg / kg~4.0x10 14 , or approximately 1.25x10 14 vg / kg~1.0x10 15 , or approximately 1.25x10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.25x10 14 vg / kg~approx.2.75x10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25x10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25x10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 1.25x10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5x10 14 vg / kg ~ approx. 1.0x10 15 vg / kg, or approximately 1.5x10 14 vg / kg~6.0x10 14 , or about 1.5x1014 vg / kg~5.0x10 14 , or about 1.5x10 14 vg / kg~4.0x10 14 , or about 1.5x10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.3.25x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.2.75x10 14 vg / kg, or approximately 1.5x10 14 vg / kg ~ approx. 2.5x10 14 vg / kg, or approximately 1.5x10 14 vg / kg~approx.2.0x10 14 vg / kg, or 1.75x10 14 vg / kg ~ approx. 1.0x10 15 vg / kg, or approximately 1.75x10 14 vg / kg~6.0x10 14 , or approximately 1.75x10 14 vg / kg~5.0x10 14 , or approximately 1.75x10 14 vg / kg~4.0x10 14 , or approximately 1.75x10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.3.25x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.3.0x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.2.75x10 14 vg / kg, or approximately 1.75x10 14 vg / kg ~ approx. 2.5x10 14vg / kg, or approximately 1.75x10 14 vg / kg~approx.2.25x10 14 vg / kg, or approximately 1.75x10 14 vg / kg~approx.2.0x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg~1.0x10 15 , or approximately 2.0x10 14 vg / kg~6.0x10 14 , or approximately 2.0x10 14 vg / kg~5.0x10 14 , or approximately 2.0x10 14 vg / kg ~ approx. 4.0x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg~approx.3.75x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg~approx.3.5x10 14 vg / kg, or approximately 2.0 x 10 14 vg / kg~approx.3.25x10 14 The rAAV comprises a dose of rAAV in an amount of 1000 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0118] In one embodiment, the composition of the present disclosure is formulated for intravenous administration and contains about 2.0 x 10 14 In another embodiment, the composition of the present disclosure is formulated for intravenous administration and comprises a dose of about 5.0x10 vg / kg of rAAV. 12 vg / kg, or approximately 6.0 x 10 12 vg / kg, or approximately 7.0x10 12vg / kg, or approximately 8.0 x 10 12 vg / kg, or approximately 9.0x10 12 vg / kg, or approximately 1.0 x 10 13 vg / kg, or approximately 1.25x10 13 vg / kg, or approximately 1.5x10 13 vg / kg, or approximately 1.75x10 13 vg / kg, or approximately 2.25x10 13 vg / kg, or approximately 2.5x10 13 vg / kg, or approximately 2.75x10 13 vg / kg, or approximately 3.0 x 10 13 vg / kg, or approximately 3.25x10 13 vg / kg, or approximately 3.5x10 13 vg / kg, or approximately 3.75x10 13 vg / kg, or approximately 4.0 x 10 13 vg / kg, or approximately 5.0 x 10 13 vg / kg, or approximately 6.0 x 10 13 vg / kg, or approximately 7.0x10 13 vg / kg, or approximately 8.0 x 10 13 vg / kg, or approximately 9.0x10 13 vg / kg, or approximately 1.0 x 10 14 vg / kg, or approximately 1.25x10 14 vg / kg, or approximately 1.5x10 14 vg / kg, or approximately 1.75x10 14 vg / kg, or approximately 2.25x10 14 vg / kg, or approximately 2.5x10 14 vg / kg, or approximately 2.75x10 14 vg / kg, or approximately 3.0 x 10 14 vg / kg, or approximately 3.25x10 14 vg / kg, or approximately 3.5x10 14 vg / kg, or approximately 3.75x10 14 vg / kg, or approximately 4.0 x 10 14 vg / kg, or approximately 5.0 x 10 14 vg / kg, or approximately 6.0 x 10 14 vg / kg, or approximately 1x10 15The rAAV comprises a dose of rAAV in an amount of 1000 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In another embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0119] In any of the compositions of the present disclosure, the dose of rAAV is delivered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the composition comprises a dose of rAAV delivered at about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In another embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0120] The composition of the present disclosure is formulated for administration by injection, infusion or implantation.For example, the composition is formulated for administration by infusion over about 1 hour.In addition, the composition of the present disclosure is formulated for intravenous administration through peripheral limb veins, such as peripheral arm veins or peripheral leg veins.Alternatively, the infusion can be administered over about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0121] Any of the compositions of the disclosure include an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7, or an rAAV comprising the AAVrh74.MHCK7 microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0122] In particular, compositions of the present disclosure are for treating Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present disclosure provides a composition for treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, the composition comprising a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin, the composition formulated for administration by intravenous infusion over about 1 hour, and the dose of rAAV administered is about 2×10 14 vg / kg, and the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6.

[0123] In another embodiment, the present disclosure also provides a composition comprising an rAAV vector for reducing fibrosis in a subject in need thereof. Additionally, the present disclosure provides a composition comprising an rAAV vector for preventing fibrosis in a subject suffering from muscular dystrophy.

[0124] The present disclosure also provides compositions comprising an rAAV for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy. In a further aspect, the present disclosure provides compositions comprising any of the rAAVs of the present disclosure for the treatment of muscular dystrophy.

[0125] In other embodiments of any of the compositions of the present disclosure, after administration of the composition to a human subject in need of treatment for muscular dystrophy, the serum CK level in the subject is reduced by a percentage level selected from the group consisting of: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46, 55, 70, or 85% by 270 days post-dose; c) at least 72, 73, 74, or 95% by 180 days post-dose; d) at least 87, 99, 93, or 95% by 90 days post-dose; e) at least 70% by 270 days post-dose; f) 70–95% by 90, 180, or 270 days after administration; g) at least 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration; and h) 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration.

[0126] In another embodiment, the present disclosure provides a use of a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin for the preparation of a medicament for the treatment of muscular dystrophy in a human subject in need thereof, wherein the medicament is formulated for a systemic route of administration, and the dose of rAAV is about 1 x 10 14 vg / kg ~ approx. 4×10 14 vg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0127] For example, the drug is approximately 5.0 x 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 2.0×10 13 vg / kg, or approximately 5.0 × 10 12 vg / kg ~ approx. 1.0×10 13 vg / kg, or 1.0 x 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 x 10 13 vg / kg ~ approx. 1.0×1014 vg / kg, or approximately 1.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 13 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 1.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 x 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg~1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 1.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 5.0×10 14 vg / kg, or approximately 5.0 × 10 13 vg / kg ~ approx. 6.0×10 14 vg / kg, or 5.0 × 10 13 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 x 10 14 vg / kg ~ approx. 6.0×10 14 vg / kg, or 1.0 x 10 14 vg / kg ~ approx. 5.0×10 14 vg / kg, or 1.0 x 10 14vg / kg ~ approx. 4.0×10 14 vg / kg, or 1.0 x 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or 1.0 x 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.0 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or 1.0 x 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg~6.0×10 14 , or approximately 1.25 × 10 14 vg / kg~5.0×10 14 , or approximately 1.25 × 10 14 vg / kg~4.0×10 14 , or approximately 1.25 × 10 14 vg / kg~1.0×10 15 , or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.25 x 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or 1.5 × 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.5 × 10 14 vg / kg~6.0×10 14 , or approximately 1.5 × 1014 vg / kg~5.0×10 14 , or approximately 1.5 × 10 14 vg / kg~4.0×10 14 , or approximately 1.5 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.5×10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or 1.75 x 10 14 vg / kg ~ approx. 1.0×10 15 vg / kg, or approximately 1.75 × 10 14 vg / kg~6.0×10 14 , or approximately 1.75 × 10 14 vg / kg~5.0×10 14 , or approximately 1.75 × 10 14 vg / kg~4.0×10 14 , or approximately 1.75 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 3.0×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.75×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.5×10 14vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.25×10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg ~ approx. 2.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg~1.0×10 15 , or approximately 2.0 × 10 14 vg / kg~6.0×10 14 , or approximately 2.0 × 10 14 vg / kg~5.0×10 14 , or approximately 2.0 × 10 14 vg / kg ~ approx. 4.0×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.75×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.5×10 14 vg / kg, or approximately 2.0 × 10 14 vg / kg ~ approx. 3.25×10 14 The rAAV comprises a dose of rAAV in an amount of 1000 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0128] In one embodiment, the agent of the present disclosure is administered by intravenous route and at a dose of rAAV of about 2.0×10 14 In another embodiment, the medicament of the present disclosure is formulated for systemic administration, wherein the systemic administration route is intravenous, and the dose of rAAV is about 5.0 x 10 12vg / kg, or approximately 6.0 × 10 12 vg / kg, or approximately 7.0 × 10 12 vg / kg, or approximately 8.0 × 10 12 vg / kg, or approximately 9.0 × 10 12 vg / kg, or approximately 1.0 × 10 13 vg / kg, or approximately 1.25 × 10 13 vg / kg, or approximately 1.5 × 10 13 vg / kg, or approximately 1.75 × 10 13 vg / kg, or approximately 2.25 × 10 13 vg / kg, or approximately 2.5 × 10 13 vg / kg, or approximately 2.75 × 10 13 vg / kg, or approximately 3.0 × 10 13 vg / kg, or approximately 3.25 × 10 13 vg / kg, or approximately 3.5 × 10 13 vg / kg, or approximately 3.75 × 10 13 vg / kg, or approximately 4.0 × 10 13 vg / kg, or approximately 5.0 × 10 13 vg / kg, or approximately 6.0 × 10 13 vg / kg, or approximately 7.0 × 10 13 vg / kg, or approximately 8.0 × 10 13 vg / kg, or approximately 9.0 × 10 13 vg / kg, or approximately 1.0 × 10 14 vg / kg, or approximately 1.25 × 10 14 vg / kg, or approximately 1.5 × 10 14 vg / kg, or approximately 1.75 × 10 14 vg / kg, or approximately 2.25 × 10 14 vg / kg, or approximately 2.5 × 10 14 vg / kg, or approximately 2.75 × 10 14 vg / kg, or approximately 3.0 × 10 14 vg / kg, or approximately 3.25 × 10 14 vg / kg, or approximately 3.5 × 10 14 vg / kg, or approximately 3.75 × 10 14 vg / kg, or approximately 4.0 × 10 14 vg / kg, or approximately 5.0 × 10 14 vg / kg, or approximately 6.0 × 10 14vg / kg, or approximately 1 × 10 15 The rAAV is formulated for systemic administration at a dose of rAAV that is 0.1 mg / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin of SEQ ID NO:5, nucleotides 56-4820.

[0129] In any of the uses of the present disclosure, 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, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In certain embodiments, the dose of rAAV is about 10 mL / kg. In one embodiment, the rAAV is AAVrh74.MHCK7.microdystrophin. In one embodiment, the AAVrh74.MHCK7.microdystrophin is AAVrh74.MHCK7.microdystrophin of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6. In one embodiment, the rAAV is AAVrh74.MCK.microdystrophin. In one embodiment, the AAVrh74.MCK.microdystrophin is AAVrh74.MCK.microdystrophin from nucleotides 56 to 4820 of SEQ ID NO:5.

[0130] In any of the uses of the present disclosure, the agent is formulated for administration by injection, infusion, or implantation. For example, the agent is formulated for administration by infusion over about 1 hour. In addition, the agent is formulated for intravenous administration via a peripheral limb vein, such as a peripheral arm vein or a peripheral leg vein. Alternatively, the infusion can be administered over about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0131] In any of the uses of the disclosure, the agent comprises an rAAV comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7, or the AAVrh74.MHCK7 microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0132] A particular use of the present disclosure is for the preparation of a medicament for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present disclosure provides the use of a dose of recombinant adenovirus-associated (rAAV) rAAV.MHCK7.microdystrophin 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 1 hour, and the dose of rAAV administered is about 2×10 14 vg / kg, and the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 to 5021 of SEQ ID NO:3, nucleotides 1 to 4977 of SEQ ID NO:8, or nucleotides 56 to 5022 of SEQ ID NO:6.

[0133] In a further embodiment, the present disclosure provides a use of rAAV for the preparation of a medicament for reducing fibrosis in a subject in need thereof. For example, the subject in need may be suffering from a muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0134] In another embodiment, the present disclosure provides a use of rAAV for the preparation of a medicament for preventing fibrosis in a subject suffering from muscular dystrophy.

[0135] Additionally, the present disclosure 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.

[0136] The present invention also provides the use of rAAV for the preparation of a medicament for the treatment of muscular dystrophy.

[0137] The disclosure provides for the use of an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7 for the preparation of a medicament for the treatment of muscular dystrophy, or the use of an rAAV vector comprising the AAVrf74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the treatment of muscular dystrophy.

[0138] In any other embodiment of the uses of the present disclosure, the serum CK level in the subject is reduced after administration of the rAAV to the subject, compared to the serum CK level before administration of the rAAV, by a percent level selected from the group consisting of: a) at least 78% by 90, 180, or 270 days after administration; b) at least 46, 55, 70, or 95% by 270 days post-dose; c) at least 72, 73, 74, or 95% by 180 days post-dose; d) at least 87, 88, 93, or 95% by 90 days post-dose; e) at least 70% by 270 days post-dose; f) 70–95% by 90, 180, or 270 days after administration; g) at least 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration; and h) 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% by 90, 180, or 270 days after administration.

[0139] In any of the combination therapies, compositions for treating muscular dystrophy, or drugs for treating muscular dystrophy, the level of microdystrophin gene expression in the subject's cells increases after administration of the composition or drug. Expression of the microdystrophin gene in cells is detected by measuring microdystrophin protein levels by Western blot in muscle biopsied before and after administration of the composition or drug. In particular, the level of microdystrophin protein increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of the composition or drug, compared to the level of microdystrophin before administration of the composition or drug. For example, the level of microdystrophin protein is increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition compared to the level of microdystrophin before administration of the composition or agent.

[0140] Additionally, intracellular microdystrophin gene expression is detected by measuring microdystrophin protein levels by immunohistochemistry in muscle biopsies before and after administration of the composition or agent. Microdystrophin protein levels are increased by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90% after administration of the rAAV compared to microdystrophin levels before administration of the composition or agent. For example, microdystrotrophin protein levels are increased by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85% after administration of the composition or agent compared to microdystrophin levels before administration of the composition or agent.

[0141] In any of the combination therapies, compositions for treating muscular dystrophy, and uses of drugs for treating muscular dystrophy, serum CK levels in a subject are reduced after administration of rAAV compared to serum CK levels before administration of the composition or drug. For example, serum CK levels in a 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% by 60 days after administration of rAAV compared to serum CK levels before administration of the composition or drug. In particular, in any of the combination therapies, compositions, and uses of drugs for treating muscular dystrophy of the present disclosure, serum CK levels in a subject are reduced by about 87% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV. In any of the combination therapies, compositions for treating muscular dystrophy, or drugs for treating muscular dystrophy of the present disclosure, serum CK levels in the subject are reduced by about 72% by 60 days after administration of rAAV compared to serum CK levels before administration of the composition or drug, or in any of the combination therapies, compositions for treating muscular dystrophy, or drugs for treating muscular dystrophy of the present disclosure, serum CK levels in the subject are reduced by about 73% by 60 days after administration of rAAV compared to serum CK levels before administration of rAAV, or In either the use of the composition for treating dystrophy or the agent for treating muscular dystrophy, serum CK levels in the subject are reduced by about 78% by 60 days after administration of rAAV compared to serum CK levels before administration of the combination therapy, composition, or agent; or in either the use of the combination therapy, composition for treating muscular dystrophy, or agent for treating muscular dystrophy of the present disclosure, serum CK levels in the subject are reduced by about 95% by 60 days after administration of rAAV compared to serum CK levels before administration of the combination therapy, composition, or agent.In any combination therapy, composition for treating muscular dystrophy, or drug for treating muscular dystrophy, the number of microdystrophin-positive fibers in the muscle tissue of a subject increases after administration of the combination therapy, composition, or drug, compared with the number of microdystrophin-positive fibers before administration of rAAV.For example, the number of microdystrophin-positive fibers is detected by measuring microdystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the combination therapy, composition, or drug.

[0142] In any combination therapy, composition for treating muscular dystrophy, or drug for treating muscular dystrophy, administration of the composition or drug upregulates the expression of DAPC proteins such as alpha-sarcoglycan or beta-sarcoglycan. For example, the level of alpha-sarcoglycan in a subject increases after administration of rAAV compared to the level of alpha-sarcoglycan before administration of the combination therapy, composition, or drug. The level of alpha-sarcoglycan is detected by measuring the level of alpha-sarcoglycan protein or beta-sarcoglycan protein by Western blot or immunohistochemistry in muscle biopsies before and after administration of rAAV.

[0143] In any of the combination therapies, compositions for treating muscular dystrophy, or drug use for treating muscular dystrophy, disease progression in a subject can be monitored by a 6-minute walk test, stand-up time, four-step climb, four-step climb, North Star Ambulatory Delay after administration of rAAV as measured by any of the following: National Standing Order Assessment (NSAA), 10-meter timed test, 100-meter timed test, Handheld Dynamometry (HHD), Timed Up and Go, and / or Gross Motor Subtest Scaled (Bayley-III) score.

[0144] For example, after administration of any of the combination therapies, compositions for treating muscular dystrophy, or uses of agents for treating muscular dystrophy, the subject has an improvement of at least 6 points in NSAA score at least 270 days after administration of the composition or agent, compared to the NSAA score before administration of rAAV. Further, in any of the methods, combination therapies, compositions for treating muscular dystrophy, or uses of agents for treating muscular dystrophy, the subject has an improvement of at least about 0.8 seconds in rise time at least 270 days after administration of rAAV, compared to the rise time before administration of the combination therapy, composition, or agent. Additionally, in any of the methods, combination therapies, compositions, or uses of the present disclosure, the subject has an improvement of at least about 1.2 seconds in the four-step rise test time at least 270 days after administration of rAAV, compared to the four-step rise test time before administration of the combination therapy, composition, or agent. Additionally, in any of the methods, combination therapies, compositions or uses disclosed herein, the subject has an improvement of at least about 7 seconds in the 100m timed test at least 270 days after administration of rAAV compared to the 100m timed test before administration of rAAV.

[0145] In another embodiment, the disclosure provides a composition for expressing a micro-dystrophin gene in patient cells comprising the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In a further embodiment, the disclosure provides use of a dose of the composition AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, for the preparation of a medicament for expressing a micro-dystrophin gene in patient cells. For example, expression of the micro-dystrophin gene in patient cells is detected by measuring micro-dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.micro-dystrophin construct. In addition, the expression of the micro-dystrophin gene is measured in patients by detecting a higher number of vector genomes per nucleus, where one vector genome per nucleus is approximately 50% micro-dystrophin expression, and more than one copy per nucleus is consistent with the micro-dystrophin expression level. 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.

[0146] In further embodiments, the disclosure provides a composition for reducing serum CK levels in a patient in need thereof, the composition comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. Additionally, the disclosure provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6 for the preparation of a medicament for reducing serum CK levels in a patient in need thereof. For example, the serum CK level in the patient is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%, by 60 days after administration of the composition or drug, compared to the serum CK level before administration of the composition or drug. In particular, the serum CK level in the subject is reduced by about 87% by 60 days after administration of the composition or agent compared to the serum CK level before administration of the composition or agent, or is reduced by about 72% by 60 days after administration of the composition or agent compared to the serum CK level before administration of the composition or agent, or is reduced by about 73% by 60 days after administration of the composition or agent compared to the serum CK level before administration of the composition or agent, or is reduced by about 78% by 60 days after administration of the composition or agent compared to the serum CK level before administration of the composition or agent, or is reduced by about 95% by 60 days after administration of the composition or agent compared to the serum CK level before administration of the composition or agent.

[0147] The present disclosure also provides a composition for increasing microdystrophin-positive fibers in patient muscle tissue, comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. Additionally, the present disclosure provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, for the preparation of a medicament for increasing microdystrophin-positive fibers in patient muscle tissue. For example, the number of microdystrophin-positive fibers is detected by measuring dystrophin protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or medicament. In addition, the expression of the micro-dystrophin gene is measured in patients by detecting a higher number of vector genomes per nucleus, where one vector genome per nucleus is approximately 50% micro-dystrophin expression, and more than one copy per nucleus is consistent with the micro-dystrophin expression level. 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.

[0148] In another embodiment, the present disclosure provides a composition for increasing alpha-sarcoglycan expression in a patient in need thereof, comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. The present disclosure also provides use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, for the preparation of a medicament for increasing alpha-sarcoglycan expression in a patient in need thereof. For example, alpha-sarcoglycan levels are detected by measuring alpha-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or medicament.

[0149] Additionally, the present disclosure provides a composition for increasing beta-sarcoglycan expression in a patient in need thereof, comprising the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6. The present disclosure also provides use of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9, nucleotides 55-5021 of SEQ ID NO: 3, nucleotides 1-4977 of SEQ ID NO: 8, or nucleotides 56-5022 of SEQ ID NO: 6 for the preparation of a medicament for increasing beta-sarcoglycan expression in a patient in need thereof. For example, beta-sarcoglycan levels are detected by measuring beta-sarcoglycan protein levels by Western blot or immunohistochemistry in muscle biopsies before and after administration of the composition or medicament.

[0150] The disclosure also provides for the use of a dose of the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6, for the preparation of a medicament for treating a patient with Duchenne muscular dystrophy or Becker muscular dystrophy, wherein administration of the medicament results in disease progression in the patient, as measured by any of the 6-minute walk test, stand time, 4-step climb, 4-step climb, North Star Ambulatory Assessment (NSAA), timed 10-meter test, timed 100-meter test, handheld dynamometry (HHD), Timed Up and Go, and / or Gross Motor Subtest Scaled (Bayley-III) score.

[0151] For example, the subject has at least a 6-point improvement in NSAA score at least 270 days after administration of the rAAV, compared to the NSAA score before administration of the rAAV. Furthermore, the subject has at least about a 0.8-second improvement in climb time at least 270 days after administration of the rAAV, compared to the climb time before administration of the rAAV. Additionally, the subject has at least about a 1.2-second improvement in the 4-step climb test at least 270 days after administration of the rAAV, compared to the 4-step climb test time before administration of the composition or agent. Additionally, the subject has at least about a 7-second improvement in the 100-meter timed test at least 270 days after administration of the rAAV, compared to the 100-meter timed test before administration of the composition or agent. The present invention provides, for example, the following items. (Item 1) 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated virus (rAAV) and an anti-inflammatory steroid, wherein the rAAV is selected from the group consisting of rAAV.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05. (Item 2) Item 1. The method of item 1, wherein the muscular dystrophy is Duchenne muscular dystrophy and the rAAV is rAAV.MHCK7.microdystrophin. (Item 3) 2. The method of claim 1, wherein the muscular dystrophy is limb-girdle muscular dystrophy and the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05. (Item 4) 4. The method of any one of items 1 to 3, wherein the anti-inflammatory steroid is administered about 24 hours prior to administration of the rAAV. (Item 5) 4. The method of any one of items 1 to 3, wherein the anti-inflammatory steroid is administered at least once daily for about 14 days prior to administration of the rAAV. (Item 6) 6. The method of any one of items 1 to 5, wherein the anti-inflammatory steroid is administered prior to administration of the rAAV and the anti-inflammatory steroid is administered at least once daily from day 1 to day 30 after administration of the rAAV or at least once daily from day 1 to day 60 after administration of the rAAV. (Item 7) 7. The method according to any one of items 1 to 6, wherein the anti-inflammatory steroid is administered orally. (Item 8) 8. The method according to any one of items 1 to 7, wherein the anti-inflammatory steroid is a glucocorticoid. (Item 9) Items 1 to 7, wherein the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort. 10. The method according to any one of the preceding claims. (Item 10) 10. The method of any one of items 1 to 9, further comprising administering an anti-CD20 specific antibody prior to administration of the rAAV. (Item 11) 11. The method of claim 10, wherein the anti-CD20 specific antibody is administered at least 7 days prior to administration of the rAAV. (Item 12) 12. The method of claim 10 or 11, wherein the anti-CD20 specific antibody is administered at least 14 days prior to administration of the rAAV. (Item 13) 11. The method of claim 10, wherein the anti-CD20 specific antibody is administered about 14 days before administration of the rAAV, about 7 days before administration of the rAAV, and about 24 hours before administration of the rAAV. (Item 14) 14. The method of any one of items 10 to 13, further comprising the step of administering an anti-CD20 specific antibody after administration of the rAAV. (Item 15) 15. The method according to any one of items 10 to 14, wherein the anti-CD20 specific antibody is rituximab, ocrelizumab or ofatumumab. (Item 16) 16. The method of any one of items 1 to 15, further comprising administering an immunosuppressant macrolide. (Item 17) 17. The method of claim 16, wherein the immunosuppressant macrolide is administered at least once daily for at least three days prior to administration of the rAAV. (Item 18) 18. The method of claim 16 or 17, wherein the immunosuppressant macrolide is administered after administration of the rAAV. (Item 19) 19. The method according to any one of items 16 to 18, wherein the immunosuppressive macrolide is administered orally. (Item 20) 20. The method according to any one of items 16 to 19, wherein the immunosuppressant macrolide is tacrolimus, pinecromus or sirolimus. (Item 21) 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated virus (rAAV) and an immunosuppressive regimen, wherein the immunosuppressive regimen comprises administering one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide, and the rAAV is selected from the group consisting of AAVrh.74.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05. (Item 22) 22. The method of claim 21, wherein the muscular dystrophy is Duchenne muscular dystrophy and the rAAV is rAAV.MHCK7.microdystrophin. (Item 23) The muscular dystrophy is limb-girdle muscular dystrophy, and the rAAV is AAVr 22. The method of claim 21, wherein the vector is h.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. (Item 24) 24. The method of any one of items 21 to 23, wherein the immunosuppressive regimen comprises administering an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide. (Item 25) 25. The method of any one of items 21 to 24, wherein the anti-inflammatory steroid is administered about 24 hours prior to administration of the rAAV. (Item 26) 26. The method of any one of items 21 to 25, wherein the anti-inflammatory steroid is administered prior to administration of the rAAV, and the anti-inflammatory steroid is administered at least once daily from day 1 to day 30 after administration of the rAAV. (Item 27) 27. The method according to any one of items 21 to 26, wherein the anti-inflammatory steroid is administered orally. (Item 28) 28. The method according to any one of items 21 to 27, wherein the anti-inflammatory steroid is a glucocorticoid. (Item 29) 29. The method according to any one of items 21 to 28, wherein the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone or deflazacort. (Item 30) 296. The method of any one of items 21 to 295, wherein the anti-CD20 specific antibody is administered prior to administration of the rAAV. (Item 31) 31. The method of claim 30, wherein the anti-CD20 specific antibody is administered at least 14 days prior to administration of the rAAV. (Item 32) 31. The method of claim 30, wherein the anti-CD20 specific antibody is administered about 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and about 24 hours prior to administration of the rAAV. (Item 33) 33. The method of any one of items 21 to 32, wherein the immunosuppressive regimen further comprises administering an anti-CD20 specific antibody after administration of the rAAV. (Item 34) 34. The method of any one of items 21 to 33, wherein the anti-CD20 antibody is administered by intravenous infusion. (Item 35) 35. The method according to any one of items 21 to 34, wherein the anti-CD20 specific antibody is rituximab, ocrelizumab or ofatumumab. (Item 36) 36. The method of any one of items 21 to 35, wherein the immunosuppressant macrolide is administered at least once daily for at least 3 days prior to administration of the rAAV. (Item 37) 37. The method of any one of items 21 to 36, wherein the immunosuppressive regimen further comprises administering an immunosuppressant macrolide after administration of the rAAV. (Item 38) 38. The method according to any one of items 21 to 37, wherein the immunosuppressant macrolide is administered orally. (Item 39) 39. The method according to any one of items 21 to 38, wherein the immunosuppressive macrolide is tacrolimus, pinecrolimus or sirolimus. (Item 40) 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) selected from the group consisting of AAVrh.74.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and an immunosuppressive regimen, wherein the immunosuppressive regimen is i) orally administering an anti-inflammatory steroid about 24 hours before administration of the rAAV and administering the anti-inflammatory steroid at least once daily from day 1 to day 30 after administration of the rAAV, or from day 1 to day 60 after administration of the rAAV; ii) intravenously administering an anti-CD20 antibody about 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and about 24 hours after administration of the rAAV, and optionally administering the anti-CD20 antibody after administration of the rAAV; iii) orally administering an immunosuppressant macrolide at least once daily for at least three days prior to administration of the rAAV, and optionally administering the immunosuppressant macrolide after administration of the rAAV. (Item 41) 41. The method of claim 40, wherein the muscular dystrophy is Duchenne muscular dystrophy and the rAAV is AAVrh.74.MHCK7.microdystrophin. (Item 42) 41. The method of claim 40, wherein the muscular dystrophy is limb-girdle muscular dystrophy and the rAAV is AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. (Item 43) 43. The method according to any one of items 40 to 42, wherein the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone or deflazacort. (Item 44) 44. The method according to any one of items 40 to 43, wherein the anti-CD20 specific antibody is rituximab, ocrelizumab or ofatumumab. (Item 45) 43. The method according to any one of items 40 to 42, wherein the immunosuppressant macrolide is tacrolimus, pinecromus or sirolimus. (Item 46) 43. The method of any one of items 40 to 42, wherein the anti-inflammatory steroid is prednisolone, the anti-CD20 antibody is rituximab, and the immunosuppressive macrolide is sirolimus. (Item 47) 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising administering to a human subject an AAVrh.74.MHCK7.microdystrophin vector, an AAVrh.74.tMCK.CAPN3 vector, an rAAVrh.74.MHCK7.DYSF vector, an scAAVr vector, or an AAVrh.74.tMCK.CAPN3 vector. A method comprising subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administration of a second dose of a recombinant adenovirus-associated (rAAV) selected from the group consisting of h.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, wherein the subject received a first dose of rAAV prior to being subjected to TPE. (Item 48) 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising: a) administering a first dose of a recombinant adenovirus associated with a human adenovirus selected from the group consisting of AAVrh.74.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05; b) subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE); c) administering a second dose or rAAV. (Item 49) 49. The method of claim 47 or 48, wherein the muscular dystrophy is Duchenne muscular dystrophy and the rAAV is AAVrh.74.MHCK7.microdystrophin. (Item 50) 49. The method of claim 47 or 48, wherein the muscular dystrophy is limb-girdle muscular dystrophy and the rAAV is AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. (Item 51) 51. The method of any one of items 47 to 50, wherein the subject's plasma is subjected to at least two TPEs or at least three TPEs prior to administration of the second dose or rAAV. (Item 52) 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising: a) subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administering a recombinant adenovirus-associated (rAAV); b) administering the rAAV; The method, wherein the rAAV is rAAV.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. (Item 53) 53. The method of claim 52, wherein the subject's plasma is subjected to at least two TPEs, at least three TPEs, at least four TPEs, at least five TPE rAAVs, at least six TPEs, or at least seven TPEs prior to administration. (Item 54) 54. The method of claim 52 or 53, wherein the subject's plasma is subjected to TPE at least 9 days, at least 7 days, 5 days, or 2 days prior to administration of the rAAV. (Item 55) Items 52 to 54, wherein the subject's plasma is subjected to TPE on the day the rAAV is administered. 10. The method according to any one of the preceding claims. (Item 56) 56. The method of any one of items 52 to 55, wherein the subject's plasma is subjected to at least two TPEs, with about 48 hours between the TPEs. (Item 57) 57. The method of any one of items 52 to 56, wherein the subject is administered an anti-inflammatory steroid about 24 hours prior to administration of the rAAV. (Item 58) 58. The method of any one of items 52 to 57, wherein the subject is administered an anti-inflammatory steroid at least once daily from day 1 to day 60 after administration of the rAAV. (Item 59) 59. The method of claim 57 or 58, wherein the anti-inflammatory steroid is administered orally. (Item 60) 60. The method according to any one of items 57 to 59, wherein the anti-inflammatory steroid is a glucocorticoid. (Item 61) 61. The method according to any one of items 57 to 60, wherein the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone or deflazacort. (Item 62) 62. The method of any one of items 1 to 61, wherein the rAAV is administered by a systemic route. (Item 63) The systemic administration route is an intravenous route, and the dose of the rAAV administered is about 2×10 14 Item 63. The method of item 62, wherein the saturation is 0.05 to 0.15 vg / kg. (Item 64) 64. The method of any one of items 1 to 63, wherein the dose of rAAV is administered at a concentration of about 10 mL / kg. (Item 65) 65. The method of any one of items 1 to 64, wherein the rAAV is administered by injection, infusion or implantation. (Item 66) 66. The method of any one of items 1 to 65, wherein the rAAV is administered by infusion over about 1 hour. (Item 67) 66. The method of any one of items 1 to 65, wherein the rAAV is administered by an intravenous route via a peripheral limb vein. (Item 68) 68. The method of any one of items 1 to 67, wherein the rAAV is of the serotype AAVrh.74. (Item 69) 69. The method of any one of items 1 to 68, wherein the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO: 1. (Item 70) 69. The method of any one of items 1 to 69, wherein the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO: 2 or SEQ ID NO: 7. (Item 71) 71. The method of any one of items 1 to 70, wherein the rAAV comprises the nucleotide sequence of SEQ ID NO: 44. (Item 72) 69. The method of any one of paragraphs 1 to 68, wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9 or nucleotides 55 to 5021 of SEQ ID NO: 3. (Item 73) The human subject has Duchenne muscular dystrophy, and the rAAV is administered at a dose of about 2×10 14 69. The method of any one of paragraphs 1 to 68, wherein the rAAV is administered by intravenous infusion over about 1 hour at a dose of 1000 mg / kg of rAAV, and wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 9 or nucleotides 55 to 5021 of SEQ ID NO: 3. (Item 74) 69. The method of any one of items 1 to 68, wherein the muscular dystrophy is LGMD2E and the rAAV is scAAVrh74.MHCK7.HSGCB. (Item 75) The human subject is afflicted with LGMD2E, and the rAAV is administered in an amount of about 2×10 1469. The method of any one of paragraphs 1 to 68, wherein the rAAV is administered by intravenous infusion at a dose of 0.5 mg / kg, and wherein the rAAV is an rAAV that is scAAVrh74.MHCK7.HSGCB comprising the nucleotide sequence of SEQ ID NO: 44. (Item 76) 76. The method of any one of items 1 to 75, wherein the level of microdystrophin gene expression in cells of the subject is increased after administration of the rAAV compared to the level of microdystrophin gene expression before administration of the rAAV. (Item 77) 77. The method of any one of items 1 to 76, further comprising determining the presence of anti-AAVrh.74 antibodies in the serum or plasma of the subject. (Item 78) 78. The method of claim 77, wherein the determining step is performed before the administering step of the immunosuppressive regimen or TPE. (Item 79) 79. The method of claim 78, wherein the determining step is performed prior to administration of any AAV to the subject. (Item 80) 80. The method of claim 79, wherein the determining step is performed before administering aAAVrh.74 to the subject. (Item 81) 81. The method of any one of items 77 to 80, wherein the determination is quantitative. (Item 82) 82. The method of claim 81, further comprising the step of comparing the level of anti-AAVrh.74 antibodies in the serum or plasma of the subject with a positive control. (Item 83) 83. The method of claim 82, wherein the positive control utilizes an anti-AAVrh.74 monoclonal antibody. (Item 84) 84. The method of any one of items 77 to 83, wherein the determining step comprises utilizing an anti-AAVrh.74 monoclonal antibody. (Item 85) 85. The method of any one of items 77 to 84, wherein the determining step comprises utilizing an immunofluorescence assay, an immunohistochemical assay, a Western blot, a direct enzyme-linked immunosorbent assay (ELISA), an indirect ELISA, a sandwich ELISA, a competitive ELISA, a reverse ELISA, a chemiluminescence assay, a radioimmunoassay, or an immunoprecipitation assay. (Item 86) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a VH CDR1 amino acid sequence selected from the group consisting of NYGMN (SEQ ID NO: 20), DYGMN (SEQ ID NO: 22), YTFTNYGMN (SEQ ID NO: 21), and YTFTKYGMN (SEQ ID NO: 23). (Item 87) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a VH CDR2 amino acid sequence selected from the group consisting of WINTYTGEPTYADDFKG (SEQ ID NO: 24), WINTNTGEPTYGDDFKG (SEQ ID NO: 25), and WMGWINTYTGEPTY (SEQ ID NO: 26). (Item 88) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a VH CDR3 amino acid sequence selected from the group consisting of GVAHYSDSRFAFDY (SEQ ID NO: 27), GNAHPGGSAFVY (SEQ ID NO: 28), RGSYYYDSSPAWFAY (SEQ ID NO: 29), RGVDSSGYGAFAY (SEQ ID NO: 30), and TRGTSTMISTFAFVY (SEQ ID NO: 31). (Item 89) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a VL CDR1 amino acid sequence selected from the group consisting of SVSSSVSYMH (SEQ ID NO: 32), SASSGVTYMH (SEQ ID NO: 33), SSVSYMH (SEQ ID NO: 34), and SSVRYMH (SEQ ID NO: 35). (Item 90) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a VL CDR2 amino acid sequence selected from the group consisting of YTSNLAS (SEQ ID NO: 36), RTSNLAS (SEQ ID NO: 37), LWIYSTSNLAS (SEQ ID NO: 38), and VWIYSTSNLAS (SEQ ID NO: 39). (Item 91) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a VH CDR3 amino acid sequence selected from the group consisting of QQRSSYPFT (SEQ ID NO: 40), QQRSTYPF (SEQ ID NO: 41), QQRSFYPF (SEQ ID NO: 42), and QQRTYYPF (SEQ ID NO: 43). (Item 92) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a variable heavy chain (VH) sequence shown in SEQ ID NO: 10, 12, 14, 16, or 18. (Item 93) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a variable light chain (VL) sequence shown in SEQ ID NO: 11, 13, 15, 17, or 19. (Item 94) 86. The method of any one of Items 83 to 85, wherein the monoclonal antibody comprises a variable heavy chain (VH) sequence set forth in SEQ ID NO: 10, 12, 14, 16, or 18, and a variable light chain (VL) sequence set forth in SEQ ID NO: 11, 13, 15, 17, or 19. (Item 95) 86. The method of any one of items 83 to 85, wherein the determination is quantitative, the subjects are identified as seropositive for anti-AAVrh.74 based on the quantification, and the immunosuppressive regimen or TPE is selectively administered to the seropositive subjects. [Brief explanation of the drawings]

[0152] [Figure 1]The rAAV.MHCK7.microdystrophin construct is shown. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). The construct features an in-frame rod deletion (R4-R23) that maintains the hinges 1, 2, and 4 (H1, H2, and H4) and cysteine-rich domain to produce a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) is driven by the MHCK7 promoter (795 bp). The intron and 5' UTR are derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette has a consensus Kozak sequence immediately before the ATG start and a small 53 bp synthetic poly(A) signal for mRNA termination. As previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)), the human microdystrophin cassette contained the (R4-R23 / Δ71-78) domain. [Figure 2-1] The nucleic acid sequence (SEQ ID NO: 3) rAAVrh74.MHCK7.microdystrophin is provided. [Figure 2-2] The nucleic acid sequence (SEQ ID NO: 3) rAAVrh74.MHCK7.microdystrophin is provided. [Figure 2-3] The nucleic acid sequence (SEQ ID NO: 3) rAAVrh74.MHCK7.microdystrophin is provided. [Figure 2-4] The nucleic acid sequence (SEQ ID NO: 3) rAAVrh74.MHCK7.microdystrophin is provided. [Figure 3] The pNLREP2-Caprh74 AAV helper plasmid map is provided. [Figure 4] The Ad Helper plasmid pHELP is provided. [Figure 5] rAAV.MCK.microdystrophin plasmid construct is shown. [Figure 6-1] The nucleic acid sequence (SEQ ID NO: 5) rAAVrh74.MCK.microdystrophin is provided. [Figure 6-2]The nucleic acid sequence (SEQ ID NO: 5) rAAVrh74.MCK.microdystrophin is provided. [Figure 6-3] The nucleic acid sequence (SEQ ID NO: 5) rAAVrh74.MCK.microdystrophin is provided. [Figure 6-4] The nucleic acid sequence (SEQ ID NO: 5) rAAVrh74.MCK.microdystrophin is provided. [Figure 7-1] The nucleic acid sequence (SEQ ID NO: 9) rAAVrh74.MHCK7.microdystrophin is provided. [Figure 7-2] The nucleic acid sequence (SEQ ID NO: 9) rAAVrh74.MHCK7.microdystrophin is provided. [Figure 7-3] The nucleic acid sequence (SEQ ID NO: 9) rAAVrh74.MHCK7.microdystrophin is provided. [Figure 8] The AAVrh74.MHCK7.microdystrophin plasmid construct is shown. [Figure 9-1] 1 provides the nucleic acid sequence of the rAAVrh74.MHCK7.microdystrophin plasmid construct containing the kanamycin resistance gene (SEQ ID NO:8). [Figure 9-2] 1 provides the nucleic acid sequence of the rAAVrh74.MHCK7.microdystrophin plasmid construct containing the kanamycin resistance gene (SEQ ID NO:8). [Figure 9-3] 1 provides the nucleic acid sequence of the rAAVrh74.MHCK7.microdystrophin plasmid construct containing the kanamycin resistance gene (SEQ ID NO:8). [Figure 9-4] 1 provides the nucleic acid sequence of the rAAVrh74.MHCK7.microdystrophin plasmid construct containing the kanamycin resistance gene (SEQ ID NO:8). [Figure 10] A schematic diagram of the therapeutic β-sarcoglycan transgene cassette is provided. This self-complementary AAV vector contains the codon-optimized human β-sarcoglycan gene (hSGCB). The muscle-specific MHCK7 promoter drives expression. The cassette also contains a chimeric intron that enhances processing and polyadenylation signals for stability. [Figure 11]

[0023] Figure 1 provides a graph of antibody titers to AAVrh74 in NHPs after re-administration of rAAVrh74.MHCK7.micro-dystrophin. The dotted line represents the selection criteria for total AAVrh.74 antibody titer levels, which was a threshold of 1:400 against AAVrh.74. [Figure 12] 1 provides the fold change in micro-dystrophin protein expression after TPE compared to before TPE in NHPs re-administered with rAAVrh74.MHCK7.micro-dystrophin after TPE. [Figure 13] Provide the course of plasma antibodies after plasma volume (PV) removal over a 10-day course of TPE. DETAILED DESCRIPTION OF THE INVENTION

[0153] The present disclosure relates to identifying doses, durations, and immunosuppressive regimens for enhancing gene expression following intravascular delivery of rAAV.rh74.MHCK7.micro-dystrophin, and identifying techniques for enhancing gene expression after administration or re-administration of rAAV, in conjunction with using TPE to remove pre-existing AAV antibodies, and evaluating re-administration without TPE. The methods of the invention include administering an immunosuppressive regimen and / or TPE prior to administration of any of rAAV.rh74.MHCK7.micro-dystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05 described herein. The method includes administering an immunosuppressive regimen and / or TPE prior to re-administration of rAAV.rh74.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05 described herein.

[0154] The present disclosure provides gene therapy vectors, e.g., rAAV vectors, that overexpress human microdystrophin, as well as methods for reducing and preventing fibrosis in patients with muscular dystrophy. Muscle biopsies taken at the earliest age of diagnosis of DMD reveal significant connective tissue proliferation. Muscle fibrosis is detrimental in multiple ways. It reduces the normal passage of nutrients through the connective tissue barrier in the endomysium, reducing blood flow and depriving muscles of vascular nutrients, contributing to early loss of ambulation due to limb contractures. Over time, treatment challenges increase as a result of significant muscle fibrosis. This can be observed in muscle biopsies comparing connective tissue proliferation at successive time points. This process continues to worsen, leading to loss of ambulation and accelerating loss of control, especially in wheelchair-dependent patients.

[0155] Without early treatment, including a parallel approach to alleviate fibrosis, the full benefits of exon skipping, stop codon readthrough, or gene replacement therapy are unlikely to be achieved. Even small molecule or protein replacement strategies may fail without an approach to alleviate muscle fibrosis. Previous studies in aged mdx mice with pre-existing fibrosis treated with AAV.microdystrophin demonstrated that complete functional recovery could not be achieved (Liu, M., et al., Mol Ther 11, 245-256 (2005)). Progression of DMD cardiomyopathy is also known to be accompanied by scarring and fibrosis of the ventricular wall.

[0156] The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology, and recombinant DNA technology, within the skill of one of ordinary skill in the art. Such techniques are explained fully in the literature, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, Vol. I & II (D. Glover, ed.); Oligonucleotides Synthesis (N. Gait, ed., Current Edition), Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition), Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition), CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.), Fundamental Virology, 2nd Edition, vol. I & II (BNFields and DMKnipe, eds.), Freshney Culture of Animal Cells, A Manual of Basic See Technique (Wiley-Liss, Third Edition), and Ausubel et al. (1991) Current Protocols in Molecular Biology (Wiley Interscience, NY).

[0157] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0158] definition The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells, reference to a "culture" includes reference to one or more cultures and equivalents thereof known to those skilled in the art, etc. Reference to "recombinant AAV" includes a mixture of two or more rAAV virions, etc. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0159] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or unless the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or."

[0160] Throughout this application, the term "about" is used to indicate that a value includes statistical experimental error (standard deviation of error) for the device or method being employed to determine the value.

[0161] The term "vector" refers to any genetic element that, when associated with the appropriate control elements, is capable of replication and transfer of genetic sequences between cells, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. In one embodiment, the vector is a viral vector.

[0162] As used herein, the term "AAV" is a general abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York. However, since it is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, 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 they all possess three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0163] As used herein, "AAV vector" refers to one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs). Such AAV vectors can replicate and be packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products. In one embodiment, the AAV vector is a vector derived from an adeno-associated virus serotype, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh10, and AAVrh.74. AAV vectors can be deleted in whole or in part for one or more of the AAV wild-type genes, preferably the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication, and packaging of AAV virions. Thus, an AAV vector is defined herein to contain at least those sequences required in cis for viral replication and packaging (e.g., functional ITRs). The ITRs do not need to be wild-type nucleotide sequences and can be altered, for example, by nucleotide insertion, deletion, or substitution, as long as the sequences provide functional rescue, replication, and packaging.

[0164] The term "AAV helper functions" refers to AAV-derived coding sequences that can be expressed to provide AAV gene products that then function in trans for productive AAV replication. AAV helper functions thus include the major AAV open reading frames (ORFs), rep and cap. The Rep expression product has been shown to possess many functions, including, inter alia, recognition, binding, and nicking of the AAV origin of DNA replication, DNA helicase activity, and regulation of transcription from an AAV (or other heterologous) promoter. The Cap expression product supplies the necessary packaging function. AAV helper functions are used herein to complement AAV functions in trans that are missing from an AAV vector.

[0165] By "recombinant virus" is meant a virus that has been genetically altered, for example, by the addition or insertion of a heterologous nucleic acid sequence into the viral particle.

[0166] An "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector enclosed within the capsid. In one embodiment, the AAV virion comprises a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell). In some embodiments, production of an AAV viral particle includes production of an AAV vector, e.g., the vector is contained within an AAV vector particle.

[0167] The AAV genome, such as a transgene, that is delivered to a mammalian cell is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, because such vectors are contained within AAV vector particles, production of AAV vector particles necessarily includes production of AAV vectors.

[0168] For example, a wild-type (wt) AAV viral particle comprises a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat. AAV virions can be either single-stranded (ss) AAV or self-complementary (SC) AAV. In one embodiment, single-stranded AAV nucleic acid molecules of complementary sense, e.g., either the "sense" or "antisense" strand, can be packaged into an AAV virion, and both strands are equally infectious.

[0169] The term "recombinant AAV" or "rAAV" is defined herein as an infectious, replication-defective virus consisting of an AAV protein shell encapsulating a heterologous nucleotide sequence of interest flanked on both sides by AAV ITRs. In one embodiment, rAAV is produced in a suitable host cell, which has an AAV vector, AAV helper functions, and accessory functions introduced therein. In this way, the host cell is capable of encoding the AAV polypeptides required for packaging the AAV vector (containing the recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.

[0170] The term "transfection" refers to the uptake of foreign DNA by a cell; a cell is "transfected" when exogenous DNA is introduced into the cell membrane. Many transfection techniques are commonly known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York; Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier; and Chu et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. See, e.g., W. et al. (1981) Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

[0171] The term "transduction" refers to the delivery of a DNA molecule to a recipient cell either in vivo or in vitro via a replication-defective viral vector, for example, via a recombinant AAV virion.

[0172] The term "host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of AAV helper constructs, AAV vector plasmids, accessory function vectors, or other transfer DNA. The term includes the progeny of the original transfected cell. Thus, as used herein, "host cell" generally refers to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genomic or total DNA complement due to natural, accidental, or deliberate mutation.

[0173] "Muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle (e.g., skeletal and smooth muscle derived from the digestive tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0174] The term "heterologous," when referring to nucleic acid sequences, such as coding sequences and control sequences, refers to sequences that are not normally linked together and / or not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with that molecule in nature. For example, a heterologous region of a nucleic acid construct can include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from the native gene). Similarly, a cell transformed with a construct that is not normally present in the cell would be considered heterologous for purposes of the present invention. As used herein, allelic variation or naturally occurring mutational events do not give rise to heterologous DNA.

[0175] A "coding sequence," or a sequence "encoding" a particular protein, is a nucleic acid sequence that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

[0176] "Nucleic acid" sequences refer to DNA or RNA sequences. Nucleic acids include 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, and 2,6-diaminopurine.

[0177] The term DNA "control sequences" collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0178] The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region that contains DNA regulatory sequences derived from a gene that are capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcriptional promoters can include "inducible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters." In one embodiment, the promoter is a muscle-specific promoter, including, but not limited to, a human skeletal actin gene element, a cardiac actin gene element, a desmin promoter, a skeletal alpha actin (ASKA) promoter, a troponin I (TNNI2) promoter, a muscle cell-specific enhancer-binding factor (mef)-binding element, a muscle creatine kinase (MCK) promoter, a truncated MCK (tMCK) promoter, a myosin heavy chain (MHC) promoter, a hybrid α-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) promoter, a C5-12 promoter, a mouse creatine kinase enhancer element, a fast skeletal troponin c gene element, a slow cardiac troponin c gene element, a slow troponin i gene element, a hypoxia-inducible nuclear factor (HIF) response element (HRE), a steroid-inducible element, and a glucocorticoid response element (gre). In another embodiment, the promoter is an MCK promoter, a tMCK promoter, or an MHCK7 promoter.

[0179] The term "operably linked" refers to an arrangement of elements such that the components so described are configured to perform their normal function. Thus, control sequences operably linked to a coding sequence are capable of affecting the expression of the coding sequence. Control sequences need not be contiguous with the coding sequence, so long as they function to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence.

[0180] A promoter "directs the transcription" of a coding sequence in a cell when RNA polymerase binds to the promoter sequence and transcribes the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

[0181] An "expression cassette" or "expression construct" refers to an assembly capable of directing the expression of a sequence or gene of interest. An expression cassette, as described above, includes regulatory elements such as a promoter operably linked (to direct transcription) to the sequence or gene of interest, and often also includes a polyadenylation sequence. Within certain embodiments of the invention, the expression cassettes described herein may be included within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include one or more selectable markers, a signal that allows the plasmid construct to exist as single-stranded DNA, at least one multiple cloning site, and a "mammalian" origin of replication (e.g., an SV40 or adenoviral origin of replication).

[0182] "Isolated," when referring to a nucleotide sequence, means that the indicated molecule is present in the substantial absence of other nucleotide sequences, other biological macromolecules, such as chromatin material, etc. Thus, an "isolated nucleic acid molecule encoding a particular polypeptide" refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest; however, the molecule may contain some additional bases or moieties that do not adversely affect the essential characteristics of the composition.

[0183] For purposes of describing the relative position of nucleotide sequences in particular nucleic acid molecules throughout this application, such as when a particular nucleotide sequence is described as being located "upstream," "downstream," "3," or "5" relative to another sequence, it should be understood that this is the position of the sequence on the "sense" or "coding" strand of the DNA molecule, as conventionally referred to in the art.

[0184] The terms "sequence identity," "percent sequence identity," or "percent identity" in the context of nucleic acid or amino acid sequences refer to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison can be the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500-5,000 nucleotides is desirable. However, identity between smaller fragments, such as at least about 9 nucleotides, usually at least about 20-24 nucleotides, at least about 28-32 nucleotides, or at least about 36 or more nucleotides, can also be desirable. Percent sequence identity can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs such as ALIGN, ClustalW2, and BLAST. In one embodiment, when BLAST is used as the alignment tool, the following default parameters are used: genetic code=standard; filter=none; strand=both; cutoff=60; prediction=10; matrix=BLOSUM62; explanation=50 sequences; sort=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss Protein+Spupdate+PIR.

[0185] The term "subject" refers to any member of the animal kingdom, including, but not limited to, humans and non-human primates such as chimpanzees and other ape and monkey species, domestic animals such as cows, sheep, pigs, goats, and horses, domestic mammals such as dogs and cats, and laboratory animals including rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human between birth and 2 years of age, between 1 and 10 years of age, between 4 and 15 years of age, between 10 and 19 years of age, between 20 and 40 years of age, between 15 and 29 years of age, between 25 and 55 years of age, between 40 and 60 years of age, or between 50 and 60 years of age, between 65 and 70 years of age, or older.

[0186] AAV Adeno-associated virus (AAV) is a replication-deficient parvovirus, whose single-stranded DNA genome is approximately 4.7 kb long, including a 145-nucleotide inverted terminal repeat (ITR). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), as revised by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is presented in GenBank Accession No. NC_002077, the complete genome of AAV-3 is presented in GenBank Accession No. NC_1829, the complete genome of AAV-4 is presented in GenBank Accession No. NC_001829, the AAV-5 genome is presented in GenBank Accession No. AF085716, the complete genome of AAV-6 is presented in GenBank Accession No. NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are presented in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is presented in GenBank Accession No. NC_001829. The AAV-10 genome is presented in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is presented in Virology, 330(2):375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al., Journal of Translational Medicine 5, 45 (2007). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 in AAV2), drive the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins possess multiple enzymatic properties that ultimately contribute to viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites contribute to the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0187] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells do not tolerate superinfection.

[0188] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996), and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000), and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction resulted in the appearance of the transgene product 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). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.

[0189] The recombinant AAV genome of the present disclosure comprises a nucleic acid molecule of the present disclosure and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA of the rAAV genome can be derived from any AAV serotype capable of deriving a recombinant virus, 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, AAV-13, and AAVrh.74. The generation of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. AAVrh.74 can be used to promote muscle-specific expression.

[0190] The DNA plasmid of the present disclosure comprises the rAAV genome of the present disclosure. The DNA plasmid is transferred to a cell permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided in the cell, are standard in the art. rAAV production requires that the following components be present in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype from which a recombinant virus can be derived, or may be derived from an AAV serotype different from the rAAV genomic ITRs, for example, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. Production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0191] The method for generating packaging cells involves creating a cell line that stably expresses all components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene are integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by methods such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus, such as adenovirus. The advantage of this method is that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0192] 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 approaches include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mo 1. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988), Samulski et al. al., J. Virol., 63:3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365, and corresponding U.S. Pat. Nos. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), W97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13:1244-1250 (1995), Paul et al. Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996), U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis being placed on the portions of the documents relating to rAAV production.

[0193] Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 cells). In another embodiment, the packaging cells are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0194] Recombinant AAVs (i.e., infectious, encapsidated rAAV particles) of the present disclosure comprise an rAAV genome. In exemplary embodiments, both rAAV genomes lack AAV rep and cap DNA, i.e., no AAV rep or cap DNA is present between the ITRs of the genome. Examples of rAAVs that can be constructed to contain nucleic acid molecules of the present disclosure are described in International Patent Application No. PCT / US2012 / 047999 (WO2013 / 016352), the entire contents of which are incorporated herein by reference.

[0195] In an exemplary embodiment, a recombinant AAV vector of the present disclosure is produced by a triple transfection method (Xiao et al., J Virol 72, 2224-2232 (1998)) using the AAV vector plasmids rAAV.MHCK7.microdystrophin, pNLRep2-Caprh74, and pHelp; the rAAV contains a microdystrophin gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is encapsidated into AAVrh.74 virions. The plasmid contains the microdystrophin sequence and the core promoter elements of the MHCK7 enhancer and muscle-specific promoter that drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) to promote high-level gene expression, and uses the bovine growth hormone polyadenylation signal for efficient transcription termination.

[0196] pNLREP2-Caprh74 is an AAV helper plasmid encoding four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from serotype rh74. A schematic diagram of the pNLREP2-Caprh74 plasmid is shown in Figure 3.

[0197] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. This plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4 ORF6, and VA RNA (the adenovirus E1 function is provided by 293 cells). The adenovirus sequences present in this plasmid represent only approximately 40% of the adenovirus genome and do not contain cis-elements important for replication, such as the adenovirus terminal repeats. Therefore, infectious adenovirus is not expected to be generated from this production system. A schematic diagram of the pHELP plasmid is shown in Figure 4.

[0198] rAAV can be purified by standard methods 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, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0199] In another embodiment, the present disclosure contemplates a composition comprising the rAAV of the present disclosure. The composition of the present disclosure comprises the rAAV and a pharmaceutically acceptable carrier. The composition may also contain other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to recipients, preferably inert at the dosages and concentrations employed, and include buffers and surfactants such as Pluronic®.

[0200] The titer of the rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the targeted individual, and the cell type, and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 Dosages may range from 1000 mg to 1000 mg of DNase-resistant particles (DRP) or more. Dosages may be expressed in units of viral genomes (vg). One exemplary method for determining the encapsulated vector genome titer is (Pozsgai Use quantitative PCR, such as the method described in (Mol. Ther. 25(4):855-869, 2017). Unless otherwise stated, the dosages described herein correspond to doses determined by supercoiled DNA standards.

[0201] Methods of transducing target cells with rAAV in vivo or in vitro are contemplated by the present disclosure. In vivo methods include administering an effective dose or effective multiple doses of a composition comprising an rAAV of the present disclosure to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present disclosure, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the extent of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease contemplated for prevention or treatment by the methods of the present disclosure is DMD.

[0202] Combination therapies are also contemplated by the present disclosure. As used herein, combination includes both simultaneous and sequential treatments. Combination of the methods of the present disclosure with standard medical treatments (e.g., corticosteroids), including combinations with novel therapies, is particularly contemplated.

[0203] Administration of an effective dose of a composition, combination therapy, or formulation can be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The route and serotype of administration of the AAV components of the rAAV of the present disclosure (particularly the AAV ITRs and capsid proteins) can be selected and / or adapted by one skilled in the art taking into account the disease state to be infected and / or treated, and the target cells / tissues expressing the micro-dystrophin protein.

[0204] The present disclosure provides for local and systemic administration of effective doses of the rAAVs, formulations, and compositions of the present disclosure. For example, systemic administration refers to administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the digestive tract, and parenteral administration via injection, infusion, or implantation.

[0205] In particular, the actual administration of the rAAV of the present disclosure can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present disclosure includes, but is not limited to, injection into muscle and injection into the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in practicing the present disclosure. The rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0206] In one embodiment of the present disclosure, the AAVrh74.MHCK7.microdystrophin described herein is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0207] The dose of rAAV administered in the methods disclosed herein will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. The titer of each rAAV administered is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×1014 , 2 × 10 14 or about 1 x 10 15 Dosages may also range from 1 x 10 to 1 x 10 or 1 x 10 DNase-resistant particles (DRP). 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 2 × 10 14 vg, 1×10 15 Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1 x 10 10 vg / kg, 1 × 10 11 vg / kg, 1 × 10 12 vg / kg, 1 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1.25 × 10 14 vg / kg, 1.5 × 10 14 vg / kg, 1.75 × 10 14 vg / kg, 2.0 × 10 14 vg / kg, 2.25 × 10 14 vg / kg, 2.5 × 10 14 vg / kg, 2.75 × 10 14 vg / kg, 3.0 × 10 14 vg / kg, 3.25 × 10 14 vg / kg, 3.5 × 10 14 vg / kg, 3.75 × 10 14 vg / kg, 4.0 × 10 14 vg / kg, 1 × 10 15 AAV may be expressed in units of vg / kg. Methods for titrating AAV are described by Clark et al. al., Hum. Gene Ther., 10:1031-1039 (1999).

[0208] In particular, the actual administration of the rAAV of the present disclosure can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present disclosure includes, but is not limited to, injection into muscle and injection into the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in practicing the present disclosure. The rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0209] For intramuscular injection, solutions in adjuvants such as sesame or peanut oil, or aqueous propylene glycol solutions, and sterile aqueous solutions can be used. Such aqueous solutions can be buffered, if necessary, and the liquid diluent is first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary storage and use conditions, these formulations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed are readily available by standard techniques well known to those skilled in the art.

[0210] Pharmaceutical carriers, diluents, or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0211] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, along with various other ingredients as listed above, as needed, followed by filter sterilization. Generally, dispersions are prepared by mixing the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of the active ingredient plus any additional desired ingredients from their previously sterile-filtered solution.

[0212] Transduction with rAAV can also be performed in vitro. In one embodiment, desired target muscle cells are removed from a subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used if they do not generate an inappropriate immune response in the subject.

[0213] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in an appropriate medium and screening for cells carrying the DNA of interest using conventional techniques such as Southern blot and / or PCR, or using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, and the composition can be introduced into a subject by a variety of techniques, including intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle, for example, using a catheter.

[0214] Transduction of cells with the rAAV of the present disclosure results in sustained expression of the micro-dystrophin protein. Thus, the present disclosure provides methods for administering / delivering rAAVs expressing the micro-dystrophin protein to animals, preferably humans. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of the present disclosure. Transduction can be performed with a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present disclosure provides a method for transducing muscle cells and muscle tissue directed by muscle-specific regulatory elements, including, but not limited to, those from the actin and myosin gene families, e.g., 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)), a regulatory element from the human skeletal actin gene (Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)), the cardiac actin gene, and the muscle creatine kinase sequence element (Johnson et al. al., Mol. Cell. Biol., 9:3393-3399 (1989)), as well as regulatory elements from the mouse creatine kinase enhancer (mCK) element, the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene, hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA 88:5680-5684 (1991)), steroid-inducible elements and promoters, including glucocorticoid response elements (GREs) (Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), and other regulatory elements.

[0215] Because muscle tissue is not a vital organ and is easily accessible, it is an attractive target for in vivo DNA delivery. The present disclosure contemplates sustained expression of micro-dystrophin from transduced muscle fibers.

[0216] Thus, the present disclosure provides methods of administering an effective dose of rAAV encoding micro-dystrophin (or essentially simultaneous or spaced doses) to a patient in need thereof.

[0217] Immunosuppressive regimens The present disclosure provides methods for treating muscular dystrophy in a subject receiving an immunosuppressive regimen. The term immunosuppressive regimen refers to a treatment method that suppresses or modulates a subject's immune system. The regimen includes the administration of one or more immunosuppressive agents. In any of these methods, the immunosuppressive regimen includes at least one immunosuppressive agent, or at least two immunosuppressive agents, or at least three immunosuppressive agents, or at least four immunosuppressive agents, or at least five immunosuppressive agents.

[0218] The immunosuppressive regimen is administered prophylactically in that it is administered before administration of gene therapy or before the initiation of an immune response to the rAAV in the subject after administration of gene therapy. The immune response includes an adverse immune response or inflammatory reaction to the administered rAAV. The immune response may be the production of antibodies in the subject in response to the administered rAAV, for example, anti-AAVrh.74 antibodies.

[0219] Prophylactic administration includes administration of an immunosuppressive regimen concurrently with the administration of gene therapy, e.g., within 24 hours of the administration of gene therapy, or within 12 hours of the administration of gene therapy, or within 6 hours of the administration of gene therapy, or within 5 hours of the administration of gene therapy, or within 4 hours of the administration of gene therapy, or within 3 hours of the administration of gene therapy, or within 2 hours of the administration of gene therapy, or within 1 hour of the administration of gene therapy. An immunosuppressant is any agent that inhibits a subject's immune system, reduces the effectiveness of a subject's immune system, or modulates the activity or effectiveness of a subject's immune system.

[0220] In other embodiments, the immunosuppressive regimen is administered therapeutically. For example, the immunosuppressant is administered after the initiation of an immune response to the rAAV in the subject following administration of gene therapy. The immune response in the subject includes an adverse immune response or inflammatory reaction following or caused by administration of the rAAV to the subject. The immune response can be the production of antibodies in the subject in response to the administered rAAV, e.g., anti-AAVrh.74 antibodies.

[0221] In other embodiments, the immunosuppressive regimen is administered before administering the second dose of gene therapy. In some embodiments, the second dose is administered after therapeutic plasma exchange (TPE).

[0222] Exemplary immunosuppressants include glucocorticosteroids, Janus kinase inhibitors, calcineurin inhibitors, mTOR inhibitors, purine analogs, cytostatics such as methotrexate and cyclophosphamide, inosine monophosphate dehydrogenase (IMDH) inhibitors, biologics such as monoclonal antibodies or fusion proteins and polypeptides, and polypeptides.

[0223] The immunosuppressant may be an anti-inflammatory steroid, which is a steroid that reduces inflammation and suppresses or modulates the subject's immune system. Exemplary anti-inflammatory steroids are glucocorticoids such as prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, budesonide, or prednisone.

[0224] Janus kinase inhibitors are inhibitors of the JAK / STAT signaling pathway by targeting one or more of the Janus kinase family of enzymes. Exemplary Janus kinase inhibitors include tofacitinib, baricitinib, upadacitinib, peficitinib, and oclacitinib.

[0225] Calcineurin inhibitors bind to cyclophilin and inhibit the activity of calcineurin. Exemplary calcineurin inhibitors include cyclosporine, tacrolimus, and picecrolimus.

[0226] mTOR inhibitors reduce or inhibit the serine / threonine-specific protein kinase mTOR. Exemplary mTOR inhibitors include sirolimus, everolimus, and temsirolimus.

[0227] Immunosuppressants include immunosuppressant macrolides. The term "immunosuppressant macrolide" refers to a macrolide drug that suppresses or modulates a subject's immune system. Macrolides are a class of drugs that contain a large macrocyclic lactone ring to which one or more deoxy sugars, such as cladinose or desoamine, are attached. The lactone ring is usually 14, 15, or 16 members. Macrolides belong to the polyketide class of drugs and can be natural products. Examples of immunosuppressant macrolides include tacrolimus, pimecrolimus, and sirolimus.

[0228] Purine analogs block nucleotide synthesis and include IMDH inhibitors. Exemplary purine analogs include azathioprine, mycophenolic acid, and lefunomide.

[0229] Exemplary immunosuppressive biologics include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, belatacept, and daclizumab.

[0230] In particular, the immunosuppressant is an anti-CD20 antibody. The term anti-CD20 specific antibody refers to an antibody that specifically binds to CD20 or inhibits or reduces the expression or activity of CD20. Exemplary anti-CD20 antibodies include rituximab, ocrelizumab, or ofatumumab.

[0231] Additional examples of immunosuppressive antibodies include anti-CD25 antibodies (or anti-IL2 or anti-TAC antibodies) such as basiliximab and daclizumab, as well as anti-CD3 antibodies such as muromonab-CD3, otelixizumab, teplizumab and visilizumab, and anti-CD52 antibodies such as alemtuzumab.

[0232] In an embodiment of the present invention, immunosuppressants (immunosuppressant antibodies) utilized in one or more of the methods of the present disclosure may be administered as nanoparticles. Methods for making and / or formulating nanoparticles and nanoparticles that can be used in the methods of the present disclosure include, for example, nanoparticles formulated as polymers (Patil et al., Pharmaceutical Nanotechnol. 367:195-203, 2009; Yang et al., ACS Appl. Mater. Interfaces, doi:10.1021 / acsami.6b16556, 2017; Perepelyuk et al., Mol. Ther. Nucleic Acids 6:259-268, 2017), liposomes (Buyens et al., J. Control Release 158(3):362-370, 2012; Scarabel et al., Expert Opin. Drug Deliv. 17:1-14, 2017), micelles (Tangsangasaksri et al., Bio Macromolecules 17:246-255, 2016; Wu et al., Nanotechnology, doi:10.1088 / 1361-6528 / aa6519, 2017), as microemulsions (WO 11 / 004395), as nanoemulsions or solid lipid nanoparticles (Sahay et al., Nature Biotechnol. 31:653-658, 2013; and Lin et al., Nanomedicine 9(1):105-120,2014), as well as WO2008 / 066965, WO2011 / 143201, WO2008 / 014478, WO2020 / 081938, WO2013 / 016058, WO2013 / 086373, WO2019 / 177550, WO2013 / 016126, WO2 019 / 089828, WO99 / 39741, WO2017 / 117528, WO2017 / 004143, WO2017 / 075531, WO2015 / 199952, WO2014 / 008334, WO2013 / 086373, WO2013 / 086322, WO201 Nos. 3 / 016058, WO2013 / 086373, WO2011 / 141705 and WO2001 / 07548, U.S. Patent Application Nos. 2004 / 0142025, 2007 / 0042031, 2016 / 0199485, 2016 / 0009637, 201 5 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2 014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2 013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 0295832, No. 2012 / 0183581 , 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 031158 No. 3, No. 2011 / 0311582, No. 2011 / 0262527, No. 2011 / 0216622, No. 2011 / 01171 No. 25, No. 2011 / 0091525, No. 2011 / 0076335, No. 2011 / 0060032, No. 2010 / 013 No. 0588, No. 2007 / 0042031, No. 2006 / 0240093, No. 2006 / 0083780, No. 2006 / 00 Nos. 08910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076, and U.S. Pat.569,256 and 5,965,542, the entire contents of which are incorporated herein by reference.

[0233] therapeutic plasma exchange Therapeutic plasma exchange (TPE) is an extracorporeal blood purification technique designed to remove high-molecular-weight substances, such as antibodies. After initial systemic administration of a dose of rAAV, a subject may develop antibodies against AAV serotypes; for example, a subject may develop antibodies against AAVrh74 after administration of rAAV.MHCK7.microdystrophin. Using TPE to remove these antibodies allows for safe and effective re-administration of rAAV vectors. In the TPE process, whole blood is removed via vascular access and then spun in a centrifuge within an apheresis machine, where plasma (antibodies) is removed. Red blood cells are delivered back along with replacement fluid (human albumin) for the subject's maintenance fluid.

[0234] The amount of plasma exchanged in TPE session is determined in relation to the subject's estimated plasma volume (EPV).Many formulas can be used to calculate EPV (see, for example, Inkley et al., J.Lab Clin.Med.45:841-850,1955; Retzlaff et al., Blood 33:649-887,1969; Feldschuh et al., Circulation 56:605-612,1977; Spenger et al., Predication of patient's plasma volume in plasma exchange therapy, In: Smeby et al. ed. Immune and Metabolic Aspects of Therapeutic Blood Purification Systems. Basel, Switzerland: Krager.1986, pp.394-402). An exemplary method for estimating EPV is calculated using a subject's body weight and hematocrit according to the following formula as described in Kaplan et al. Kidney Intl. 38:160-166, 1990, which is incorporated herein by reference in its entirety: EPV = [0.065 x body weight (kg)] x [1 - hemocrit]

[0235] The protocol described in Example 5 was developed based on the kinetics of IgG antibodies, which show that there is a rebound after 48 hours, with approximately 50-60% of the antibodies removed from the previous procedure rebounding. For the purposes of the methods described herein, in one embodiment, it was determined that antibody titers must be reduced to AAVrh.74 antibody levels <1:100 to enable gene delivery.

[0236] For example, a method of subjecting a subject's plasma to TPE removes at least about 50% of the anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 55% of the anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 60% of the anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 63% of the anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 64% of the anti-rAAV antibodies, or removes at least about 6 .... Removes at least about 69% of rAAV antibodies, or removes at least about 70% of anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 74% of anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 75% of anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 85% of anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 90% of anti-rAAV antibodies in the intravascular space of the subject, or removes at least about 95% of anti-rAAV antibodies in the intravascular space of the subject.

[0237] The disclosed methods include subjecting a subject's plasma to at least one TPE session, or at least two TPE sessions, or at least three TPE sessions, or at least five TPE sessions, or at least six TPE sessions, or at least seven TPE sessions, or at least eight TPE sessions, or at least nine TPE sessions, or at least ten TPE sessions. Additionally, the TPE sessions are performed once per day for about 1-5 days, or about 1-10 days, or about 5-10 days, or about 5-7 days, or about 7-10 days. The TPE sessions are performed once per day for two consecutive days, or once per day for three consecutive days, or once per day for four consecutive days, or once per day for five consecutive days, or once per day for six consecutive days, or once per day for seven consecutive days, or once per day for seven consecutive days, or once per day for eight consecutive days, or once per day for nine consecutive days, or once per day for ten consecutive days.

[0238] TPE is performed using techniques implemented using blood bank procedures using membrane plasma separation (MPS) using highly permeable filters and dialysis equipment as described by Gurland et al., Nephron 36:173-182, 1984, and selective cell removal (cytopheresis) as described by Gurland et al., Int. J. Artif Organs 7:35-38, 1984. Additional methods of performing TPE are described by Sowada et al. (Available Removal Systems: State of the Art. In Nydegger UE, editor. Therapeutic Hemapheresis in the 1990s. Current Studies in Hematology and Blood Transfusions, Vol. 57. Basalt, Switzerland: Karger. 1990 pp. 57-113). These references are incorporated herein by reference in their entirety.

[0239] Embodiments of the present invention include treating a subject with (rAAV)rAAV.MHCK7.micro-dystrophin gene therapy comprising (rAAV)rh74MHCK7.micro-dystrophin, and further treating the subject with an immunosuppressive regimen, TPE, or both, recognizing that subjects, including human patients, may contain pre-existing anti-AAVrh.74 antibodies and therefore may be identified as seropositive for AAVrh.74 prior to receiving any gene therapy treatment, and determining the presence of anti-AAVrh.74 antibodies in the serum or plasma of the relevant subject. It is further recognized that subjects, including human patients, may become seropositive upon receiving AAV-based gene therapy. Accordingly, the determination can be used to monitor the presence and level of anti-AAVrh.74 antibodies in such subjects' serum or plasma, which can be further used in determining whether an immunosuppressive regimen, TPE, or both should be administered to such subjects prior to treatment with rAAV.MHCK7.micro-dystrophin comprising AAVrh74.MHCK7.micro-dystrophin. In this context, the rAAV.MHCK7.micro-dystrophin comprising AAVrh74.MHCK7.micro-dystrophin can be an initial gene therapy treatment or an additional gene therapy treatment, including re-administration treatment. In this context, determining seropositivity in such serum or plasma is used to determine whether a subject is suitable for initial gene therapy treatment with rAAV.MHCK7.micro-dystrophin comprising AAVrh74.MHCK7.micro-dystrophin, whether the subject requires an immunosuppressive regimen and / or TPE, and whether treatment with an immunosuppressive regimen has resulted in the subject clearing serum or plasma from anti-AAVrh74 antibodies to a level sufficient to make the subject suitable for treatment or re-treatment with rAAV.MHCK7.micro-dystrophin comprising AAVrh74.MHCK7.micro-dystrophin.Methods and compositions useful for such determination of the presence of anti-AAVrh.74 antibodies in a subject's serum or plasma include those described in Griffin et al., Adeno-associated Virus Serotype rh74 Prevalence in Muscular Dystrophy Population, American Society of Gene and Cell Therapy, 22nd annual meeting, 2019, and International Patent Application PCT / US2021 / 037314, filed June 15, 2021, corresponding to U.S. Patent Application No. 63 / 038957, the entire contents of which are incorporated herein by reference.

[0240] Examples of antibodies utilized in determining the presence of anti-AAVrh.74 antibodies in a subject's serum or plasma include the following or are otherwise described herein: [Table 2]

[0241] Further examples of antibodies useful in determining the presence of anti-AAVrh.74 antibodies in a subject's serum or plasma include those having a VH CDR1 amino acid sequence selected from the group consisting of NYGMN (SEQ ID NO:20), DYGMN (SEQ ID NO:22), YTFTNYGMN (SEQ ID NO:20), YTFTKYGMN (SEQ ID NO:23), and YTFTNYGMN (SEQ ID NO:21), and / or a VH CDR2 amino acid sequence selected from the group consisting of WINTYTGEPTYADDFKG (SEQ ID NO:24), WINTNTGEPTYGDDFKG (SEQ ID NO:25), and WMGWINTYTGEPTY (SEQ ID NO:26), and / or a VH CDR2 amino acid sequence selected from the group consisting of GVAHYSDSRFAFDY (SEQ ID NO:27), GNAHPGGSAFVY (SEQ ID NO:28), RGSYYYDSSPAWFAY (SEQ ID NO:29), RGVDSSGYGAFAY (SEQ ID NO:30), and TRGTSTMISTFAFVY (SEQ ID NO:31). and / or a VL CDR1 amino acid sequence selected from the group consisting of SVSSSVSYMH (SEQ ID NO: 32), SASSGVTYMH (SEQ ID NO: 33), SSVSYMH (SEQ ID NO: 34), SSVSYMH (SEQ ID NO: 34), and SSVRYMH (SEQ ID NO: 35); and / or a VL CDR2 amino acid sequence selected from the group consisting of YTSNLAS (SEQ ID NO: 36), RTSNLAS (SEQ ID NO: 37), LWIYSTSNLAS (SEQ ID NO: 38), and VWIYSTSNLAS (SEQ ID NO: 39); and / or a VH CDR3 amino acid sequence selected from the group consisting of QQRSSYPFT (SEQ ID NO: 40), QQRSTYPF (SEQ ID NO: 41), QQRSFYPF (SEQ ID NO: 42), and QQRTYYPF (SEQ ID NO: 43).

[0242] Treatment of muscular dystrophies, including Duchenne muscular dystrophy ("DMD") or Becker muscular dystrophy ("BMD"), with rAAV containing microdystrophins The present invention encompasses methods of treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated virus (rAAV) comprising a heterologous nucleotide sequence encoding microdystrophin, and further comprising administering an immunosuppressive regimen. In various embodiments of the present invention, the method comprises administering a recombinant adenovirus-associated virus (rAAV) comprising a heterologous nucleotide sequence encoding microdystrophin, and further comprises administering an anti-inflammatory steroid, including, for example, prednisone.

[0243] In all embodiments of the present invention directed to treating muscular dystrophy with an rAAV comprising microdystrophin, further comprising administering an immunosuppressive regimen or an anti-inflammatory steroid, rAAV or microdystrophin encoding nucleotide sequences that can be utilized in the methods of the present invention include those described in WO-2020 / 123645, WO-2019 / 209777, WO-2019 / 195362, WO-2016 / 115543, WO-2019118806, and WO-2017 / 221145, the contents of each of which are incorporated herein by reference, including SGT-001, zildistrogene, These include varoparvovec, and PF-06939926.

[0244] Treatment of limb-girdle muscular dystrophy Treatment of limb-girdle muscular dystrophy ("LGMD") is an aspect of the present invention. It is recognized that the methods of the present invention described herein can be utilized to treat limb-girdle muscular dystrophy by employing rAAV vectors useful for treating such dystrophies. Such rAAV vectors include those described in PCT / US2019 / 039893 (WO2020 / 06458), including AAVrh.74.tMCK.CAPN3; U.S. Patent Application No. 63 / 024,338, including rAAVrh.74.MHCK7.DYSF.DV; PCT / US2019 / 015779 (WO2019 / 15474), including scAAVrh.74.MHCK7.hSGCG; and AAVrh74.tMCK.CAPN3. These include those described in PCT / US2020 / 47339, including scAAVrh74.MHCK7.HSGCB; PCT / US2020 / 019892 (WO2020 / 176614), including scAAVrh74.MHCK7.HSGCB; and PCT / US2016 / 061703 (WO2017 / 083776), including rAAVrh.74.MHCK7.huAN05, the contents of each of which are incorporated herein by reference.

[0245] Combination therapy for treating muscular dystrophy The present disclosure provides a combination therapy for treating muscular dystrophy in a human subject in need thereof, including treating DMD, Becker muscular dystrophy, and limb-girdle muscular dystrophy, the combination therapy comprising rAAV and an anti-inflammatory steroid. The present disclosure also provides a use of a combination therapy comprising rAAV and an anti-inflammatory steroid for the preparation of a medicament for treating muscular dystrophy, including treating DMD, Becker muscular dystrophy, and limb-girdle muscular dystrophy.

[0246] The present disclosure provides combination therapies and medicaments comprising an rAAV rAAV.MHCK7.microdystrophin and an anti-inflammatory steroid administered in combination, including simultaneously, sequentially, or at different times. The present disclosure provides combination therapies and medicaments comprising an rAAV selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, in combination with an anti-inflammatory steroid, including simultaneously, sequentially, or at different times. For example, the anti-inflammatory steroid is a glucocorticoid. In some embodiments, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort. In some embodiments, the anti-inflammatory steroid is formulated for oral administration. In the case of combination therapies and agents, the anti-inflammatory steroid may be administered both before and after administration of rAAV. Alternatively, the anti-inflammatory steroid is administered only before or only after administration of rAAV.

[0247] In some combination therapies and agents, the anti-inflammatory steroid is administered about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or about 96 hours before administration of rAAV. In some combination therapies and agents, the anti-inflammatory steroid is administered about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 30 days before administration of rAAV.

[0248] In some combination therapies and medicaments, the anti-inflammatory steroid is administered at least once daily for about 7 days prior to administration of the rAAV, or at least once daily for about 14 days prior to administration of the rAAV, or at least once daily for 21 days, or at least once daily for about 28 days prior to administration of the rAAV, or at least once daily for about 30 days prior to administration of the rAAV, or at least once daily for about 45 days prior to administration of the rAAV, or at least once daily for about 60 days prior to administration of the rAAV. In some compositions and medicaments, the anti-inflammatory steroid is administered 30 to 60 days prior to administration of the rAAV.

[0249] In exemplary combination therapies and agents, the anti-inflammatory steroid is administered prior to administration of rAAV, and the anti-inflammatory steroid is administered at least once daily from day 1 to day 30 after administration of rAAV, or at least once daily from day 1 to day 60 after administration of rAAV, or at least once daily from day 1 to day 7 after administration of rAAV, or at least once daily from day 1 to day 14 after administration of rAAV, or at least once daily from day 1 to day 21 after administration of rAAV, or at least once daily from day 1 to day 24 after administration of rAAV, or at least once daily from day 1 to day 28 after administration of rAAV, or at least from day 1 to day 30 after administration of rAAV, or at least from day 30 to day 60 after administration of rAAV.

[0250] In any of the combination therapies and medicaments disclosed herein, the combination therapy or medicament may also include an anti-CD20 specific antibody administered in combination with the rAAV and an anti-inflammatory steroid. The anti-CD20 specific antibody is administered prior to administration of the rAAV. In some compositions, the anti-CD20 specific antibody is administered at least 7 days prior to administration of the rAAV. Exemplary anti-CD20 antibodies include rituximab, ocrelizumab, or ofatumumab.

[0251] In some combination therapies and agents, the anti-CD20-specific antibody is administered about 60 days prior to administration of the rAAV, or about 45 days prior to administration of the rAAV, or about 30 days prior to administration of the rAAV, or about 14 days prior to administration of the rAAV, or about 7 days prior to administration of the rAAV, and within about 24 hours of administration of the rAAV. In some compositions, the anti-CD20 antibody is administered 30 to 60 days prior to administration of the rAAV. In some combination therapies and agents, the anti-CD20-specific antibody is administered after administration of the rAAV. For example, the anti-CD20-specific antibody is administered both before and after administration of the rAAV. Alternatively, the anti-CD20-specific antibody is administered before administration of the rAAV, or the anti-CD20-specific antibody is administered after administration of the rAAV.

[0252] Additionally, in any of the disclosed combination therapies and medicaments, an immunosuppressant macrolide is administered to a subject in combination with rAAV and an anti-inflammatory steroid, and optionally, an anti-CD20 antibody. Examples of immunosuppressant macrolides include tacrolimus, pimecrolimus, and sirolimus. In some combination therapies and medicaments, the immunosuppressant macrolide is formulated for oral administration. In some combination therapies and medicaments, the immunosuppressant macrolide may be administered both before and after administration of rAAV. Alternatively, the immunosuppressant macrolide is administered before administration, or the rAAV or the immunosuppressant macrolide is administered after administration of rAAV.

[0253] In some combination therapies and agents, the immunosuppressant macrolide is administered at least once daily for at least 3 days prior to administration of rAAV, or at least 4 days prior to administration of rAAV, or at least 5 days prior to administration of rAAV, or at least 6 days prior to administration of rAAV, or at least 7 days prior to administration of rAAV, or at least 10 days prior to administration of rAAV, or at least 14 days prior to administration of rAAV, or at least 30 days prior to administration of rAAV, or at least 45 days prior to administration of rAAV, or at least 60 days prior to administration of rAAV. In some combination therapies and agents, the immunosuppressant macrolide is administered 30-60 days prior to administration of rAAV.

[0254] The present disclosure also provides a combination therapy for treating muscular dystrophy in a human subject in need thereof, comprising co-administering rAAV and an immunosuppressive regimen, wherein the rAAV and one or more components of the immunosuppressive regimen are administered simultaneously, sequentially, or at different times, including treating DMD, Becker muscular dystrophy, and limb-girdle muscular dystrophy. In addition, the present disclosure also provides a use of a combination therapy comprising rAAV and an immunosuppressive regimen for the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, including treating DMD, Becker muscular dystrophy, and limb-girdle muscular dystrophy, wherein the rAAV and one or more components of the immunosuppressive regimen are administered simultaneously, sequentially, or at different times. For example, the present disclosure provides a combination therapy for treating muscular dystrophy comprising rAAV.MHCK7.microdystrophin and an immunosuppressive regimen, wherein the immunosuppressive regimen comprises one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide, and wherein the rAAV and one or more of the components of the immunosuppressive regimen are administered simultaneously, sequentially, or at different times. For example, the present disclosure provides a combination therapy for treating muscular dystrophy, comprising an rAAV selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and an immunosuppressive regimen, wherein the immunosuppressive regimen comprises one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide, and wherein the rAAV and one or more of the components of the immunosuppressive regimen are administered simultaneously, sequentially, or at different times.

[0255] For example, the present disclosure provides the use of a combination therapy comprising rAAV.MHCK7.microdystrophin and an immunosuppressive regimen for the preparation of a medicament for treating muscular dystrophy, wherein the immunosuppressive regimen comprises one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide, and wherein the rAAV and one or more of the components of the immunosuppressive regimen are administered simultaneously, sequentially, or at different times. For example, the disclosure provides the use of an rAAV and an immunosuppressive regimen for treating muscular dystrophy, wherein the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and the immunosuppressive regimen includes one or more of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide, and wherein the rAAV and one or more of the components of the immunosuppressive regimen are administered simultaneously, sequentially, or at different times.

[0256] The term immunosuppressive regimen refers to a therapeutic method for suppressing or modulating a subject's immune system. The regimen includes the administration of one or more immunosuppressive agents. In some embodiments, the immunosuppressive regimen includes the administration of an anti-inflammatory steroid, an anti-CD20 antibody, and an immunosuppressive macrolide.

[0257] In an exemplary combination therapy or agent, the immunosuppressive regimen includes an anti-inflammatory steroid administered about 24 hours prior to administration of rAAV. In another exemplary combination therapy or agent, the immunosuppressive regimen includes an anti-inflammatory steroid administered prior to administration of rAAV, wherein the anti-inflammatory steroid is administered at least once daily from day 1 to day 30 after administration of rAAV, or the anti-inflammatory steroid is administered at least once daily from day 1 to day 60 after administration of rAAV. In another embodiment, a glucocorticoid anti-inflammatory steroid, such as prednisone, is administered for at least 60 days after administration of rAAV at 1 mg / kg.

[0258] In any of the disclosed combination therapies and medications, in the immunosuppressive therapy, the anti-inflammatory steroid is a glucocorticoid such as prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort. In some combination therapies, the anti-inflammatory steroid is formulated for oral administration.

[0259] In further exemplary combination therapies and agents, the immunosuppressive regimen includes an anti-CD20 specific antibody administered prior to administration of the rAAV. For example, the anti-CD20 antibody is formulated for administration by intravenous infusion. Exemplary anti-CD20 specific antibodies include rituximab, ocrelizumab, or ofatumumab.

[0260] In some combination therapies and agents, the anti-CD20-specific antibody is administered at least 14 days prior to administration of the rAAV. In other embodiments, the anti-CD20-specific antibody is administered about 60 days prior to administration of the rAAV, about 45 days prior to administration of the rAAV, about 30 days prior to administration of the rAAV, 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, and within about 24 hours of administration of the rAAV. Additionally, the anti-CD20-specific antibody is administered 30 to 60 days prior to administration of the rAAV. The disclosed immunosuppressive regimens also include administering the anti-CD20-specific antibody after administration of the rAAV.

[0261] Additionally, the disclosed immunosuppressive regimens include an immunosuppressant macrolide administered at least once daily for at least three days prior to administration of the rAAV. The immunosuppressant regimen may also include an immunosuppressant macrolide administered after administration of the rAAV. In any of the disclosed immunosuppressive regimens, the immunosuppressant macrolide is formulated for oral administration. Exemplary immunosuppressant macrolides include tacrolimus, pinecrolimus, or sirolimus.

[0262] In some embodiments, the disclosed immunosuppressive regimens are administered 30 to 60 days prior to administration of the rAAV. Additionally, the immunosuppressive regimen is administered about 60 days prior to administration of the rAAV, about 45 days prior to administration of the rAAV, about 30 days prior to administration of the rAAV, about 14 days prior to administration of the rAAV, about 7 days prior to administration of the rAAV, about 24 hours prior to administration of the rAAV, or about 12 hours prior to administration of the rAAV.

[0263] In certain embodiments, the present disclosure provides a combination therapy for treating muscular dystrophy in a human subject in need thereof, the combination therapy comprising rAAV and an immunosuppressive regimen comprising: i) an anti-inflammatory steroid administered orally about 24 hours before administration of the rAAV and administered at least once daily from day 1 to day 30 after administration of the rAAV, or administered at least once daily from day 1 to day 60 after administration of the rAAV; ii) an anti-CD20 antibody administered intravenously about 14 days before administration of the rAAV, about 7 days before administration of the rAAV, and within about 24 hours of administration of the rAAV (and optionally comprising an anti-CD20 antibody administered after administration of the rAAV); and iii) an immunosuppressive macrolide administered orally once daily for at least 3 days before administration of the rAAV (and optionally comprising an immunosuppressive macrolide administered after administration of the rAAV). For example, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort, the anti-CD20 specific antibody is rituximab, ocrelizumab, or ofatumumab, or more of the anti-inflammatory steroid, anti-CD20 antibody, and immunosuppressive macrolide, and the immunosuppressive macrolide is tacrolimus, pinecrolimus, or sirolimus. In an exemplary embodiment, the immunosuppressive regimen includes the anti-inflammatory steroid prednisone or prednisolone, the anti-CD20 antibody rituximab, and the immunosuppressive macrolide sirolimus.

[0264] In certain embodiments, the disclosure provides use of a combination therapy comprising an rAAV and an immunosuppressive regimen for treating limb-girdle muscular dystrophy in a human subject in need thereof, wherein the rAAV is selected from the group consisting of AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, and rAAVrh.74.MHCK7.huAN05, and the combination therapy comprises the rAAV and an immunosuppressive regimen, the immunosuppressive regimen being administered i) about 24 hours prior to administration of the rAAV. and an orally administered anti-inflammatory steroid, and an anti-inflammatory steroid administered at least once daily from day 1 to day 30 after administration of rAAV, or an anti-inflammatory steroid administered at least once daily from day 1 to day 60 after administration of rAAV; ii) an anti-CD20 antibody administered intravenously about 14 days before administration of rAAV, about 7 days before administration of rAAV, and about 24 hours after administration of rAAV (and optionally including an anti-CD20 antibody administered after administration of rAAV); and iii) an immunosuppressive macrolide administered orally once daily for at least 3 days before administration of rAAV (and optionally including an immunosuppressive macrolide administered after administration of rAAV). For example, the anti-inflammatory steroid is prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort, the anti-CD20 specific antibody is rituximab, ocrelizumab, or ofatumumab, or more of the anti-inflammatory steroid, anti-CD20 antibody, and immunosuppressive macrolide, and the immunosuppressive macrolide is tacrolimus, pinecrolimus, or sirolimus. In an exemplary embodiment, the immunosuppressive regimen includes the anti-inflammatory steroid prednisone or prednisolone, the anti-CD20 antibody rituximab, and the immunosuppressive macrolide sirolimus.

[0265] The present disclosure also provides a combination therapy for treating muscular dystrophy in a human subject in need thereof, the combination therapy comprising a dose, e.g., a second dose, of rAAV, wherein the subject's plasma has been subjected to at least one therapeutic plasma exchange (TPE) prior to administration of the second dose of recombinant adenovirus-associated (rAAV), and the subject received the first dose of rAAV prior to TPE. For example, the rAAV is rAAV.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. The muscular dystrophy is DMD, Becker muscular dystrophy, or limb-girdle muscular dystrophy.

[0266] Additionally, the present disclosure provides use of a combination therapy for the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, the combination therapy including a dose of rAAV, e.g., a second dose of rAAV, wherein the subject's plasma has been subjected to at least one therapeutic plasma exchange (TPE) prior to administration of the second dose of recombinant adenovirus-associated (rAAV), and the subject received the first dose of rAAV prior to TPE. For example, the rAAV is rAAV.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. The muscular dystrophy is DMD, Becker muscular dystrophy or limb-girdle muscular dystrophy.

[0267] In any of the disclosed combination therapies and uses, the subject's plasma is subjected to at least two TPEs or at least three TPEs before administration of the second dose or rAAV. In some embodiments, the subject's plasma is subjected to at least four TPEs before administration of the second dose of rAAV, or the subject's plasma is subjected to five TPEs before administration of the second dose of rAAV, or the subject's plasma is subjected to six TPEs before administration of the second dose of rAAV, or the subject's plasma is subjected to seven TPEs before administration of the second dose of rAAV.

[0268] The present disclosure provides a combination therapy for treating muscular dystrophy in a human subject in need thereof, the combination therapy comprising an rAAV administered to the subject, the subject's plasma having been subjected to at least one therapeutic plasma exchange (TPE) prior to administering the rAAV, and the rAAV rAAV.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. The muscular dystrophy is DMD, Becker muscular dystrophy, or limb-girdle muscular dystrophy.

[0269] The present disclosure provides use of a combination therapy for the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, the combination therapy comprising an rAAV administered to the subject, the subject's plasma having been subjected to at least one therapeutic plasma exchange (TPE) prior to administering the rAAV, and the rAAV rAAV.MHCK7.microdystrophin, AAVrh.74.tMCK.CAPN3, rAAVrh.74.MHCK7.DYSF, scAAVrh.74.MHCK7.hSGCG, AAVrh74.tMCK.hSCGA, scAAVrh74.MHCK7.HSGCB, or rAAVrh.74.MHCK7.huAN05. The muscular dystrophy is DMD, Becker muscular dystrophy, or limb-girdle muscular dystrophy.

[0270] In any of the disclosed combination therapies and uses, the subject's plasma is subjected to at least two TPEs before administering rAAV, at least three TPEs before administering rAAV, at least four TPEs before administering rAAV, at least five TPEs, at least six TPEs before administering rAAV, or at least seven TPEs before administering rAAV. In these disclosed combination therapies and uses, the subject is administered an anti-inflammatory steroid about 24 hours before administering rAAV. Additionally, in some embodiments, the subject is administered an anti-inflammatory steroid at least once daily from day 1 to day 60 after administration of rAAV. For example, the anti-inflammatory steroid is formulated for oral administration. Additionally, the anti-inflammatory steroid is a glucocorticoid such as prednisone, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, or deflazacort.

[0271] In any of the disclosed combination therapies and uses, the subject's plasma is subjected to TPE at least 9 days, at least 7 days, 5 days, or 2 days prior to administration of rAAV. In addition, there is about 24 to about 48 hours between TPE sessions performed on the subject's plasma prior to administration of rAAV. In certain embodiments, the subject's plasma is subjected to at least two TPE sessions prior to administration of rAAV, with about 48 hours between TPE sessions.

[0272] In any of the combination therapies and uses described herein, the subject has an anti-AAVrh.74 antibody level of about 1:400 or less upon administration of rAAV. For example, the subject has an anti-AAVrh.74 antibody level of about 1:100 to about 1:400 upon administration of rAAV, or an anti-AAVrh.74 antibody level of about 1:100 to 1:300, or an anti-AAVrh.74 antibody level of about 1:100 to 1:200, or an anti-AAVrh.74 antibody level of about 1:250 to 1:500, or an anti-AAVrh.74 antibody level of about 1:200 to 1:400. The antibody titer is determined as the total antibody binding titer. In any of the disclosed combination therapies and uses for treating muscular dystrophy, the presence of anti-AAVrh.74 antibodies is determined in the subject's serum or plasma before administration of rAAV, after administration of rAAV, before an immune response or adverse event is observed, or after an immune response or adverse event is observed. In addition, the presence of anti-AAVrh.74 antibodies is determined before administering an immunosuppressive regimen or TPE. For example, the presence of anti-AAVrh.74 antibodies is determined before any administration of any combination therapy or drug containing AAV to the subject, or before administration of any combination therapy or drug containing AAVrh.74 to the subject.

[0273] Additionally, in the disclosed combination therapies and uses, the level of anti-AAVrh.74 antibodies in the subject's serum or plasma is used as a positive control, e.g., the positive control utilizes an anti-AAVrh.74 monoclonal antibody, such as any of the anti-AAVrh.74 monoclonal antibodies described herein.

[0274] In any of the disclosed methods and uses, the presence of anti-AAVrh.74 antibodies in a subject is determined using a quantitative method, the subject is identified as seropositive for anti-AAVrh.74 antibodies based on the quantification, and the immunosuppressive regimen or TPE is selectively administered to seropositive subjects.

[0275] The present disclosure provides the following additional aspects.

[0276] Claim 1. A method for treating muscular dystrophy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated virus (rAAV) and an anti-inflammatory steroid, wherein the rAAV is of serotype AAVrh.74, and the rAAV comprises an expression cassette of SEQ ID NO:9.

[0277] Claim 2. The rAAV is 1.33 x 10 14 1. The method of claim 1, wherein the antibody is administered at a dose of 1.33×10 vg / kg. 14 The dose is 2 x 10 as determined by supercoiled qPCR DNA standard for titer determination. 14 Determined using a linear qPCR DNA standard corresponding to

[0278] Claim 3. The method of claim 1 or 2, wherein the subject has Duchenne muscular dystrophy, has not previously received rAAVrh.74-based gene therapy, and has been determined to be seropositive for rAAV.rh74 antibodies.

[0279] Claim 4. The method of claim 3, wherein the subject is determined to be seropositive for rAAVrh.74 antibodies based on an ELISA immunoassay, and the subject exhibits an absorbance ratio of 2.00 at a serum dilution of 1:400 or greater.

[0280] Claim 5. The method of any one of claims 1 to 3, further comprising the step of determining the presence of anti-AAVrh.74 antibodies in the serum or plasma of the subject prior to any administration of rAAVrh74 to the subject.

[0281] Claim 6. The method of claim 5, wherein determining the presence of anti-AAVrh.74 antibodies is determined by ELISA immunoassay and the subject exhibits an absorbance ratio of 2.00 or greater at a serum dilution of 1:400.

[0282] Claim 7. The method of any one of claims 1 to 6, wherein the anti-inflammatory steroid is administered orally.

[0283] Claim 8. The method of any one of claims 1 to 7, wherein the anti-inflammatory steroid is administered about 12 hours prior to administration of the rAAV.

[0284] Claim 9. The method of any one of claims 1 to 7, wherein the anti-inflammatory steroid is administered at least 12 hours prior to administration of the rAAV.

[0285] 10. The method of any one of claims 1 to 7, wherein the anti-inflammatory steroid is administered at least 12 hours prior to administration of the rAAV.

[0286] 11. The method of any one of claims 1 to 7, wherein the anti-inflammatory steroid is administered at least 24 hours prior to administration of the rAAV.

[0287] Claim 12. The method of any one of claims 1 to 11, wherein the anti-inflammatory steroid is administered at least once daily from day 1 to about 30 days after administration of rAAV, or at least once daily from day 1 to about 60 days after administration of rAAV.

[0288] Claim 13. The method of any one of claims 1 to 11, wherein the anti-inflammatory steroid is administered at least once daily for at least 30 days after administration of the rAAV.

[0289] Claim 14. The method of any one of claims 1 to 11, wherein the anti-inflammatory steroid is administered at least once daily for at least 60 days after administration of the rAAV.

[0290] Claim 15. The method of any one of claims 1 to 14, wherein the anti-inflammatory steroid is a glucocorticoid.

[0291] 16. The method of claim 13, wherein the immune cell is prednisone.

[0292] In addition, the present disclosure provides the following additional aspects:

[0293] Claim 1. A method of treating muscular dystrophy in a human subject in need thereof, comprising: a) subjecting the subject's plasma to at least one therapeutic plasma exchange (TPE) prior to administering a recombinant adenovirus-associated (rAAV); b) administering an rAAV, wherein the rAAV is of serotype rhAAVrh.74 and comprises an expression cassette of SEQ ID NO:9.

[0294] Claim 2. The method of claim 1, wherein the subject's plasma is subjected to at least two TPEs, at least three TPEs, at least four TPEs, at least five TPEs rAAV, at least six TPEs, or at least seven TPEs prior to administration.

[0295] Claim 3. The method of claim 1 or 2, wherein the subject's plasma is subjected to TPE at least 9 days prior to administration, at least 7 days prior to administration, 5 days prior to administration, or 2 days prior to administration of the rAAV.

[0296] Claim 4. The method of any one of claims 1 to 3, wherein the subject's plasma is subjected to TPE on the day the rAAV is administered.

[0297] Claim 5. The method of any one of claims 1-4, wherein the subject's plasma is subjected to at least two TPEs with about 48 hours between TPEs.

[0298] Claim 6. The rAAV is 1.33 x 10 14 The method according to any one of claims 1 to 5, wherein the dose is 1.33 x 10 14 The dose is 2 x 10 as determined by supercoiled qPCR DNA standard for titer determination. 14 Linear qPCR corresponding to It is determined using a DNA standard.

[0299] Claim 7. The method of any one of claims 1-6, wherein the subject is afflicted with Duchenne muscular dystrophy and the subject has been determined to be seropositive for rAAVrh.74 antibodies.

[0300] Claim 8. The method of claim 7, wherein the subject has been administered AAVrh.74 at least once prior to the administering step of claim 1b.

[0301] 9. The method of claim 7, wherein the subject is determined to be seropositive for rAAVrh7 antibodies based on an ELISA immunoassay, and the subject exhibits an absorbance ratio of 2.00 or greater at a serum dilution of 1:100.

[0302] 10. The method of any one of claims 1 to 9, further comprising the step of determining the presence of anti-AAVrh.74 antibodies in the serum or plasma of the subject prior to administration of rAAVrh.74.

[0303] 11. The method of claim 10, wherein determining the presence of anti-AAVrh.74 antibodies is determined by ELISA immunoassay and the subject exhibits an absorbance ratio of 2.00 or greater at a serum dilution of 1:100.

[0304] 12. The method of any one of claims 1 to 11, further comprising administering an anti-inflammatory steroid.

[0305] Claim 13. The method of claim 12, wherein the anti-inflammatory steroid is administered orally.

[0306] 14. The method of claim 12 or 13, wherein the anti-inflammatory steroid is administered about 12 hours prior to administration of the rAAV.

[0307] 15. The method of claim 12 or 13, wherein the anti-inflammatory steroid is administered at least 12 hours prior to administration of the rAAV.

[0308] 16. The method of claim 12 or 13, wherein the anti-inflammatory steroid is administered at least 12 hours prior to administration of the rAAV.

[0309] 17. The method of claim 12 or 13, wherein the anti-inflammatory steroid is administered at least 24 hours prior to administration of the rAAV.

[0310] 18. The method of any one of claims 12 to 17, wherein the anti-inflammatory steroid is administered at least once daily from day 1 to about 30 days after administration of the rAAV, or at least once daily from day 1 to about 30 days after administration of the rAAV.

[0311] Claim 19. The method of any one of claims 12 to 17, wherein the anti-inflammatory steroid is administered at least once daily for at least 30 days after administration of the rAAV.

[0312] Claim 20. The method of any one of claims 12-17, wherein the anti-inflammatory steroid is administered at least once daily for at least 60 days after administration of the rAAV.

[0313] Claim 21. The method of any one of claims 12 to 17, wherein the anti-inflammatory steroid is a glucocorticoid.

[0314] 22. The method of claim 21, wherein the immune cell is prednisone.

[0315] The following examples are offered by way of illustration and not by way of limitation: Numerical ranges recited include each integer value within each range, including the stated integer minimum and maximum. [Example]

[0316] Example 1 A) Generation of the AAVrh74.MHCK7.microdystrophin control The AAVrh74.MHCK7.microdystrophin plasmid contains a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 1). The microdystrophin construct features an in-frame rod deletion (R4-R23) that maintains hinges 1, 2, and 4, as well as the cysteine-rich domain, producing a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) was driven by the MHCK7 promoter (795 bp). The plasmid was constructed from the rAAV.MCK.microdystrophin plasmid by removing the MCK promoter and inserting the MHCK7 promoter. Following the core promoter, 53 bp of endogenous mouse MCK Exon 1 (untranslated) is present, followed by the SV40 late 16S / 19S splice signal (150 bp) and a small 5' UTR (61 bp), allowing for efficient transcription initiation. The intron and 5' UTR were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contained a consensus Kozak sequence immediately before the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contained the (R4-R23 / Δ71-78) domain, as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)). Complementary DNA was codon-optimized for human use and synthesized by GenScript (Piscataway, NJ) (Mol Ther 18, 109-117 (2010)). The only viral sequence included in this vector was the AAV2 inverted terminal repeat sequence, required for both viral DNA replication and packaging. The microdystrophin cassette contained a small 53-bp synthetic poly(A) signal for mRNA termination.

[0317] Previous studies have demonstrated cardiac expression using the MHCK7 promoter (Salva et al. Mol Ther 15, 320-329 (2007)), and AAVrh.74 achieved expression in skeletal muscle, diaphragm, and cardiac muscle (Sondergaard et al. Annals of Clinical and Transl Neurology 2, 256-270 (2015)). The construct sequence in Figure 1 was encapsidated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a lymph node of a rhesus macaque and is discussed in Rodino-Klapac et al. Journal of Translational Medicine 5, 45 (2007). [Table 3]

[0318] B) Generation of plasmids encoding AAVrh74.MHCK7.microdystrophin control and kanamycin (Kan) resistance Cloning of MHCK7μDys.KAN was achieved by isolating the MHCK7.μDys fragment from the MHCK7.μDys.AMP plasmid and kanamycin backbone and annealing them using the NEBuilder cloning workflow. The MHCK7.μDys fragment was isolated by restriction enzyme digestion with SnaBI. Digestion was carried out in a 50 μL total reaction in 1x CutSmart Buffer (NEB) and 1 μL of SnaBI at 37°C for 1 hour. The resulting fragment was isolated by electrophoresis using a 1% agarose gel run at 105 volts for 1.5 hours. The band corresponding to the MHCK7μDys insert was excised and purified using a gel purification kit (Macherey-Nagel). The DNA concentration of the resulting fragment was 10 ng / μL. The Kan backbone fragment was isolated by XbaI restriction enzyme digestion in a 50 μL reaction using 1x CutSmart Buffer (NEB) and 1 μL XbaI at 37°C for 1 hour. The resulting fragment was isolated by electrophoresis using a 1% agarose gel run at 105 volts for 1.5 hours. The band corresponding to the Kan backbone was excised and purified using a gel purification kit (Macherey-Nagel). The DNA concentration of the resulting fragment was 8.1 ng / μL. The two fragments were annealed using the NEB Builder cloning workflow, which has the ability to join two fragments with overlapping sequences. The NEBuilder cloning reaction was performed according to manufacturer automation at 50°C for 15 minutes using a ratio of MHCK to kanamycin backbone of 7.0 μg / µL Dys in 1x NEBuilder HiFi DNA Assembly Master Mix for a total reaction volume of 20 μL. The resulting clones were then transformed into NEB® Stable Competent E. coli (C3040) by adding 2.5 μL of the cloning product to the cells for 30 minutes on ice, then 30 seconds at 42° C. and an additional 5 minutes on ice. After transformation, 950 μL of growth medium was added to the cells and grown for 1.5 hours at 30° C. with shaking at 225 rpm.After growth, 450 μL of these cells were plated onto 50 μg / mL kanamycin LB agar plates and incubated overnight at 30°C in a dry incubator. Colonies were picked from the plates and grown overnight in LB containing 50 μg / mL kanamycin. DNA was isolated from 3 mL of this culture using a QIAprep® Spin Miniprep Kit (Qiagen). This DNA was used to verify the cloning product. The cloning product was confirmed by restriction enzyme digestion with PmeI, MscI, and SmaI followed by gel electrophoresis. The cloning product was further verified by sequencing. The resulting plasmid is shown in SEQ ID NO:8 and is presented in Figures 8 and 9. The sequence of the construct in Figure 7, which corresponds to the sequence of SEQ ID NO:9, and nucleotides 1 to 4977 of SEQ ID NO:8, were encapsidated into the AAVrh.74 virions described above.

[0319] C) Generation of pAAV.MCK.microdystrophin construct The pAAV.MCK.microdystrophin plasmid was constructed by inserting an MCK expression cassette driving a codon-optimized human microdystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski et al., J. Virol. 61(10):3096-3101). To drive muscle-specific gene expression, the construct included a muscle-specific regulatory element. This regulatory element consisted of the mouse MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). Following the core promoter, the construct included 53 bp of the endogenous mouse MCK exon 1 (untranslated) for efficient transcription initiation, followed by the SV40 late 16S / 19S splice signal (97 bp) and a small 5' UTR (61 bp). The intron and 5' UTR were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contains a consensus Kozak site immediately before the ATG start and a small 53-bp synthetic poly(A) signal for mRNA termination. As previously described by Harper et al. Nat. Med. 8(3):253-61, 2002, the human microdystrophin cassette contains the (R4-R23 / Δ71-78) domain.

[0320] The pAAV.MCK.microdystrophin plasmid contained a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 5). This sequence was encapsidated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a rhesus macaque lymph node and described in Rodino-Klapac et al. Journal of Tran. Med. 45 (2007).

[0321] D) Vector production The vectors for the studies described herein were produced using a triple transfection method in HEK293 cells under research-grade conditions. Post-production vector characterization included titer determination by qPCR with supercoiled standards, endotoxin level measurement (EU / mL), and sterility assessment. The produced vectors were analyzed by SDS-PAGE to verify consistency of the banding pattern with the expected rAAV vector. The surrogate vector used in these studies, rAAVrh74.MCHK7.uDYS.FLAG, was constructed as described above with the addition of a C-terminal FLAG tag.

[0322] Example 2 Systemic gene delivery of rAAVrh74.MCHK7.microdystrophin with immunosuppression in non-human primate studies The primary goal of this study was to identify the optimal dose, duration, and immunosuppressive regimen to optimize gene expression after intravascular delivery of rAAV.rh74.MHCK7.microdystrophin. The study began with five cohorts of rhesus macaques (n = 3 per cohort) with varying durations of immunosuppression before and after vector administration (Table 1). Rhesus macaques are referred to herein as "non-human primates" or NHPs. [Table 4]

[0323] In cohort 1, control monkeys received 2 × 10 rAAVrh74.MHCK7.microdystrophin injections into the cephalic or saphenous vein without immunosuppressant therapy. 14 In all other cohorts, rhesus monkeys received 2 × 10 mAb delivered into the cephalic or saphenous vein along with immunosuppression. 14 They also received an intravenous injection of rAAVrh74.MHCK7.microdystrophin at 1000 mg / kg.

[0324] In cohort 2, oral prednisone (2 mg / kg / day) was administered from 1 day before systemic gene transfer until 30 days after gene transfer (n = 3). In cohort 3, oral prednisone (2 mg / kg / day) was administered from 1 day before systemic gene transfer until 60 days after gene transfer (n = 3). In cohort 4, oral prednisone (2 mg / kg / day) was administered from 14 days before systemic gene transfer until 60 days after gene transfer (n = 3).

[0325] In cohort 5 (n = 3), a triple immunosuppression regimen was investigated. In this cohort, rituximab (750 mg / m²) was administered by intravenous infusion in two administration sessions, 14 and 7 days before vector administration, with the third administration given on the day of vector administration before gene transfer. If antibodies do not respond to the first three doses, rituximab can be administered for a fourth dose after the infusion. Sirolimus (4 mg / m²) was administered three days before vector administration. 2 Prednisone was administered orally (2 mg / kg / day) from 1 day before vector administration until 30 days after vector administration.

[0326] After treatment, all cohorts underwent needle biopsies obtained from the tibialis anterior (TA) and / or gastrocnemius (GN) muscles. Biopsies were collected up to three times: before gene transfer and at 6, 8, and 12 weeks after gene transfer. Blood samples for immunology, CBC, and chemistry were collected at least every other week.

[0327] The following hematology measurements were performed on blood samples: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin, concentration, red blood cell distribution width, absolute reticulocyte count, platelet count blood count, absolute basophil count, absolute large unstained cell count, and blood smear.

[0328] The following clinical chemistry measurements were performed on blood samples: glucose, urea nitrogen, total protein, albumin, globulin, albumin:globulin ratio, total bilirubin, alanine aminotransferase, glutamate dehydrogenase, cholesterol, gamma glutamyltransferase, aspartate aminotransferase, and alkaline phosphatase.

[0329] Safety profile and transduction efficiency Total antibody responses to AAVrh.74 (anti-AAVrh.74 antibodies) were similar across cohorts, with no evidence of abnormal observations, except for one NHP from Cohort 2 (NHP_03), who did not develop an antibody response to AAVrh.74. In addition, an NHP from Cohort 5, despite being treated with a triple immunosuppressive regimen, demonstrated an antibody response to AAVrh.74 similar to that observed in NHPs from Cohorts 1-4. Adverse effects experienced by NHPs from Cohorts 1-4 included transient elevations of alanine transaminase (ALT) and aspartate transaminase (AST) liver enzymes. Two NHPs from Cohort 1 (NHP_12, NHP_13), one NHP from Cohort 3 (NHP_06), and one NHP from Cohort 4 (NHP_07) demonstrated elevated ALT and AST liver enzymes 12 weeks after gene transfer. Regarding transduction efficiency, no statistically significant differences in vector genome copies (vg copies / μg DNA) were observed between NHP cohorts 1–5 at 12 weeks post-transduction (P>0.05).

[0330] Example 3 Administration by therapeutic plasma exchange (TPE) The primary goal of this study was to optimize both the technique and gene expression using therapeutic plasma exchange (TPE) to remove pre-existing AAV antibodies and evaluate rechallenge with rAAVrh74.MHCK7.dystrophin after TPE.

[0331] In the study described in Example 2, non-human primates (from cohorts 2, 3, and 4) pre-injected with rAAVrh74.MHCK7.microdystrophin underwent two to three TPEs during a single apheresis procedure. Four and two weeks before TPE, primates underwent a maximum blood draw (10% of the primate's body weight). Blood was stored in ACDA solution for up to 30 days and used on the day of TPE to prime the apheresis machine to prevent excessive blood loss during the procedure. In addition, titers of AAVrh.74-binding antibodies were determined to confirm that the titer exceeded 1:400, the threshold for inclusion in current clinical trials. After TPE, 2 × 10 14 vg / kg of rAAVrh74.MHCK7.microdystrophin or rAAVrh74.MHCK7.microdystrophin.FLAG was delivered systemically via the saphenous or cephalic vein.

[0332] These NHPs also received prednisone (2 mg / kg) once daily from 1 day before TPE until 30 days after TPE and gene transfer. Blood samples for chemistry, CBC, ELISA, and ELISpot assays were collected before TPE, after TPE before rechallenge, and at least every other week until endpoint, which occurred between 8 and 12 weeks after the second gene transfer and included a complete necropsy. Western blot and qPCR for vector genome and FLAG immunofluorescence were performed to assess efficacy.

[0333] Additionally, non-human primates (Cohort 5) previously injected with the immunosuppressive regimen described in Example 2 were re-administered with rAAVrh74.MHCK7.microdystrophin without TPE to reduce pre-existing antibodies directed against AAVrh.74. Specifically, rituximab (750 mg / m 2 / day) delivered intravenously (IV) 7 and 14 days before gene transfer, once on the day of injection, and once after gene transfer, and sirolimus (4 mg / m 2100 mg / day) was delivered 3 days before gene transfer and continued until the end of the study. Sirolimus levels were monitored by blood sampling, varying between 3 and 14 ng / mL. Blood samples for chemistry, CBC, sirolimus blood concentration, ELISA, and ELISpot assays were collected at least every other week until the endpoint, which occurred between 8 and 12 weeks after the second gene transfer and included a complete necropsy. Observations of each animal were performed daily. NHP body weights were monitored every other week, and immunosuppressant dosages were adjusted accordingly.

[0334] Therapeutic Plasma Exchange Procedure: In the TPE process, whole blood was withdrawn via vascular access and subsequently centrifuged in a centrifuge within the apheresis machine to remove plasma (antibodies). Red blood cells were returned along with a replacement fluid (human albumin) for the primate's maintenance fluid. Due to the small size of nonhuman primates (<10 kg), the apheresis machine was primed with stored blood prior to plasma exchange to ensure safety and reduce the amount of blood withdrawn from the primate. Maximum blood draws (10% of the nonhuman primate's circulating blood) were performed 28 and 14 days before apheresis. The collected whole blood was preserved and stored in an anticoagulant acid-citrate-dextrose (ACDA) solution at 4°C for up to 30 days. Additionally, NHPs were provided with extra iron-rich supplements and concentrates. On the day of apheresis, nonhuman primates were intramuscularly sedated with Telazol (3–6 mg / kg), intubated, and secured to a heated procedure table. Maintenance of anesthesia was achieved with 1-4% isoflurane in oxygen. Vascular catheters were placed in both legs (saphenous veins), with one access port for whole blood collection and another in the contralateral leg for redelivery of red blood cells and replacement fluids. An additional catheter was placed in the arm (cephalic vein) to support fluid and blood collection throughout the procedure. After vascular access was obtained, animals were administered heparin (50-100 U / kg) to maintain adequate blood flow and prevent clotting during apheresis. NHPs were monitored using temperature, ECG, and respiration to determine the appropriate anesthetic plane.

[0335] As described above, NHPs were connected to a COBE Spectra apheresis device via a catheter, and the device was primed with previously collected blood. One total plasma exchange corresponds to the removal and replacement of the entire circulating blood volume in one session. Two to three plasma exchanges were performed to achieve an estimated antibody removal rate of 98%. Blood was collected after each completed exchange for blood chemistry analysis and serum antibody testing. Immediately after plasma exchange, the NHPs were disconnected from the apheresis unit and re-administered systemically with rAAVrh74.MHCK7.microdystrophin. After vector delivery, all catheters were removed, and pressure was applied to control bleeding. The animals were monitored until fully ambulatory.

[0336] Autopsy analysis At necropsy, NHPs were administered Euthasol (1 mL / 10 lb) at endpoint (which could occur between 8 and 12 weeks after rechallenge). Blood was collected and whole blood was sent for complete blood count (CBC) analysis, sirolimus test levels, and serum chemistry. Tissues were then collected and sent for analysis by an independent veterinary histopathologist, and gene and protein expression was analyzed to assess efficacy and toxicity.

[0337] To evaluate gene expression optimization, 2 x 10 14Muscle biopsies were collected before and after intravenous administration of rAAVrh74.MHCK7.microdystrophin or rAAVrh74.MHCK7.microdystrophin.FLAG at 100 mg / kg of rAAVrh74.MHCK7.microdystrophin.FLAG from NHPs. DNA from pre- and post-injection muscle tissues was extracted for real-time quantitative qPCR to detect specific sequences of vector DNA. Proteins from all collected muscles were extracted, and Western blots were performed to detect microdystrophin protein (138 kD) compared with pre-biopsy tissue. Additionally, naive full-length dystrophin (427 kD) was used as a normal control and quantitatively compared with microdystrophin protein as a measure of gene expression outcomes. Immunofluorescence staining to observe the presence of FLAG expression was performed in primates injected with rAAVrh74.MHCK7.microdystrophin.FLAG.

[0338] Blood samples were collected at baseline and every other week until endpoint to assess the safety of immunosuppressive regimens, vector administration, and rechallenge. Serum chemistries, CBCs, and sirolimus levels were monitored every other week throughout both arms of the study. ELISpot analysis was used to assess T cell responses to both the AAVrh.74 peptide and the microdystrophin peptide. Finally, anti-AAVrh.74 antibody responses were monitored every other week throughout both arms of the study.

[0339] The following hematology measurements were performed on blood samples: red blood cell (erythrocyte) count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin, concentration, red blood cell distribution width, absolute reticulocyte count, platelet count blood count, absolute basophil count, absolute large unstained cell count, and blood smear.

[0340] The following clinical chemistry measurements were performed on blood samples: glucose, urea nitrogen, total protein, albumin, globulin, albumin:globulin ratio, total bilirubin, alanine aminotransferase, glutamate dehydrogenase, cholesterol, gamma glutamyltransferase, aspartate aminotransferase, and alkaline phosphatase.

[0341] result Total antibody titers against AAVrh.74 in NHPs before and after TPE (before rechallenge with rAAVrh74.MHCK7.micro-dystrophin) are shown in the table below. Figure 11 provides antibody titers against AAV74 in NHPs after rechallenge with rAAVrh74.MHCK7.micro-dystrophin. The number of TPC cycles that can be performed in NHPs is limited due to a lack of available donor blood. In humans, multiple rounds of TPE can be administered. Titers detected in Example 2 were obtained 12 weeks after the initial gene transfer (*). Titers detected in Example 3 were obtained before rechallenge injection of rAAVrh74.MHCK7.micro-dystrophin ( + NHP_03 was re-administered without prior TPE due to lack of antibody response to AAVrh.74. NHP_06 received only 0.5 cycles of TPE due to its small size and poor vascular access. [Table 5]

[0342] The TPE procedure was generally well tolerated. There were no abnormal immunological observations as assessed by IFN-γ spot-forming cell (SCF) levels in response to AAVrh.74 and microdystrophin peptides derived from peripheral blood mononuclear cells. Rechallenge after TPE resulted in increased liver enzyme levels (ALT / AST) in the following NHPs: NHP_01 and NHP_02, cohort 2; NHP_04, cohort 3; NHP_08 and NHP_09, cohort 4). This was resolved by continuing daily administration of prednisone.

[0343] NHPs from cohort 5 did not undergo TPE due to incompatibility with previous treatment with rituximab, and two NHPs (NHP_10, NHP_11) were rechallenged. Cohort 5 had total antibody titers to AAVrh.74 of >1:51,200 before rechallenge. The rechallenged NHP (cohort 5) with high antibody titers experienced the following adverse events: increased heart rate and ventilation rate, vomiting, rash near the delivery site, pallor, and shallow breathing; these events resolved after administration of diphenhydramine and dexamethasone.

[0344] Seven NHPs underwent two to three consecutive cycles of TPE, resulting in a decline in circulating antibody levels to AAVrh.74. Immediately after TPE, the NHPs were successfully re-administered with rAAVrh74.MHCK7.microdystrophin. Two NHPs from Cohort 4 (NHP_08 and NHP_09) achieved antibody titers of 1:200.

[0345] As shown in Figure 12, increased microdystrophin protein expression was observed in tissue samples from all NHPs re-administered with rAAVrh74.MHCK7.micro-dystrophin after TPE when compared to pre-TPE expression from 12-week biopsies (described in Example 2). Increased micro-dystrophin protein expression was observed in skeletal muscle (abdominal muscle), heart, and diaphragm.

[0346] Example 4 Alternatively, the tests and studies described in Examples 2 and 3 above are performed utilizing the rAAVrh74.MHCK7.microdystrophin construct set forth in SEQ ID NO:9, nucleotides 1-4977 set forth in SEQ ID NO:8, and nucleotides 56-5022 set forth in SEQ ID NO:6.

[0347] Example 5 Gene therapy for DMD with pre-existing AAVrh.74 antibody after therapeutic plasma exchange (TPE) A Phase 1 clinical trial will be conducted in humans to investigate gene therapy of DMD in patients with pre-existing AAVrh.74 antibodies following therapeutic plasma exchange (TPE). It is hypothesized that five cycles of TPE will reduce antibody binding to AAVrh.74 (also referred to herein as "anti-AAVrh.74 antibodies"), enabling safe and efficient muscle transduction using AAVrh74.MHCK7.microdystrophin to achieve mean expression levels >50% compared to baseline.

[0348] The objective and primary outcome of the study is the safe delivery of rAAV carrying the microdystrophin gene (AAVrh74.MHCK7.microdystrophin). Secondary objectives are expression of the microdystrophin gene in muscle of subjects with DMD and clinical improvement using the North Star Ambulatory Assessment for Muscular Dystrophy (NSAA) as a functional outcome measure.

[0349] The treatment plan is a two-phase (weekly) protocol combining safety and efficacy (Table 1), initially reducing AAVrh.74 antibodies by apheresis over a 10-day schedule, followed by intravenous delivery of AAVrh74.MHCK7.microdystrophin. In week 1, TPE is administered on alternate days: Monday (day 9, following the day of gene therapy infusion), Wednesday (day 7), and Friday (day 5). In week 2, TPE is administered every other day for two days, followed by intravenous infusion of AAVrh74.MHCK7.microdystrophin on Monday (day 2) and Wednesday (day 1), followed by delivery of AAVrh74.MHCK7.microdystrophin on the same day of TPE (day 1). Patients are transported to the outpatient apheresis unit according to the schedule in Table 1. Patients are admitted to the pediatric intensive care unit (PICU) for inpatient gene therapy infusion on day 1 and are scheduled for discharge on day 2. [Table 6]

[0350] This protocol has been developed based on the kinetics of IgG antibodies, which have been shown to have a rebound after 48 hours, recovering approximately 50-60% of the antibodies removed in the previous procedure, and therefore supports TPE every other day over a 10-day course, followed by gene delivery on the final day of TPE (designated day 1 for gene therapy) (Figure 13 and Exchange). (See Volumes 1 and 2). The timing of these procedures follows a similar course to other known antibody-mediated pathologies (Padmanabhan et al., J Clin Apheresis 34:171-354, 2019).

[0351] Because this protocol is being developed for a new indication, it is appropriate to follow existing established guidelines recommending repeated treatments of 5–6 (Padmanabhan et al., J Clin Apheresis 34:171–354, 2019) to 5–7 (Pham et al., Transfusion and Apheresis Science 58:237–246, 2019) treatments. Additional support for subjects in this protocol taking prednisone for immunosuppression is provided by the following assumption: A negligible rate of immunosuppression is assumed as a result of concurrent immunosuppressive therapy, and an extravascular equilibration rate relative to intravascular equilibration rate of approximately 1–2 percent per hour. Then, five separate procedures over a 7–10 day period are required to remove 90% of the body's initial immunoglobulin load. Additional treatments may be required if new antibody production occurs. (Fridey & Kaplan Therapeutic apheresis(plasma exchange or cytapheresis):Indications and technology.American Society for Apheresis Guidelines UPTODATE May 2020(available on-line)(Kaplan et al.J Clin Apheresis 28:3-10,2013)

[0352] protocol Patient characteristics for this protocol: Selection Criteria Ambulatory male subjects, of any ethnicity, aged 4 to 10 years (inclusive) at screening - Confirmed DMD frameshift or premature termination codon gene mutation CK>1000U / L Below the 95th percentile predicted for age in the 100m walk test, indicating symptomatic disease Ability to participate in motor skill assessment tests Weakness indicated by a history of difficult running, jumping, and stair climbing · Stable oral corticosteroid equivalent dose for at least 12 weeks prior to screening, with dose expected to remain constant throughout the study (except for potential changes in response to weight changes). Patients with AAVrh.74 antibody titers >1:400 as determined by ELISA immunoassay at baseline screening (Days 40-10) Exclusion criteria Signs of cardiomyopathy including ECHO with LVEF less than 40% Serological evidence of human immunodeficiency virus (HIV) infection or hepatitis B or C infection Diagnosis of (or ongoing treatment for) an autoimmune disease Laboratory abnormalities considered clinically significant (gamma-glutamyltransferase >3 times the upper limit of normal, bilirubin >3 mg / dL, creatinine >1.8 mg / dL, hemoglobin <8 g / dL or >18 g / dL, white blood cell count >18,500 / cm², platelets <50,000 / microliter). · Concomitant illness or need for chronic medication that, in the opinion of the PI, creates unnecessary risk of gene transfer. · Severe infection within the last 4 weeks (e.g. pneumonia, pyelonephritis, meningitis). Received any investigational drug (other than a corticosteroid) or exon-skipping drug (including EXONDYS51®) within 6 months of screening. Have received any type of gene therapy, cell-based therapy (e.g., stem cell transplant), or CRISPR / Cas9 therapy. · Family members do not want their patients' research participation disclosed by their primary care physicians and other health care providers.

[0353] Screening / Baseline Period The screening / baseline period is up to 4 weeks prior to Day 9. After obtaining informed consent, patients are assessed for eligibility. Screening includes collection of demographics and medical history, vital sign measurements, physical examination, electrocardiogram (ECG), echocardiogram (ECHO), and cardiac MRI. Blood and urine samples are collected for clinical and safety laboratory evaluations. Blood samples are also collected for hepatitis B and C, human immunodeficiency virus (HIV), and antibodies to AAVrh.74 and antigen-specific T cells against AAVrh.74 capsid and microdystrophin. A pre-treatment muscle biopsy, including the gastrocnemius muscle or a muscle selected by the primary investigator (PI), is performed after eligibility and prior to Day 9. Parents / caregivers are asked to complete the PROMIS questionnaire. Physical functionality assessment includes the North Star Ambulatory System for Muscular Dystrophy. The assessment includes a Timed Functional Assessment (NSAD) and timed functional tests including floor rising, standard four-step climb, 10-meter walk / run test, and 100-meter walk / run test.

[0354] therapeutic plasma exchange TPE involves removing a patient's plasma and replacing it with 5% albumin. Fresh frozen plasma (FFP) may be used during the TPE procedure if necessary for patient safety. Plasma removed during plasma exchange is not used for transfusion to another individual, in accordance with U.S. Food and Drug Administration (FDA) regulations.

[0355] protocol TPE is performed via a peripheral vein when possible. Patients more likely to have a tunneled central line placed by an interventional radiologist. Parents are instructed on how to care for the catheter between appointments.

[0356] Exchange volume - Plasma exchange of 1.0 to 1.5 plasma volumes is performed per procedure. A single plasma exchange reduces plasma macromolecular levels by 63% (J Clin Apheresis 2019;34:171-354). IgG antibodies, which distribute to both the intravascular and extravascular compartments, require multiple exchanges to reduce total body stores, typically performed every other day to allow for redistribution between both compartments. The first 1.0 to 1.5 plasma volume exchange removes the greatest amount of target substances, and the amount removed decreases with each subsequent exchange. The same volume of replacement fluid is used for each single plasma exchange. In this protocol, 5% albumin is used as the replacement fluid.

[0357] An immunosuppressive regimen is recommended to achieve a sustained response. Although rebound after exchange is possible in the absence of immunosuppression, all DMD boys participating in this program receive glucocorticoids as standard treatment for their disease. During the experimental protocol for plasma exchange, patients receive glucocorticoids (1 mg / kg prednisone or equivalent corticosteroid) one day before TPE. Patients remain on this dose for at least 60 days after gene delivery unless the PI determines that an early tapering is in the patient's best interest. (Table 1)

[0358] For further patient consideration, an alternative plan (called a "substitute schedule") is provided that may delay gene delivery and allow TPE #7 to proceed with gene therapy. If the target AAVrh.74 titer is not reached on day 1 following apheresis #4, TPE #5 is performed but does not proceed with gene delivery. AAV antibody levels are obtained on day 2 (Thursday), followed by apheresis, TPE #6, and gene delivery on Friday. However, if the titer does not reach the target of 1:100 on Thursday (day 2), do not proceed with TPE or gene delivery on Friday.

[0359] ***Gene delivery This is a 52-week, open-label clinical trial. Six to 12 patients who meet eligibility requirements will be enrolled and administered IV AAVrh74.MHCK7.micro-dystrophin. On Day 1, as an outpatient, a physical examination will be performed, and vital signs, blood samples, and urine samples will be collected. On Day 1, patients will be admitted to the hospital. A fifth and final TPE will be performed in the morning, and later that same day, patients will receive AAVrh74.MHCK7.micro-dystrophin administered IV over 1-2 hours, according to the study operations manual used for previous IRB- and FDA-approved protocols. On the day after the infusion (Day 2), patients will undergo a physical examination, have vital signs collected, and provide blood and urine samples before discharge.

[0360] Patients will be followed for 452 weeks and then progress to a 5-year long-term follow-up. Patients will complete follow-up visits after gene delivery at weeks 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 (relative to day 1 infusion) weeks. In addition, at weeks 3 and 5, patients will complete follow-up visits for evaluation of liver function tests. All patients will have a muscle biopsy performed at week 12. The biopsy will include the gastrocnemius muscle or a muscle of the PI's choice. Safety will be assessed by monitoring treatment-emergent adverse events (TEAEs), SAEs, and selected laboratory evaluations.

[0361] Example 6 Sandwich ELISA determination of antibodies in serum The materials for the sandwich ELISA assay are as follows: Capture antibody = anti-AAVrh.74 mAb Test sample = serum or plasma, useful for detection Antigen = AAVrh.74 capsid Blocking solution: 5% dry milk, 1% goat serum, 100 mL PBS Washing buffer = 0.05% PBS-Tween® Positive control = serum known to have anti-AAVrh.74 antibodies Secondary antibody = Anti-human HRP-conjugated antibody ·Substrate=TMB ·Stop liquid = sulfuric acid

[0362] method All wells of a 96-well plate are coated overnight with capture antibody diluted in carbonate buffer at 4°C. The contents are discarded, and the plate is blocked with blocking solution at 37°C for 1 hour. The blocking solution is discarded, and two AAVrh.74 capsids are added to the capture antibody-coated wells. Additionally, carbonate buffer is added to duplicate wells to determine background values. Unbound capsid is discarded, and test serum is added at a starting dilution of 1:25 in blocking solution and serially diluted. A positive control is diluted at a 1:400 dilution in blocking solution. The plate is washed with wash buffer, followed by a secondary incubation at a 1:10,000 dilution in blocking solution. The plate is washed, the buffer is discarded, and substrate is added, followed by termination of the assay with sulfuric acid. The absorbance of the plate is read at 450 nm.

[0363] Analysis and Results The absorbance ratio is determined by subtracting the mean optical density (OD) of the antigen-coated wells from the mean OD of the antigen-uncoated wells and dividing by the mean OD of the antigen-uncoated wells. A ratio of 2.00 or greater is considered a positive antibody response. The endpoint titer is determined by identifying the last serum dilution that results in a ratio of 2.00 or greater. The antibody cutoff is defined as a serum dilution greater than 1:400.

[0364] Example 7 Indirect ELISA: Determination of anti-AAVrh.74 antibodies in serum material Antigen = AAVrh.74 capsid Blocking solution: 5% dry milk, 1% goat serum, 100 mL of calcium- and magnesium-free PBS Washing buffer = 0.05% PBS-Tween® Primary antibody = serum or plasma for human testing Positive control = anti-AAVrh74 mAb, or optionally serum (or plasma) containing anti-rh74 antibody Negative control: Serum from a subject without anti-AAVrh74 antibodies can be included as a negative control. Secondary antibody = anti-human antibody Substrate = 3,3',5,5'-tetramethylbenzidine (TMB) ·Stop liquid = sulfuric acid

[0365] method 2x10 diluted in carbonate buffer 9Duplicate wells in a 96-well plate are coated overnight with antigen at a concentration of 1000 mg / well. Additionally, carbonate buffer is added to duplicate wells to determine background. The antigen is discarded, and the wells are blocked with blocking solution. The blocking solution is discarded, and the primary antibody (test serum) is added at a starting dilution of 1:25 in blocking solution and serially diluted. A positive control is diluted in blocking solution at a dilution of 1:400 if serum is used, or at...

Claims

[Claim 1] The invention described in the specification.

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