Recombinant AAV vectors for treating muscular dystrophy

Abstract

The present disclosure provides gene therapy vectors, such as recombinant adeno-associated viruses (rAAV) for expression of human micro anti-amyotrophin genes. The disclosure also provides compositions and methods for treating muscular dystrophy (e.g., Duchenne muscular dystrophy) using these rAAVs. The disclosure also provides for genotyping the DMD gene in a subject to determine if rAAV gene therapy should be tabounded.

Description

[0001] Reference to an electronically submitted sequence listing

[0002] The content of the electronically submitted sequence listing (name: 4140_062PC04_Seqlisting_ST26.xml; size: 55,294 bytes; creation date: September 18, 2023) submitted with this application is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of gene therapy. More specifically, the present disclosure provides gene therapy vectors for expressing a miniaturized human mini-dystrophin gene, such as adeno-associated virus (AAV) vectors. The present disclosure also provides methods for using these vectors to express mini-dystrophin in skeletal muscle (including the diaphragm and heart muscle) and to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in subjects with muscular dystrophy. Background Art

[0004] The importance of muscle mass and strength for daily activities (such as movement and breathing) and for whole-body metabolism is undeniable. Muscle function deficits lead to muscular dystrophy (MD), which is characterized by muscle weakness and wasting and has a severe impact on quality of life. The best-characterized MDs are caused by mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs are caused by membrane fragility associated with loss of the myofiber membrane-cytoskeleton tethering caused by DAPC. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in every 5,000 male newborns.

[0005] DMD is caused by mutations in the DMD gene, which lead to reduced mRNA and a lack of dystrophin, a 427kD sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51:919-28, 1987). The DAPC consists of multiple proteins at the muscle sarcolemma, which form structural linkages between the extracellular matrix (ECM) and the cytoskeleton via dystrophin, an actin-binding protein, and alpha-dystroglycan, a laminin-binding protein. These structural linkages serve to stabilize the muscle cell membrane during contraction and prevent contraction-induced damage. With the loss of dystrophin, membrane fragility leads to sarcolemmal tearing and calcium influx, triggering calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol. 10:168-75 (1997)). This uncontrolled cycle of muscle degeneration and regeneration ultimately depletes the muscle stem cell population (Sacco et al., Cell 143:1059-1071 (2010); Wallace et al., Annu Rev Physiol 71:37-57 (2009)), resulting in progressive muscle weakness, endomysial inflammation, and fibrotic scar formation.

[0006] Without the membrane-stabilizing effects of dystrophin or microdystrophin, DMD presents as an uncontrolled cycle of tissue damage and repair, ultimately replacing lost muscle fibers with fibrotic scar tissue through connective tissue hyperplasia. Fibrosis is characterized by the excessive deposition of ECM matrix proteins, including collagen and elastin. ECM proteins are primarily produced by cytokines such as TGFβ, which are released by activated fibroblasts in response to stress and inflammation. Although the primary pathological feature of DMD is muscle fiber degeneration and necrosis, fibrosis has an equal impact as a pathological consequence. In DMD patients, the overproduction of fibrotic tissue limits muscle regeneration and leads to progressive muscle weakness. 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 68:762-767 (2009)). These results point to fibrosis as a major contributing factor to DMD muscle dysfunction and highlight the need for early intervention before significant fibrosis occurs.

[0007] International Publication No. WO 2019 / 245973 A1 (incorporated herein by reference in its entirety) describes the delivery of a microdystrophin gene by an AAV vector to treat muscular dystrophy (e.g., DMD) in human subjects. International Application No. PCT / US2022 / 029328, filed May 13, 2022 (incorporated herein by reference in its entirety), describes the production of recombinant AAV vectors for treating muscular dystrophy (e.g., DMD) in human subjects. However, there remains a continuing need in the art for improved methods of treating DMD with AAV vectors, e.g., by genotyping a patient's human dystrophin (DMD) gene prior to treatment to identify patient populations that may benefit from or be contraindicated for AAV gene therapy as described herein. Summary of the Invention

[0008] The present disclosure relates to gene therapy vectors, such as AAV vectors, produced by the suspension seed process described herein, which express the human microdystrophin gene in skeletal muscle (including the diaphragm and heart muscle) to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis. The present disclosure also relates to compositions and methods for treating muscular dystrophy (e.g., Duchenne muscular dystrophy) using these AAV vectors.

[0009] The present disclosure provides a method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.microdystrophin in adherent mammalian cells by a suspension seed process, comprising: (a) culturing the cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing a lower concentration of serum than the first medium; (d) culturing the cells in suspension in the N-1 container; and (e) inoculating the cells from step (d) into a third medium in a bioreactor.

[0010] In some aspects, the rAAV used in the methods described herein comprises the human microdystrophin nucleotide sequence of SEQ ID NO:1. In some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO:7. In some aspects, the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:7.

[0011] In some aspects, the suspension seeding method further comprises: (f) transfecting the adherent cells with a transgenic plasmid comprising the rAAVrh74.MHCK7.minidystrophin construct, a plasmid comprising the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

[0012] In some aspects, the transgenic plasmid comprising the rAAVrh74.MHCK7.minidystrophin construct comprises: the nucleic acid sequence of SEQ ID NO:9; nucleotides 55 - 5021 of SEQ ID NO:3; or nucleotides 1 - 4977 of SEQ ID NO:8. In some aspects, the plasmid comprising the AAV rep gene and the AAV cap gene comprises the AAV2 rep gene and the rAAVrh74 cap gene. In some aspects, the adenovirus helper plasmid comprises the adenovirus 5E2A, E4ORF6, and VA RNA genes.

[0013] In some aspects, the suspension seeding method further comprises: (g) lysing the adherent cells. In some aspects, the adherent cells are lysed by freeze - thaw, solid shearing, hypertonic and / or hypotonic lysis, liquid shearing, sonication, high - pressure extrusion, detergent lysis, or a combination thereof.

[0014] In some aspects, the suspension seeding method further comprises: (h) purifying the rAAV by at least one column chromatography step. In some aspects, the at least one column chromatography step comprises anion - exchange chromatography, size - exclusion chromatography, or a combination thereof.

[0015] In some aspects, the suspension seeding method further comprises culturing the cells in the first growth medium in an N - 3 container. In some aspects, the suspension seeding method further comprises culturing the cells in the first growth medium in an N - 4 container.

[0016] In some aspects, the bioreactor is an adherent bioreactor. In some aspects, the rAAV is purified from the culture produced in the adherent bioreactor.

[0017] In some aspects, the third medium in the bioreactor comprises at least one factor promoting cell adhesion. In some aspects, the at least one factor promoting cell adhesion is selected from serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non - proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some aspects, the third medium in the bioreactor comprises DMEM and 10% FBS.

[0018] In some aspects, the adherent cells are cultured under suspension conditions for about 48 - 72 hours.

[0019] In some aspects, the N-1 container is a suspension shake flask.

[0020] In some aspects, the adherent cells are selected from HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells. In some aspects, the adherent cells are HeLa cells or HEK-293 cells. In some aspects, the adherent cells are HEK-293 cells. In some aspects, the adherent cells are not suspension-adapted. In some aspects, culturing the cells under suspension conditions does not change the adherent-dependence of the cells. In some aspects, the culturing does not change the cells to produce a new cell line.

[0021] The present disclosure also provides a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, wherein the rAAV is produced by any of the methods described herein. In some aspects, the composition comprises: a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8.

[0022] In some aspects, the present disclosure provides a composition for treating muscular dystrophy in a human subject in need thereof, comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured under suspension conditions in an N-1 container. In some aspects, the rAAV comprises the human minidystrophin nucleotide sequence of SEQ ID NO:1. In some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO:7. In some aspects, the rAAV comprises the MHCK7 promoter sequence of SEQ ID NO:7 and the human minidystrophin nucleotide sequence of SEQ ID NO:1.

[0023] In some aspects, the composition comprises: (a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; (b) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; (c) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; (d) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; (e) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8; and / or (f) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8.

[0024] The present disclosure also provides a method for treating muscular dystrophy in a human subject in need thereof, which comprises administering to the human subject a composition comprising the rAAV described herein.

[0025] In some aspects, the systemic administration route is used and the rAAV is administered at a dose of about 5.0×10 12 vg / kg to about 1.0×10 15 vg / kg. In some aspects, the systemic administration route is the intravenous route, and the administration dose of the rAAV is about 2×10 14 vg / kg. In some aspects, the systemic administration route is the intravenous route, and the administration dose of the rAAV is about 1.33×10 14 vg / kg

[0026] In some aspects, the rAAV of the dose is administered at a concentration of about 10 mL / kg. In some aspects, the rAAV is administered by injection, infusion or implantation. In some aspects, the rAAV is administered by infusion within about one hour. In some aspects, the rAAV is administered via a peripheral limb vein by the intravenous route.

[0027] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0028] In some aspects, compared with the level of micro-dystrophin gene expression before administering the rAAV, the level of micro-dystrophin gene expression in the cells of the subject increases after administering the rAAV. In some aspects, the expression of the micro-dystrophin gene in the cells is determined by measuring the level of micro-dystrophin by Western blot of biopsy muscle before and after administering the rAAV. In some aspects, the expression after administering the rAAV is at least 55.4% compared with that before administration.

[0029] In some aspects, compared with the number of micro-dystrophin positive fibers before administering the rAAV, the average percentage of micro-dystrophin positive fibers in the muscle tissue of the subject increases after administering the rAAV. In some aspects, the average percentage of micro-dystrophin positive fibers is at least 70.5%, and the average intensity is at least 116.9%, which are detected by immunofluorescence (IF) in muscle biopsies before and after administering the rAAV. In some aspects, the micro-dystrophin transduction by vector genome counting is at least 3.87 average vector genome copies per cell nucleus.

[0030] In some aspects, the compositions as described herein are administered to a subject who has been genotyped for at least one mutation in their human dystrophin (DMD) gene. In some aspects, at least one of the mutations in the DMD gene is a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the absence of expression of the human dystrophin. In some aspects, at least one mutation in exons 18 - 79 of the subject's DMD gene has been genotyped.

[0031] In some aspects, at least one mutation in exons 9 - 13 of the subject's DMD gene has been genotyped. In some aspects, at least one of the mutations in exons 9 - 13 is a deletion. In some aspects, the deletion completely encompasses exons 9 - 13 of the DMD gene.

[0032] In some aspects, at least one mutation in exon 8 and / or exon 9 of the subject's DMD gene has been genotyped. In some aspects, at least one of the mutations in exon 8 and / or exon 9 of the DMD gene is a deletion. In some aspects, the deletion is in exon 8 of the DMD gene. In some aspects, the deletion is in exon 9 of the DMD gene. In some aspects, the deletion is in both exon 8 and exon 9 of the DMD gene.

[0033] In some aspects, the method of treating muscular dystrophy further comprises genotyping the DMD gene of the human subject before administering the composition to the human subject.

[0034] In some aspects, genotyping detects at least one mutation in exons 18 to 79 of the DMD gene. In some aspects, at least one of the mutations is a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the absence of expression of the human dystrophin.

[0035] In some aspects, the genotyping detects at least one mutation in exons 9 - 13 of the DMD gene. In some aspects, at least one of the mutations in exons 9 - 13 is a deletion. In some aspects, the deletion completely encompasses exons 9 - 13 of the DMD gene.

[0036] In some aspects, the genotyping detects at least one mutation in exon 8 and / or exon 9 of the DMD gene. In some aspects, at least one of the mutations in exon 8 and / or exon 9 is a deletion. In some aspects, the deletion is in exon 8 of the DMD gene. In some aspects, the deletion is in exon 9 of the DMD gene. In some aspects, the deletion is in both exon 8 and exon 9 of the DMD gene.

[0037] The present disclosure also provides the use of the compositions described herein for treating muscular dystrophy in human subjects in need thereof. In some aspects, the present disclosure also provides the use of the compositions described herein in the preparation of a medicament for treating muscular dystrophy.

[0038] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0039] The present disclosure also provides a method for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles, the AAV viral particles encapsulating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion of exons 9-13 of the DMD gene in its entirety.

[0040] The present disclosure also provides a method for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles, the AAV viral particles encapsulating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion in exon 8 and / or exon 9 of the human dystrophin (DMD) gene.

[0041] In some aspects, the DMD gene of the subject is genotyped prior to treatment.

[0042] In some aspects, the AAV viral particles are rh74 serotype viral particles.

[0043] In some aspects, the rAAV vector is administered as a composition comprising: a) rh74 serotype AAV viral particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rh74 serotype AAV viral particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rh74 serotype AAV viral particles encapsulating nucleotides 1-4977 of SEQ ID NO:8.

[0044] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) administering to the human subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, provided that genotyping does not identify a deletion that completely encompasses exons 9-13 of the DMD gene; wherein the composition comprises: a) rAAV particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsulating nucleotides 1-4977 of SEQ ID NO:8.

[0045] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) administering to the subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, provided that genotyping does not identify a deletion in exon 8 and / or 9 of the DMD gene; wherein the composition comprises: a) rAAV particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsulating nucleotides 1-4977 of SEQ ID NO:8.

[0046] In some aspects, the human subject treated according to any of the methods described herein is ambulant.

[0047] In some aspects, the human subject treated according to any of the methods described herein is non-ambulant.

[0048] In some aspects, the human subject treated according to any of the methods described herein is 2 to 3 years old. In some aspects, the human subject treated according to any of the methods described herein is 4 to 5 years old.

[0049] In some aspects, the human subject treated according to any of the methods described herein has been non-ambulant for at least 9 months.

[0050] In some aspects, the human subject treated according to any of the methods described herein has a stable forced vital capacity (FVC) of less than 40% of predicted value and / or requires nocturnal ventilator support.

[0051] In some aspects, the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin used in the treatment methods described herein is made according to any of the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Shows the rAAV.MHCK7.minidystrophin construct. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). The construct is characterized by an in-frame rod deletion (R4-R23), while hinges 1, 2, and 4 (H1, H2, and H4) and the cysteine-rich domain are retained, resulting in a 138 kDa protein. The expression of minidystrophin (3579 bp) is directed by the MHCK7 promoter (795 bp). The intron and 5'UTR are derived from plasmid pCMVβ (Clontech). The minidystrophin cassette has a consensus Kozak immediately preceding the ATG start, and a small 53 bp synthetic polyA signal for mRNA termination. As previously described by Harper et al. (Nature Medicine 8:253-261 (2002)), the human minidystrophin cassette contains the (R4-R23 / Δ71-78) domain.

[0053] Figure 2 Provides the nucleic acid sequence of AAVrh74.MHCK7.minidystrophin (SEQ ID NO:3).

[0054] Figure 3 Provides the plasmid map of the pNLREP2-Caprh74 AAV helper plasmid.

[0055] Figure 4 Provides the Ad helper plasmid pHELP.

[0056] Figure 5 Shows the rAAV.MCK.minidystrophin plasmid construct.

[0057] Figure 6 Provides the nucleic acid sequence of rAAVrh74.MCK.minidystrophin (SEQ ID NO:5).

[0058] Figure 7 Shows minidystrophin gene expression in muscle fibers of gastrocnemius muscle biopsies measured by immunocytochemistry.

[0059] Figures 8A - 8C Provides a Western blot showing minidystrophin expression at the corrected molecular weight. In Figure 8A and Figure 8BIn this study, Western blot analysis detected micro-dystrophin expression in Subject 1 (5 years old), Subject 2 (4 years old), and Subject 3 (6 years old). In Figure 8C In this study, due to exceeding the ULDQ (>80%) in the initial analysis, the Subject 4 sample (*) was diluted 1:4 (to the linear range), and the average value was multiplied by the dilution correction factor to obtain the final value relative to the normal value. Compared with the normal value, the average micro-dystrophin expression was 182.7% in Method 1 and 222.0% in Method 2.

[0060] Figures 9A - 9C Shown is the expression of the DAPC proteins α-sarcoglycan and β-sarcoglycan in Subject 1 ( Figure 9A ), Subject 2 ( Figure 9B ), and Subject 3 ( Figure 9C ) following administration of rAAVrh74.MHCK7.micro-dystrophin.

[0061] Figure 10 Provided is a graph showing a continuous and significant decrease in creatine kinase (CK) values following administration of rAAVrh74.MHCK7.micro-dystrophin.

[0062] Figure 11 Provided is a graph showing the change in mean creatine kinase (CK) from baseline to day 270. This data indicates that CK decreased significantly over time following administration of rAAVrh74.MHCK7.micro-dystrophin.

[0063] Figure 12 Provided is a graph showing the change in mean NSAA and mean CK from baseline to day 270. This data indicates that NSAA increased significantly over time following administration of rAAVrh74.MHCK7.micro-dystrophin.

[0064] Figure 13 Provided is the 4977-base nucleic acid sequence (SEQ ID NO:9) of the AAVrh74.MHCK7.micro-dystrophin construct. The following molecular elements are depicted: 5' ITR (bases 1 - 145); MHCK7 promoter (190 - 981 (792 bases)); intron (991 - 1140 (150 bases)); human micro-dystrophin sequence (1151 - 4729 (3579 bases)); Poly A tail (4732 - 4784 (53 bases)); and 3' ITR (4833 - 4977 (145 bases)).

[0065] Figure 14 Shown is the AAVrh74.MHCK7.micro-dystrophin plasmid construct.

[0066] Figure 15Provide the nucleic acid sequence (SEQ ID NO. 8) of the AAVrh74.MHCK7. micro - dystrophin plasmid construct, which contains a kanamycin resistance gene.

[0067] Figure 16 Is an intuitive representation of the hybrid seeding chain - amplification method described herein.

[0068] Figure 17 Is an intuitive representation of generating AAV particles using the hybrid seeding chain - amplification method described herein.

[0069] Figures 18A - 18B Provide a graph showing the viability of HEK - 293 cells cultured according to the hybrid seeding chain - amplification method described herein ( Figure 18A ) and only under adherent conditions ( Figure 18B ).

[0070] Figures 19A - 19B Provide a graph showing the viable cell density of HEK - 293 cells cultured according to the hybrid seeding chain - amplification method described herein ( Figure 19A ) and only under adherent conditions ( Figure 19B ).

[0071] Figure 20 Is a graph showing the average NSAA score of Group 1 (the first 11 patients treated with rAAVrh74.MHCK7. micro - dystrophin) described in Example 7. The first 11 patients had a 3 - point improvement from baseline. Patients aged 6 to 7 years (n = 9) had a 2.9 - point improvement from baseline. Each time point represents the 11 patients.

[0072] Figures 21A - 21C Show, compared to saline ( Figure 21A ), the expression of micro - dystrophin (immunofluorescence) in the skeletal muscle and cardiac muscle of DMD Figure 21B rats at 12 weeks ( Figure 21C ) and 24 weeks ( mdx ) after treatment with delandistrogene moxeparvovec, as described in Example 11. Keywords: LTA = left tibialis anterior muscle; HRT = cardiac muscle.

[0073] Figures 22A - 22B The bar graph of mdx shows the expression of micro - dystrophin (immunofluorescence) ( Figure 22A ) and vector transduction (vector genome copy number) ( Figure 22B ) in the muscle tissue of DMD mdx rats at 12 weeks and 24 weeks after treatment with delandistrogene moxeparvovec.Quantification as described in Example 11. Keywords: TA = tibialis anterior muscle; HRT = heart muscle; MG = medial gastrocnemius muscle; LG = lateral gastrocnemius muscle; DIA = diaphragm muscle; TRI = triceps muscle; PSO = psoas major muscle.

[0074] Figures 23A - 23B The bar graph shows that compared with saline, at 12 weeks and 24 weeks after treatment with delandistrogene moxeparvovec, mdx the locomotion ( Figure 23A ) and vertical activity ( Figure 23B ) of DMD rats increased as described in Example 11. The movement (locomotion and vertical activity) of rats in the activity cage was measured by the number of laser beam interruptions per hour. Each point represents the value of a single animal. Data are reported as mean ± SD; *** = p < 0.001; ** = p < 0.01. SD = standard deviation.

[0075] Figures 24A - 24B The figure depicts that compared with saline, at 12 weeks and 24 weeks after delandistrogene moxeparvovec gene transfer in DMD mdx rats, muscle degeneration was significantly reduced by central nucleation analysis in skeletal muscle as described in Example 11. Figure 24A The figure depicts hematoxylin and eosin (H&E) staining of the gastrocnemius muscle. Figure 24B It is a bar graph depicting the percentage of fibers with central nuclei. Bars are reported as mean ± SD; **** = p < 0.0001. SD = standard deviation.

[0076] Figures 25A - 25B The figure depicts the analysis of collagen deposition in skeletal muscle and heart muscle, which shows that compared with saline, at 12 weeks and 24 weeks after treatment with delandistrogene moxeparvovec in DMD mdx rats, fibrosis was reduced as described in Example 11. Figure 25A The figure depicts Masson's trichrome staining at 12 weeks after treatment. Figure 25B It provides a bar graph quantifying collagen deposition in skeletal muscle and heart muscle at 12 weeks and 24 weeks after treatment. Keywords: HRT = heart muscle; MG = medial gastrocnemius muscle; DIA = diaphragm muscle. Data are reported as mean ± SD; **** = p < 0.0001; * = p < 0.05. SD = standard deviation.

[0077] Figure 26 The bar graph shows that compared with saline, in DMD mdxAt 1 week and 12 weeks after treatment of rats with delandistrogenemoxeparvovec, the serum troponin I levels in the blood did not change significantly, as described in Example 11. Bars represent mean ± SD. Each point represents the value of an individual animal.

[0078] Figures 27A - 27C Bar graph analysis of DMD at 24 weeks after treatment with delandistrogene moxeparvovec compared to saline mdx Cardiac function measured by echocardiography in DMD rats, as described in Example 11. Figure 27A Data depicting left ventricular end-systolic diameter (LVESD). Figure 27B Data depicting ejection fraction (%) (EF). Figure 27C Data depicting fractional shortening (%) (FS).

[0079] Figure 28 Histological images of show that compared to control DMD rats treated with saline, at 12 weeks, 24 weeks, and 52 weeks after treatment with delandistrogene moxeparvovec, MDX In DMD rats, the sarcolemmal localization of micro-dystrophin, as described in Example 11. MDX In DMD rats, the sarcolemmal localization of micro-dystrophin, as described in Example 11.

[0080] Figure 29 Bar graph shows that at 12 weeks, 24 weeks, and 52 weeks after treatment, compared to control DMD rats treated with saline, MDX In DMD rats treated with delandistrogene moxeparvovec, MDX The percentage of micro-dystrophin positive fibers (PDPF), as described in Example 11.

[0081] Figure 30 Bar graph shows the transgene distribution in the skeletal and cardiac muscle and liver of DMD rats treated with delandistrogene moxeparvovec at 12, 24, and 52 weeks after treatment, as described in Example 11. MDX In DMD rats treated with delandistrogene moxeparvovec, the transgene distribution in the skeletal and cardiac muscle and liver, as described in Example 11.

[0082] Figures 31A - 31B : Figure 31A Western blot shows micro-dystrophin expression in the heart (HRT), triceps (TRI), tibialis anterior (TA), and gastrocnemius (GAS) of DMD rats at 12, 24, and 52 weeks after treatment with a single dose of delandistrogenemoxeparvovec, as described in Example 11. MDX In DMD rats, micro-dystrophin expression in the heart (HRT), triceps (TRI), tibialis anterior (TA), and gastrocnemius (GAS), as described in Example 11.Figure 31B The bar graph shows DMD at 12, 24, and 52 weeks after treatment with a single dose of delandistrogene moxeparvovec MDX Quantification of microdystrophin expression in the gastrocnemius, tibialis anterior, and triceps skeletal muscles (left) and cardiac muscle (right) of DMD rats as described in Example 11.

[0083] Figure 32 The figure shows compared to control DMD treated with saline MDX rats, DMD rats treated with delandistrogenemoxeparvovec MDX Survival probability of rats, as described in Example 11.

[0084] Figure 33 The H&E histological images shown at 12, 24, and 52 weeks after treatment show that compared to control DMD treated with saline MDX rats, the gastrocnemius of DMD rats treated with delandistrogene moxeparvovec MDX as described in Example 11.

[0085] Figure 34 The bar graph shows at 12, 24, and 52 weeks after treatment that compared to control DMD treated with saline MDX rats, the percentage of CN (central nucleated) positive fibers in DMD rats treated with delandistrogene moxeparvovec MDX as described in Example 11.

[0086] Figure 35 The histological images show at 12, 24, and 52 weeks after treatment that compared to control DMD treated with saline MDX rats, the collagen staining of skeletal and cardiac muscles of DMD rats treated with delandistrogene moxeparvovec MDX as described in Example 11.

[0087] Figure 36 The bar graph shows at 12, 24, and 52 weeks after treatment that compared to control DMD treated with saline MDX rats, the fibrosis percentage of skeletal and cardiac muscles of DMD rats treated with delandistrogene moxeparvovec MDX as described in Example 11.

[0088] Figure 37The bar graph shows that at 52 weeks after treatment, compared to control DMD rats treated with saline, the cardiac performance of DMD rats treated with delandistrogene moxeparvovec was improved, as described in Example 11. MDX rats, the cardiac performance of DMD rats treated with delandistrogene moxeparvovec was improved, as described in Example 11. MDX rats was improved, as described in Example 11.

[0089] Figure 38 The graph shows that at 52 weeks after treatment, compared to control DMD rats treated with saline, MDX rats, the restored cardiomyocyte contractility and Ca MDX kinetics in DMD rats treated with delandistrogene moxeparvovec were as described in Example 11. 2+ kinetics in DMD rats treated with delandistrogene moxeparvovec were as described in Example 11.

[0090] Figure 39 The bar graph shows that at 12, 24, and 52 weeks after treatment, compared to control DMD rats treated with saline, MDX rats, the horizontal locomotor activity of DMD rats treated with delandistrogene moxeparvovec was as described in Example 11. MDX rats was as described in Example 11.

[0091] Figure 40 The bar graph shows that at 12 weeks and 52 weeks after treatment, compared to control DMD rats treated with saline, MDX rats, the serum troponin I levels of DMD rats treated with delandistrogene moxeparvovec were as described in Example 11. MDX rats were as described in Example 11.

[0092] Figures 41A - 41B The Western blot shows the expression of minidystrophin and α-actinin. Figure 41A The Western blot shown depicts the expression of minidystrophin and α-actinin in the tibialis anterior muscle, myocardium, and diaphragm of non-human primates after a single dose of delandistrogene moxeparvovec, as described in Example 11. Figure 41B The Western blot shows the expression of minidystrophin and α-actinin in the myocardium, diaphragm, and tibialis anterior muscle of non-human primates after plasmapheresis and readministration with delandistrogene moxeparvovec, as described in Example 11.

[0093] Figures 42A - 42B : Figure 42ATo show phase contrast microscopic images of in vitro human cardiomyocytes from day 1 to day 5 (when transduced with the AAVrh74-MHCK7-GFP vector) and after day 9 (fixation day), as described in Example 11. Figure 42B To show immunofluorescence images of in vitro human cardiomyocytes (day 9) transduced with the AAVrh74-MHCK7-GFP vector (4 days after transduction), as described in Example 11. Detailed Description

[0094] The present disclosure provides gene therapy vectors that express human micro-dystrophin, such as rAAV, wherein the rAAV is produced in mammalian adherent cells, and wherein the adherent cells are cultured under suspension conditions in an N-1 container. The present disclosure also provides compositions (e.g., pharmaceutical compositions) comprising the rAAV described herein, and methods of using the compositions described herein to treat muscular dystrophy (e.g., DMD). The present disclosure also provides genotyping of a subject's DMD gene prior to administration of rAAV to identify mutations in the DMD gene that are suitable for AAV gene therapy and mutations that may contraindicate AAV gene therapy (e.g., due to an increased risk of severe immune responses). The present disclosure also provides successful improvement of skeletal and cardiac muscle function in a DMD MDX rat model after treatment with the rAAV described herein.

[0095] Muscle biopsies performed at the earliest age at which DMD is diagnosed show marked connective tissue hyperplasia. Muscle fibrosis is detrimental in several respects. It reduces the normal delivery of endomysial nutrients across the connective tissue barrier, reduces blood flow and deprives the muscle of vascular-derived nutrients, and functionally causes early loss of ambulation via limb contractures. Over time, the treatment challenge multiplies due to marked fibrosis in the muscle. This can be observed in muscle biopsies comparing connective tissue hyperplasia at consecutive time points. This process continues to deteriorate, leading to loss of ambulation and accelerating out of control, especially in wheelchair-dependent patients.

[0096] In the absence of a parallel method for reducing fibrosis in early treatment, it is unlikely that the benefits of exon skipping, nonsense codon readthrough, or gene replacement therapy will be fully realized all the time. Even small molecule or protein replacement strategies may fail without a method for reducing muscle fibrosis. Previous studies in aged mdx mice with fibrosis treated with AAV.micro-dystrophin have shown that full functional recovery cannot be achieved (Liu, M. et al., Mol Ther 11:245-256 (2005)). It is also known that the progression of DMD cardiomyopathy is accompanied by scar formation and fibrosis of the ventricular wall.

[0097] Definition

[0098] 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 disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless the context requires otherwise, singular terms shall include plural and plural terms shall include singular.

[0099] In this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "polynucleotide" is to be understood as representing one or more polynucleotides. Thus, the terms "a / an", "one or more", and "at least one" may be used interchangeably herein.

[0100] In addition, "and / or" as used herein shall be taken as specifically disclosing each of the two specified features or components, whether or not accompanied by the other. Thus, the term "and / or" when used in a phrase such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" when used in a phrase such as "A, B, and / or C" is intended to embrace each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0101] The term "about" as used herein means approximately, roughly, or in the region of. When the term "about" is used in connection with a numerical range, it modifies that range by extending the upper and lower limits of the recited values. Generally, unless otherwise stated, the term "about" is used herein to modify a numerical value above and below the stated value by a deviation of up to 10% (higher or lower).

[0102] The term "at least" before a number or series of numbers shall be understood to include the number adjacent to the term "at least", as well as all subsequent numbers or integers that can logically be included, as indicated by the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in that series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18%, regardless of the number of significant digits).

[0103] Unless otherwise expressly indicated, nucleotide sequences are presented herein only as single strands in the 5′ to 3′ direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the single-letter code or the three-letter code, both in accordance with 37 CFR §1.822 and established usage.

[0104] As used herein, "polynucleotide" or "nucleic acid" means a nucleotide sequence joined by phosphodiester bonds. Polynucleotides are presented herein in the 5' to 3' direction. The polynucleotides of the present disclosure can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are represented herein by the single-letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0105] Unless otherwise indicated, as used herein, the term "polypeptide" encompasses both peptides and proteins.

[0106] The term "coding sequence" or "sequence encoding..." is used herein to mean a DNA or RNA region (transcribed region) that "encodes" a specific protein (such as insulin or glucokinase). When placed under the control of appropriate regulatory regions, such as a promoter, the coding sequence is transcribed (DNA) and translated (RNA) in vitro or in vivo into a polypeptide. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxyl) terminus. The coding sequence can include, but is not limited to: cDNA from prokaryotes or eukaryotes, genomic DNA from prokaryotes or eukaryotes, and synthetic DNA sequences. A transcription termination sequence can be located 3' of the coding sequence.

[0107] A gene can contain several operably linked segments, such as a promoter, a 5' leader sequence, introns, a coding sequence, and a 3′-untranslated sequence, e.g., containing a polyadenylation site or a signal sequence. As used herein, "expression of a gene" refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0108] As used herein, the term "promoter" refers to a nucleic acid sequence or fragment that controls the transcription of one or more genes (or coding sequences), which is located upstream of the transcriptional start site of the gene in the transcriptional direction, and is structurally identified by the presence of: a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled in the art to directly or indirectly regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated according to physiological or developmental conditions. A "tissue-specific" promoter is preferentially active in a specific type of differentiated cell / tissue.

[0109] As used herein, the term "enhancer" refers to a cis-acting element that stimulates or inhibits the transcription of an adjacent gene. An enhancer that inhibits transcription is also referred to as a "silencer". Enhancers can act in either direction (e.g., can be associated with the coding sequence), and can be located up to several thousand base pairs (kb) downstream of the coding sequence and the transcription region.

[0110] The term "operably linked" refers to positioning a regulatory element nucleotide sequence, such as a promoter nucleotide sequence, to cause the expression of the nucleotide sequence by the regulatory element.

[0111] As used herein, the term "transgene" refers to a gene (e.g., mini-dystrophin) or nucleic acid molecule introduced into a cell. An example of a transgene is a nucleic acid encoding a therapeutic polypeptide. In some aspects, the gene may be present, but in some cases, the gene is not normally expressed or is expressed at insufficient levels in the cell. In this context, "insufficient" means that although the gene is normally expressed in the cell, a condition and / or disease may still occur. In certain aspects, the transgene allows for increased or overexpression of the gene. The transgene can contain sequences native to the cell, sequences not naturally present in the cell, or it can contain a combination of both. In certain aspects, the transgene can contain sequences that can be operably linked to appropriate regulatory sequences for gene expression. In some aspects, the transgene is not integrated into the genome of the host cell.

[0112] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which some of its functions are provided by a co-infecting helper virus. There are currently 13 characterized AAV serotypes. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Volume 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully anticipated that these same principles will apply to other AAV serotypes, since it is well known that the various serotypes are very closely related structurally and functionally, even at the genetic level (see, for example, Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165-174, edited by J.R. Pattison; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further demonstrated by heteroduplex analysis revealing extensive cross-hybridization along the genome length between serotypes and the presence of similar self-annealing fragments corresponding to "inverted terminal repeats" (ITRs) at the termini. Similar infectious patterns also indicate that the replication functions of each serotype are under similar regulatory control.

[0113] As used herein, the term "adeno-associated vector" or "AAV vector" refers to a vector that contains a target polynucleotide (or "transgene", such as mini-dystrophin) flanked by one or more AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious virus particles when present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products.

[0114] As used herein, the term "AAV virion", "AAV virus particle" or "AAV vector particle" refers to a virus particle composed of at least one AAV capsid protein and an encapsulated polynucleotide AAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is usually referred to as an "AAV vector particle", or in some cases, simply as an "AAV vector". Thus, the production of AAV vector particles necessarily includes the production of AAV vectors. Thus, the vector is contained within the AAV vector particle.

[0115] As used herein, the term "expression cassette" refers to any type of genetic construct containing nucleic acids, wherein part or all of the nucleic acid coding sequences are capable of being transcribed. Typically, an expression cassette contains a promoter operably linked to a nucleic acid (e.g., a target transgene). In some aspects, an "expression cassette" includes a polynucleotide sequence encoding human mini - dystrophin.

[0116] The term "muscle - specific control element" refers to nucleotide sequences that regulate the expression of coding sequences that are specific for expression in muscle tissue. These control elements include enhancers and promoters. The present disclosure provides constructs containing the muscle - specific control elements MCKH7 promoter, MCK promoter, and MCK enhancer.

[0117] "Muscle cell" or "muscle tissue" means a cell or population of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, which are, for example, 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 cardiac myoblasts.

[0118] As used herein, the term "transduction" refers to the administration / delivery of the coding region of mini - dystrophin to recipient cells in vivo or in vitro via the replication - defective rAAV of the present disclosure, such that the recipient cells express mini - dystrophin.

[0119] As used herein, the "transfection" of a cell means the introduction of genetic material into a cell for the purpose of genetically modifying the cell. Transfection can be accomplished by a variety of methods known in the art (e.g., transduction or electroporation).

[0120] As used herein, a "vector" refers to a recombinant plasmid or virus containing a polynucleotide that will be delivered to a host cell in vitro or in vivo. "Recombinant" means different from that normally found in nature.

[0121] "Serotype", with respect to a vector or viral capsid, is defined by different immunological characteristics based on capsid protein sequences and capsid structure.

[0122] "AAV Cap" means AAV Cap proteins, VP1, VP2, and VP3 and their analogs.

[0123] "AAV Rep" means AAV Rep proteins and their analogs.

[0124] As used herein, "flanked," with respect to a sequence flanked by other elements, means that there is one or more flanking elements relative to the upstream and / or downstream of the sequence, i.e., 5' and / or 3'. The term "flanked" is not intended to mean that the sequences must be contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and the flanking elements. A sequence (e.g., a transgene) flanked by two other elements (e.g., ITRs) means that one element is located at the 5' of the sequence and the other element is located at the 3' of the sequence; however, there may be intervening sequences therebetween.

[0125] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a transgene (e.g., micro-dystrophin)) into the cells and / or tissues of an individual to treat a disease or condition. Such a transgene can be exogenous. An exogenous molecule or sequence should be understood as a molecule or sequence that is not normally present in the cells, tissues, and / or individual to be treated.

[0126] As used herein, the term "genotyping" refers to the process of determining the specific allelic composition of a cell and / or subject at one or more positions within the genome, for example, by determining the nucleic acid sequence at those positions. Genotyping refers to nucleic acid analysis and / or analysis at the nucleic acid level. Many genotyping techniques are known to those of skill in the art.

[0127] In some aspects, the human dystrophin gene (DMD) of a subject is genotyped to characterize mutations in the gene that are particularly suitable for treatment with the compositions described herein.

[0128] In some aspects, the DMD gene of a subject is genotyped to characterize mutations in the DMD gene that are contraindicated for treatment with the compositions described herein.

[0129] The term "stringent" is used to refer to conditions that are generally understood in the art to be stringent. Hybridization stringency is mainly 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 edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). More stringent conditions can also be used (e.g., higher temperature, lower ionic strength, higher formamide or other denaturing agents), however, the hybridization rate will be affected. In the case of hybridization involving deoxyoligonucleotides, other exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37 °C (for 14-base oligonucleotides), 48 °C (for 17-base oligonucleotides), 55 °C (for 20-base oligonucleotides), and 60 °C (for 23-base oligonucleotides). For the purpose of reducing non-specific and / or background hybridization, other reagents can be included in the hybridization and wash buffers. Examples are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate NaDodSO4 (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, but other suitable reagents can also be used. The concentration and type of these additives can also be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8 - 7.4, however, under typical ionic strength conditions, the hybridization rate is almost independent of the pH value. See Anderson, M.L.M. et al., Nucleic Acid Hybridisation: A Practical Approach , Chapter 4, IRL Press Limited (Oxford, England) (1998). The hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and allow DNA of different sequence relatedness to form hybrids.

[0130] As used herein, the terms "media / medium / culture medium", "cell culture medium", "tissue culture medium / tissue culture media", and "growth medium" refer to a solution containing nutrients that nourish cultured eukaryotic cells that are growing. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for the minimal growth and / or survival of the cells. The solution may also contain components that enhance growth and / or survival above the minimal rate, including hormones and growth factors. The solution is formulated to an optimal pH and salt concentration for cell survival and proliferation. The culture medium may also be a "defined medium" or "chemically defined medium" - a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. A defined medium does not contain components of animal origin, and all components have a known chemical structure. Those skilled in the art will understand that a defined medium may contain recombinant glycoproteins or proteins, such as, but not limited to, hormones, cytokines, interleukins, and other signaling molecules.

[0131] As used herein, the term "basal medium preparation" or "basal medium" refers to any cell culture medium used for culturing cells that has not been altered by supplementation or by selective removal of a component.

[0132] As used herein, the terms "culture", "cell culture", and "eukaryotic cell culture" refer to a population of eukaryotic cells that are surface-attached (i.e., adherent) or suspended and that are maintained or grown in a culture medium under conditions suitable for the survival and / or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms as used herein can refer to a combination comprising a mammalian cell population and the culture medium in which the population is suspended.

[0133] As used herein, the term "batch culture" refers to a method of culturing cells in which all of the components ultimately used to culture the cells (including the culture medium as well as the cells themselves) are provided at the beginning of the culture process. The batch culture is typically stopped at some point in time, and the cells and / or components in the culture medium are collected and optionally purified.

[0134] As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are provided to the culture at some time after the start of the culturing process. A fed-batch culture can be started with a basal medium. A medium that provides additional components to the culture at some time after the start of the culturing process is a feeding medium. The components provided typically include nutrient supplements for the cells that are depleted during the culturing process. The fed-batch culture is typically stopped at some point in time, and the cells and / or components in the medium are collected and optionally purified.

[0135] As used herein, the term "perfusion culture" refers to a method of culturing cells in which additional components are provided to the culture continuously or semi-continuously after the start of the culturing process. The components provided typically include nutrient supplements for the cells that are depleted during the culturing process. A portion of the cells and / or components in the medium are typically collected on a continuous or semi-continuous basis and optionally purified.

[0136] The "growth phase" of a cell culture refers to the period of exponential cell growth (log phase) during which cells typically divide rapidly. During this period, the cultured cells are incubated for a period of time, typically between 1 and 4 days, and under conditions that maximize cell growth. Determination of the host cell growth cycle can be determined for a particular host cell of interest without undue experimentation. "For a period of time and under conditions that maximize cell growth" and the like refer to those culture conditions that are determined to be most suitable for cell growth and division for a particular cell line. In some aspects, during the growth phase, the cells are cultured in a nutrient medium containing essential additives at a temperature typically of about 25°C - 40°C in a humidified, controlled atmosphere such that optimal growth of the particular cell line is achieved.

[0137] In some aspects, the cells are maintained in the growth phase for a period of about 1 to 7 days, such as 2 to 6 days, such as 6 days. The length of the growth phase for a particular cell can be determined without undue experimentation. For example, if a culture is maintained under growth conditions, the length of the growth phase will be the period of time sufficient to allow the particular cells to proliferate to a viable cell density within the range of about 20% - 80% of the maximum possible viable cell density. In some aspects, the "maximum growth rate" refers to the growth rate of a particular cell line / clone measured during its exponential growth phase when the cells are in fresh medium (e.g., measured at a time during the culture when nutrients are abundant and no component of the culture has any significant inhibitory effect on growth).

[0138] As used herein, the term "cell viability" refers to the ability of the cells in a culture to survive under a given set of culture conditions or experimental variations. As used herein, the term also refers to the fraction of cells that are alive at a particular time relative to the total number of live and dead cells in the culture at that time.

[0139] As used herein, the term "cell density" refers to the number of cells present in a given volume of culture medium.

[0140] As used herein, the term "bioreactor" or "culture vessel" refers to any container used for the growth of mammalian cell cultures. The bioreactor can be of any size as long as it can be used for the culture of mammalian cells.

[0141] As used herein, the term "bioreactor run" can include one or more of the lag phase, log phase, or stationary phase growth periods during a cell culture cycle.

[0142] As used herein, the term "N-l culture vessel", "N-l inoculation chain culture vessel", "N-l vessel", "N1-culture", or "N1 vessel" refers to a culture vessel that is immediately prior to the N culture vessel (production culture vessel) and is used to grow a cell culture to a high viable cell density for subsequent inoculation into the N (production) culture vessel. The cell culture to be grown in the N-l culture vessel can be obtained after culturing cells in several vessels (such as N-4, N-3, and N-2 vessels) prior to the N-l culture vessel.

[0143] As used herein, the term "N culture vessel", "production culture vessel", "N vessel", "N bioreactor", or "production bioreactor" refers to the cell culture in the bioreactor after the N-l bioreactor. The N culture is used to produce AAV.

[0144] As used herein, the term "seeding (or inoculating)" refers to the process of providing a cell culture to a bioreactor or another vessel. In one aspect, the cells have been previously proliferated in another bioreactor or vessel. In another aspect, the cells are frozen and thawed immediately before being provided to the bioreactor or vessel. The term refers to any number of cells, including a single cell.

[0145] rAAV and methods for producing rAAV (or compositions comprising rAAV) (e.g., rAAVrh74.MHCK7.minidystrophin) Figure 3

[0146] The present disclosure provides a composition comprising recombinant adeno-associated virus (rAAV) rAAV.MHCK7.minidystrophin, wherein the rAAV is produced in adherent mammalian cells, and wherein the adherent cells are cultured under suspension conditions in an N-1 vessel. In some aspects, the rAAV is serotype AAV.rh74 (such as rAAV.MHCK7.minidystrophin).

[0147] The present disclosure also provides a method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin in adherent mammalian cells by a suspension seeding method, which includes: (a) culturing cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing a lower concentration of serum than the first medium; (d) culturing the cells under suspension conditions in the N-1 container; and (e) inoculating the cells from step (d) into a third medium in a bioreactor.

[0148] In some aspects, the rAAV used in the methods described herein contains the human minidystrophin nucleotide sequence of SEQ ID NO:1. In some aspects, the rAAV contains the MHCK7 promoter sequence of SEQ ID NO:7. In some aspects, the rAAV contains the human minidystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:7.

[0149] In some aspects, the suspension seeding method further includes: (f) transfecting the adherent cells with a transgenic plasmid containing an rAAVrh74.MHCK7.minidystrophin construct, a plasmid containing an AAV rep gene and an AAV cap gene, and an adenovirus helper plasmid.

[0150] In some aspects, the transgenic plasmid containing an rAAVrh74.MHCK7.minidystrophin construct contains: the nucleic acid sequence of SEQ ID NO:9; nucleotides 55-5021 of SEQ ID NO:3; or nucleotides 1-4977 of SEQ ID NO:8. In some aspects, the plasmid containing an AAV rep gene and an AAV cap gene contains an AAV2 rep gene and an rAAVrh74 cap gene. In some aspects, the adenovirus helper plasmid contains adenovirus 5 E2A, E4 ORF6, and VA RNA genes.

[0151] In some aspects, the suspension seeding method further includes: (g) lysing the adherent cells. In some aspects, the adherent cells are lysed by freeze-thaw, solid shearing, hypertonic and / or hypotonic lysis, liquid shearing, sonication, high-pressure extrusion, detergent lysis, or a combination thereof.

[0152] In some aspects, the suspension seeding method further includes: (h) purifying the rAAV by at least one column chromatography step. In some aspects, the at least one column chromatography step includes anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0153] In some aspects, the suspension seeding method further includes culturing cells in the N-3 container with the first growth medium. In some aspects, the suspension seeding method further includes culturing cells in the N-4 container with the first growth medium.

[0154] In some aspects, the bioreactor is an adherent bioreactor. In some aspects, the rAAV is purified from the culture produced from the adherent bioreactor.

[0155] In some aspects, the third medium in the bioreactor contains at least one factor that promotes cell adhesion. In some aspects, the at least one factor that promotes cell adhesion is selected from serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some aspects, the third medium in the bioreactor contains DMEM and 10% FBS.

[0156] In some aspects, the adherent cells are cultured under suspension conditions for about 48 - 72 hours.

[0157] In some aspects, the N-1 container is a suspension flask.

[0158] In some aspects, the adherent cells are selected from HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells. In some aspects, the adherent cells are HeLa cells or HEK-293 cells. In some aspects, the adherent cells are HEK-293 cells. In some aspects, the adherent cells are not suspension-adapted. In some aspects, culturing the cells under suspension conditions does not change the cell's adhesion dependence. In some aspects, the culture does not change the cells to produce a new cell line.

[0159] In some aspects, a suspension seeding method for generating rAAVrh74.MHCK7.minidystrophin in adherent mammalian cells includes: (a) culturing the cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing a lower concentration of serum than the first medium; (d) culturing the cells in suspension conditions in the N-1 container; (e) inoculating the cells from step (d) into a third medium in a bioreactor; (f) transfecting the cells with a transgenic plasmid containing the rAAVrh74.MHCK7.minidystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid; (g) lysing the cells; and (h) purifying the rAAV by at least one column chromatography step.

[0160] In some aspects, the rAAV is described in International Publication No. WO 2019 / 245973 A1, which is hereby incorporated by reference in its entirety.

[0161] Adeno-associated virus (AAV) is a replication-defective parvovirus with a single-stranded DNA genome approximately 4.7 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). There are multiple AAV serotypes. The nucleotide sequences of the AAV serotype genomes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is shown in Srivastava et al., J Virol. 45:555-564 (1983), as amended by Ruffing et al., J Gen Virol. 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is available under GenBank accession number NC_002077; the complete genome of AAV-3 is available under GenBank accession number NC_1829; the complete genome of AAV-4 is available under GenBank accession number NC_001829; the AAV-5 genome is available under GenBank accession number AF085716; the complete genome of AAV-6 is available under GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are available under GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 regarding AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol. 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac et al., Journal of Translational Medicine 5:45 (2007).

[0162] Any AAV serotype can be used according to the present disclosure. In some aspects, the term "AAV" as used herein includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh8, AAVrh10, AAVrh74, the AAV serotypes and clades disclosed by Gao et al., J. Virol. 78: 6381 (2004) and Moris et al., Virol. 33: 375 (2004), and any other AAVs now known or later discovered. See, e.g., Knipe et al. (eds.), Fields Virology, Volume 2, Chapter 69 (4th Edition, Lippincott Williams & Wilkins Publishers) (2001).

[0163] In some aspects, the "AAV viral particle" is an AAV viral particle of any one of the above AAV serotypes.

[0164] In some aspects, the AAV viral particle is a rh74 serotype viral particle.

[0165] The cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosome integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 based on their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) - in conjunction with differential splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. Rep proteins have multiple enzymatic properties that ultimately are responsible for replicating the viral genome. The cap gene is expressed by the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-canonical translation initiation sites are responsible for 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).

[0166] AAV has unique properties that make it attractive as a vector for delivering foreign DNA into cells, for example, in gene therapy. AAV infection of cells in culture does not cause cytopathic effects, and natural infection in humans and other animals is silent and asymptomatic. In addition, AAV infects many mammalian cells, so it has the potential to target many different tissues in vivo. Furthermore, AAV transduces both dividing and non-dividing cells slowly and can persist essentially as a transcriptionally active nuclear episome (extrachromosomal element) throughout the life of these cells. The AAV proviral genome is infectious like cloned DNA in a plasmid, which makes it possible to construct recombinant genomes. In addition, since the signals that direct AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the inherently ~4.3 kb genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, the target DNA, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another remarkable feature of AAV is that it is an extremely stable and robust virus. It can easily withstand conditions used to inactivate adenoviruses (56 °C to 65 °C for several hours), making refrigeration of AAV less critical. AAV can even be lyophilized. Finally, cells infected with AAV are not resistant to reinfection.

[0167] Multiple studies have shown 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., MolTher 2:619-623 (2000) and Chao et al., Mol Ther 4:217-222 (2001). In addition, because muscle is highly vascularized, recombinant AAV transduction results in the appearance of transgenic products in the systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA 94:13921-13926 (1997). In addition, Lewis et al., J Virol.76:8769-8775 (2002) demonstrated that skeletal muscle fibers have the cytokines necessary for proper antibody glycosylation, folding, and secretion, indicating that muscle is capable of stably expressing secreted protein therapeutics.

[0168] 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 in the rAAV genome can be from any AAV serotype from which a recombinant virus can be derived, including but not limited to AAV serotypes AAVrh74, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, e.g., Marsic et al., Molecular Therapy 22(11):1900-1909 (2014). As described above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle-specific expression, AAV1, AAV6, AAV8, or AAVrh74 can be used.

[0169] The DNA plasmid of the present disclosure comprises the rAAV genome of the present disclosure. The DNA plasmid is transferred into cells permissive to infection with an AAV helper virus (such as an adenovirus, an E1-deleted adenovirus, or a herpes virus) to assemble the rAAV genome into infectious virus particles. Techniques for producing rAAV particles are standard in the art, where the AAV genome to be packaged, the rep and cap genes, and the helper virus functions are provided to the cells. The production of rAAV requires the presence in a single cell (referred to herein as a packaging cell) of the following components: the rAAV genome, the AAV rep and cap genes that are separate from the rAAV genome (i.e., not in the rAAV genome), and the helper virus functions. The AAV rep and cap genes can be from any AAV serotype from which a recombinant virus can be derived and can be from an AAV serotype different from the AAV ITR of the rAAV genome, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0170] One method of generating packaging cells is to create a cell line that stably expresses all the components necessary for AAV particle production. For example, one (or more) plasmids containing an rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separated from the rAAV genome, and an optional marker (such as a neomycin resistance gene) are integrated into the genome of the cell. The AAV genome is introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., Proc. Natl. Acad. Sci. USA 79: 2077-2081 (1982)), adding synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., Gene 23: 65-73 (1983)) or by direct blunt-end ligation (Senapathy & Carter, J. Biol. Chem. 259: 4661-4666 (1984)). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0171] The general principles of rAAV production are reviewed below, e.g., Carter, Current Opinions in Biotechnology 15: 33-539 (1992); and Muzyczka, N., Curr. Topics Microbiol. Immunol. 158: 97-129 (1992). Various methods are described below: Ratschin et al., Mol. Cell. Biol. 4: 2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984); Tratschin et al., Mo1. Cell. Biol. 5: 3251 (1985); McLaughlin et al., J. Virol., 62: 1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7: 349 (1988). Samulski et al., J. Virol., 63: 3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95 / 13365 and corresponding U.S. Patent No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4: 609-615 (1993); Clark et al. Gene Therapy 3: 1124-1132 (1996); U.S. Patent No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entirety, with particular emphasis on those portions of the documents related to rAAV production.

[0172] Accordingly, the present disclosure provides packaging cells for generating infectious rAAV. In one aspect, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (a homologous 293 line). In another aspect, the packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey embryonic lung cells).

[0173] The recombinant AAV of the present disclosure (i.e., the infectious packaged rAAV particles) contains an rAAV genome. In an exemplary aspect, the genomes of the rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAV that can be constructed to contain the nucleic acid molecules of the present disclosure are listed in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352), which is incorporated herein by reference in its entirety.

[0174] In an exemplary aspect, the recombinant AAV vector of the present invention is produced by a triple transfection method (Xiao et al., J Virol 72:2224-2232 (1998)) using the AAV vector plasmid rAAV.MHCK7.minidystrophin, pNLRep2-Caprh74, and pHELP. The rAAV contains a minidystrophin gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is packaged into the AAVrh74 virions. The plasmid contains the minidystrophin sequence and the MHCK7 enhancer and core promoter elements of a muscle-specific promoter to drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) to promote high-level gene expression, and a bovine growth hormone polyadenylation signal for efficient transcriptional termination.

[0175] pNLREP2-Caprh74 is an AAV helper plasmid that encodes four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins of the rh74 serotype. A schematic map of the pNLREP2-Caprh74 plasmid is as Figure 4 shown.

[0176] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. The plasmid contains regions of the adenovirus genome that are 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 crucial for replication, such as adenovirus terminal repeats. Thus, such a production system is not expected to generate infectious adenovirus. A schematic map of the pHELP plasmid is as Figure 1 shown.

[0177] The rAAV as described herein can be purified by standard methods in the art (e.g., by column chromatography or cesium chloride gradient). Methods for purifying rAAV vectors from helper virus are known in the art and include, for example, the methods disclosed in the following: 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.

[0178] In one aspect, the present disclosure provides an rAAV that comprises a muscle - specific control element nucleotide sequence and a nucleotide sequence encoding a micro - dystrophin. For example, the nucleotide sequence encodes a functional micro - dystrophin, wherein the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% sequence identity with SEQ ID NO:1, more typically at least 90%, 91%, 92%, 93%, or 94%, even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and wherein the protein retains micro - dystrophin activity. Micro - dystrophin provides stability to the muscle membrane during muscle contraction. For example, micro - dystrophin acts as a shock absorber during muscle contraction.

[0179] In one aspect, the rAAV is rAAVrh74.MHCK7.microdystrophin, which is in the form of viral particles of the rAAV serotype rh74 capsid encapsulating a nucleic acid expression cassette or genome, the nucleic acid expression cassette or genome comprising a microdystrophin transgene driven by the MHCK7 promoter / enhancer, and the rAAV is also referred to by the non-proprietary drug name delandistrogene moxeparvovec when administered to a subject. In some aspects, when referring to the study subjects in the examples to whom delandistrogene moxeparvovec was administered, the data (e.g., bar graphs) may be more simply designated as "treated".

[0180] In one aspect, the rAAVrh74.MHCK7.microdystrophin is the rAAVrh74.MHCK7.microdystrophin shown in any of the following: 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, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured under suspension conditions in an N - 1 container. In one aspect, the rAAV is AAVrh74.MCK.microdystrophin. In one aspect, the rAAVrh74.MCK.microdystrophin is the rAAVrh74.MCK.microdystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0181] The present disclosure 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 microdystrophin.

[0182] In one aspect, the rAAV is a non - replicating recombinant adeno - associated virus (AAV), referred to as the rAAVrh74.MHCK7.microdystrophin shown in any of the following: 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, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured under suspension conditions in an N - 1 container. The vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeats (ITRs), microdystrophin, the SV40 intron (SD / SA) and a synthetic polyadenylation (Poly A) signal, all of which are under the control of the MHCK7 promoter / enhancer. Schematic diagrams of the vector genome and the expression cassette are asFigure 1 As shown. The AAVrh74 serotype can be used to achieve efficient gene transfer in skeletal and cardiac muscles after intravenous (IV) administration.

[0183] In one aspect, the present disclosure provides an rAAV, wherein the muscle-specific control element is a human skeletal muscle actin gene element, a cardiac muscle 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 α-myosin heavy chain enhancer- / MCK enhancer-promoter (MHCK7), C5-12, a murine creatine kinase enhancer element, a fast-twitch skeletal muscle troponin c gene element, a slow-twitch cardiac muscle 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).

[0184] For example, the muscle-specific control element is the MHCK7 promoter nucleotide sequence SEQ ID NO:2 or SEQ ID NO:7, or the muscle-specific control element is the MCK nucleotide sequence SEQ ID NO:4. Additionally, in any rAAV vector of the present disclosure, the muscle-specific control element nucleotide sequence, such as the MHCK7 or MCK nucleotide sequence, is operably linked to the nucleotide sequence encoding micro-dystrophin. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO:2 or SEQ ID NO:7) is operably linked to the human micro-dystrophin coding sequence (SEQ ID NO:1), as Figure 2 or Figure 13 (SEQ ID NO:3) or Figure 5 (SEQ ID NO:9) as shown in the constructs provided. In another example, the MCK promoter (SEQ ID NO:4) is operably linked to the human micro-dystrophin coding sequence (SEQ ID NO:1), as Figure 6 or Figure 2 (SEQ ID NO:5) as shown in the constructs provided. In another aspect, the present disclosure provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, or an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:7. The present disclosure also provides an rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:4.

[0185] In another aspect, the present disclosure provides an rAAV construct contained in a plasmid, the plasmid comprising the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:8. For example, the AAVrh74.MHCK7. microdystrophin vector contains the nucleotide sequence within and including the ITR of SEQ ID NO:3, as Figure 13 shown. In another aspect, the rAAV vector contains a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, poly A, and a 3' ITR. In one aspect, the vector contains nucleotides 55 - 5021 of SEQ ID NO:3. The plasmid shown in SEQ ID NO:3 also contains ampicillin resistance and a pGEX plasmid backbone with a pBR322 origin of replication.

[0186] In another aspect, the present disclosure provides an rAAV comprising the nucleotide sequence of SEQ ID NO:9, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured under suspension conditions in an N - 1 container. For example, the AAVrh74.MHCK7. microdystrophin vector construct contains the nucleotide sequence of SEQ ID NO:9, as Figure 15 shown. The rAAV vector construct contains an MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, and poly A. In one aspect, the rAAV vector construct further contains an ITR at the 5' of the promoter and an ITR at the 3' of the poly A. In one aspect, the rAAV is AAVrh74.

[0187] In another aspect, the rAAVrh74.MHCK7. microdystrophin vector (i.e., the viral vector) contains the nucleotide sequence within and including the ITR of SEQ ID NO:8, as Figure 14 shown. The rAAV vector contains a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, poly A, and a 3' ITR. In one aspect, the vector contains nucleotides 1 - 4977 of SEQ ID NO:9. The plasmid shown in SEQ ID NO:3 also contains kanamycin resistance and a pGEX plasmid backbone with a pBR322 origin of replication.

[0188] In another aspect, the present disclosure provides a plasmid comprising an AAVrh74.MHCK7.minidystrophin construct. In one aspect, the plasmid comprises a 5' ITR, an MHCK7 promoter, a chimeric intron sequence, a coding sequence of a human minidystrophin gene, a poly A, and a 3' ITR. In one aspect, the plasmid comprises kanamycin resistance and optionally comprises a pGEX plasmid backbone having an origin of replication of pBR322. In a particular aspect, the plasmid is as described in SEQ ID NO:8 and is shown in Figure 15 and Compositions comprising rAAV and their administration .

[0189] The present disclosure provides a recombinant AAV vector comprising the human minidystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7, wherein the rAAV is produced in adherent cells and wherein the adherent cells are cultured under suspension conditions in an N-1 container. The rAAV vector is AAV serotype AAVrh.74.

[0190] The present disclosure also provides an rAAV comprising an AAVrh74.MHCK7.minidystrophin construct nucleotide sequence within and including the ITR of SEQ ID NO:3, a nucleotide sequence within and including the ITR of SEQ ID NO:8, or a nucleotide sequence as shown in SEQ ID NO:9, wherein the rAAV is produced in adherent cells and wherein the adherent cells are cultured under suspension conditions in an N-1 container. The rAAV vector is AAV serotype AAVrh.74.

[0191] The rAAV vector of the present disclosure can be any AAV serotype, for example, serotype AAVrh74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0192] The present disclosure also provides a pharmaceutical composition (or sometimes simply referred to as "composition" herein) comprising any one of the rAAV vectors of the present disclosure.

[0193] In another aspect, the present disclosure provides a method for producing rAAV vector particles, which includes culturing cells transfected with any one of the rAAV vectors of the present disclosure and recovering rAAV particles from the supernatant of the transfected cells. The present disclosure also provides virus particles comprising any one of the recombinant AAV vectors of the present disclosure.

[0194] Mixed seeding chain amplification of adherent cells

[0195] In another aspect, the present disclosure provides a composition comprising the rAAV of the present disclosure. The composition of the present disclosure comprises rAAV and a pharmaceutically acceptable carrier. The composition may further comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to the recipient, preferably inert at the doses and concentrations used, and comprise buffers and surfactants such as pluronics.

[0196] The present disclosure provides a composition for treating a subject in need thereof for muscular dystrophy (such as DMD), which comprises recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, wherein the rAAV is produced in adherent cells, and wherein the adherent cells are cultured under suspension conditions in an N-1 container.

[0197] In some aspects, the present disclosure provides a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, wherein the rAAV is produced in adherent mammalian cells by the suspension seeding method described herein. In some aspects, the composition comprises: a) rAAV particles comprising the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles comprising nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles comprising nucleotides 1-4977 of SEQ ID NO:8.

[0198] The titer of the rAAV administered in the methods of the present disclosure varies depending on, for example, the particular rAAV, the mode of administration, the treatment goal, the individual, and the one or more cell types targeted, and can be determined by standard methods in the art. The titer of rAAV can be from about 1×10 6 to about 1×10 7 to about 1×10 8 to about 1×10 9 to about 1×10 10 to about 1×10 11 to about 1×10 12 to about 1×10 13 to about 1×10 14 or more DNase-resistant particles (DRP). The dose can also be expressed in units of viral genomes (vg). An exemplary method for determining the titer of the encapsulated vector genome uses quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25:855-869 (2017)).

[0199] The present disclosure contemplates methods of transducing target cells in vivo or in vitro with rAAV. In vivo methods include the step of administering to an animal (including a human) in need thereof a single effective dose or multiple effective doses of a composition comprising the rAAV of the present disclosure. If the dose is administered prior to 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 aspects of the present disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents progression of the disorder / disease state, reduces the severity of the disease, remits (partially or fully) the disease, and / or prolongs survival. Examples of diseases contemplated for prevention or treatment by the methods of the present disclosure are DMD.

[0200] The present disclosure also contemplates combination therapies. As used herein, "combination" includes concurrent therapy and sequential therapy. Combinations of the methods of the present disclosure with standard pharmaceutical therapeutic agents (such as corticosteroids) and with novel therapies are specifically contemplated.

[0201] Administration of an effective dose of the composition can be by standard routes in the art, including but not limited to intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal routes. The route of administration and serotype of the AAV component of the rAAV of the present disclosure (specifically the AAV ITR and capsid proteins) can be selected and / or matched by those skilled in the art considering the infection and / or disease state being treated and the target cell / tissue in which the mini-dystrophin is to be expressed.

[0202] The present disclosure provides for local and systemic administration of an effective dose of the rAAV and composition of the present disclosure. For example, systemic administration is administration into the circulatory system such that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration by injection, infusion, or implantation.

[0203] In particular, the actual administration of the rAAV of the present disclosure can be accomplished 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. It has been demonstrated that simply resuspending rAAV in phosphate buffered saline is sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations on carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the normal manner of using rAAV). The capsid protein of rAAV can be modified such that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or a topical formulation for delivery to muscle by transdermal delivery. Many formulations for both intramuscular injection and transdermal delivery have been developed previously and can be used in the practice of the present disclosure. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0204] In one aspect of the present disclosure, the AAVrh74.MHCK7. micro-dystrophin described herein is formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl2), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0205] The dose of rAAV administered in the methods described herein will vary depending on, for example, the particular rAAV, the mode of administration, the treatment goal, the individual, and the one or more cell types targeted, and can be determined by standard methods in the art. The titer of each rAAV administered can be in the range of about 1×10 6 to about 1×10 7 to about 1×10 8 to about 1×10 9 to about 1×10 10 to about 1×10 11 to about 1×10 12 to about 1×10 13 to about 1×10 14 to about 2×10 14 to about 1×10 15 or more DNase-resistant particles (DRP). The dose can also be expressed in terms of the number of viral genomes (vg) (e.g., 1×10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×1013 vg, 1×10 14 vg, 2×10 14 vg, 4×10 14 vg, 6×10 14 vg, 8×10 14 vg, 1×10 15 vg, 2×10 15 vg, 3×10 15 vg, 4×10 15 vg, 5×10 15 vg, 6×10 15 vg, 7×10 15 vg, 8×10 15 vg, 9×10 15 vg, 1×10 16 vg). The dose can also be expressed as the number of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1×10 10 vg / kg, 1×10 11 vg / kg, 1×10 12 vg / kg, 1×10 13 vg / kg, 1×10 14 vg / kg, 1.25×10 14 vg / kg, 1.33×10 14 vg / kg, 1.5×10 14 vg / kg, 1.75×10 14 vg / kg, 2.0×10 14 vg / kg, 2.25×10 14 vg / kg, 2.5×10 14 vg / kg, 2.75×10 14 vg / kg, 3.0×10 14 vg / kg, 3.25×10 14 vg / kg, 3.5×10 14 vg / kg, 3.75×10 14 vg / kg, 4.0×10 14 vg / kg, 1×10 15 vg / kg). The dose can also be expressed as a total fixed dose (e.g., 9.31×10 15 vg). A "total fixed dose" is the dose administered to a subject at or above a predetermined body weight, and this dose is not adjusted according to the subject's body weight. For example, in some aspects, a dose of 1.33×10 14 vg / kg is administered to subjects <70 kg, and a dose of 9.31×10 15The total fixed dose of vg, where 1.33×10 14 vg×70 kg equals 9.31×10 15 vg. This means that the same dose (“fixed dose”) is administered to subjects over 70 kg as to subjects weighing 70 kg. A method for titrating AAV is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0206] 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. It has been demonstrated that simply resuspending rAAV in phosphate buffered saline is sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations on carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the normal manner of using rAAV). The capsid protein of rAAV can be modified such that rAAV targets a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or a topical formulation for delivery to muscle by transdermal delivery. Many formulations for both intramuscular injection and transdermal delivery have been developed previously and can be used in the practice of the present disclosure. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0207] For intramuscular injection, an adjuvant (such as sesame or peanut oil) or a solution in aqueous propylene glycol, as well as a sterile aqueous solution, can be employed. If desired, such aqueous solutions can be buffered and first made isotonic with a diluent such as saline or glucose. Solutions of rAAV in the form of the free acid (DNA contains acidic phosphate groups) or a pharmaceutically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed can be readily obtained by standard techniques well known to those skilled in the art.

[0208] Pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage, and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Appropriate fluidity can be maintained by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption (such as aluminum monostearate and gelatin).

[0209] Sterile injectable solutions can be prepared by incorporating the required amount of rAAV, as needed, into a suitable solvent together with the various other ingredients enumerated above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle that contains a base dispersion medium and the required other ingredients selected from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any other required ingredients from its pre-sterile filtered solution.

[0210] Transduction with rAAV can also be carried out in vitro. In one aspect, the desired target muscle cells are removed from a subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used, in which case these cells will not produce an inappropriate immune response in the subject.

[0211] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art. In one aspect, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in a suitable medium, and cells that carry the target DNA can be screened using conventional techniques such as Southern blotting and / or PCR or using selectable markers. The transduced cells can then be formulated into a pharmaceutical composition and introduced into the subject by various techniques such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle using, for example, a catheter.

[0212] Transducing cells with the rAAVs of the present disclosure enables persistent expression of mini - dystrophin. Accordingly, the present disclosure provides methods of administering / delivering rAAVs that express mini - dystrophin to an animal (preferably a human). These methods include transducing tissues (including, but not limited to, tissues such as muscle, myocardium, organs such as liver and brain, and glands such as salivary glands) with one or more rAAVs of the present disclosure. Transduction can be carried out using gene cassettes containing tissue - specific control elements. For example, one aspect of the present disclosure provides a method of transducing muscle cells and muscle tissue directed by a muscle - specific control element, which includes, but is not limited to, those derived from the actin and myosin gene families, such as those derived from the myoD gene family (see Weintraub et al., Science, 251:761 - 766 (1991)); the muscle - cell - specific enhancer - binding factor MEF - 2 (Cserjesi and Olson, Mol Cell Biol 11:4854 - 4862 (1991)); control elements derived from the human skeletal muscle actin gene (Muscat et al., Mol Cell Biol, 7:4089 - 4099 (1987)), the cardiac muscle actin gene, the muscle creatine kinase sequence element (see Johnson et al., Mol Cell Biol 9:3393 - 3399 (1989)) and the murine creatine kinase enhancer (mCK) element; control elements derived from the skeletal fast - twitch troponin C gene, the slow - twitch cardiac troponin C gene and the slow - twitch troponin I gene: the hypoxia - inducible nuclear factor (Semenza et al., Proc Natl Acad Sci USA 88:5680 - 5684 (1991)), the steroid - inducible element and promoters including the glucocorticoid response element (GRE) (see Mader and White, Proc.Natl.Acad.Sci.USA 90:5603 - 5607 (1993)) and other control elements.

[0213] Muscle tissue is an attractive target for in - vivo DNA delivery because it is not a vital organ and is easily accessible. The present disclosure contemplates persistent expression of mini - dystrophin by transduced muscle fibers.

[0214] Myocardial tissue is an attractive target for in - vivo DNA delivery because it is a vital organ, and persistent expression of mini - dystrophin by transduced myocardial fibers can extend the survival period of patients with muscular dystrophy.

[0215] Accordingly, the present disclosure provides methods of administering an effective dose (or multiple doses administered substantially simultaneously or multiple doses administered at intervals) of rAAV encoding micro-dystrophin to a subject in need thereof (e.g., a subject suffering from muscular dystrophy).

[0216] The present disclosure provides nucleic acid molecules comprising the nucleotide sequences of SEQ ID NO: 3, 8, or 9. The present disclosure also provides rAAV comprising the nucleic acid sequence of SEQ ID NO: 9 or nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 55-5021 of SEQ ID NO: 3, and rAAV particles comprising the nucleic acid sequence of SEQ ID NO: 9 or nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 55-5021 of SEQ ID NO: 3, wherein the rAAV is produced in adherent cells and wherein the adherent cells are cultured under suspension conditions in an N-1 container.

[0217] Another aspect of the present disclosure provides a composition comprising: a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3, 8, or 9, rAAV comprising the nucleic acid sequence of SEQ ID NO: 9 or nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 55-5021 of SEQ ID NO: 3, and rAAV particles comprising the nucleic acid sequence of SEQ ID NO: 9 or nucleotides 1-4977 of SEQ ID NO: 8 or nucleotides 55-5021 of SEQ ID NO: 3, wherein the rAAV is produced in adherent cells and wherein the adherent cells are cultured under suspension conditions in an N-1 container. Any of the methods described herein can be performed using these compositions.

[0218] Methods for producing viral vectors (e.g., AAV)

[0219] Some aspects of the present disclosure relate to a method of cell expansion comprising: (a) culturing cells in a first medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing a lower concentration of serum than the first medium; (d) culturing the cells under suspension conditions in the N-1 container; and (e) inoculating a third medium in a bioreactor with the cells from step (d). In one aspect, the second medium is a serum-free medium. In another aspect, the second medium contains a lower concentration of serum than the serum concentration in the first medium.

[0220] Some aspects of the present disclosure relate to a method of inoculation chain amplification, which includes: (a) culturing cells in a first medium containing serum in an N-3 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-2 container, the second medium being serum-free or containing serum at a concentration lower than that of the first medium; (d) culturing the cells in suspension in the N-2 container; (e) inoculating the cells from step (d) into a second medium in an N-1 container; and (f) inoculating the cells from step (d) into a third medium in a bioreactor. In one aspect, the second medium is a serum-free medium. In another aspect, the second medium contains serum at a concentration lower than the serum concentration in the first medium.

[0221] Some aspects of the present disclosure relate to a method for cell amplification of adherent cells, which includes: (a) culturing the adherent cells in a first medium containing serum under adherent conditions; (b) removing the adherent cells from the first medium; (c) suspending the adherent cells in a second medium, the second medium being serum-free or containing serum at a concentration lower than that of the first medium; (d) culturing the adherent cells in suspension; and (e) inoculating the adherent cells from step (d) into a third medium in a bioreactor. In some aspects, the method may further include passaging the adherent cells of step (a) at least once under adherent conditions. In some aspects, the method may further include passaging the adherent cells of step (d) at least once under suspension conditions. In one aspect, the second medium is a serum-free medium. In another aspect, in the N-1 container, the second medium contains serum at a concentration lower than that of the first medium.

[0222] The first, second, and third culture media can be any media suitable for the particular cells being cultured. In some aspects, the media contain, for example, inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin, and fetuin), serum (e.g., a composition containing albumin, growth factors, and growth inhibitors such as fetal bovine serum, newborn calf serum, and horse serum), trace elements (e.g., zinc, copper, selenium, and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins from plant or animal sources), and combinations thereof. The growth media can be commercially available media such as 5× concentrated DMEM / F12 (Invitrogen), CD OptiCHO Feed (Invitrogen), CD EfficientFeed (Invitrogen), Cell Boost (HyClone), BalanCD CHO Feed (Irvine Scientific), BDRecharge (Becton Dickinson), Cellvento Feed (EMD Millipore), Ex-cell CHOZN Feed (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO (Kerry), Zap-CHO (Invitria), ActiCHO (PAA / GE Healthcare), Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ([DMEM], Sigma).

[0223] In some aspects, the serum-free growth second medium that is serum-free or contains serum at a concentration lower than the first serum is substantially free (contains no more than trace levels) of calcium ions, fetal bovine serum (FBS), fibronectin, collagen, laminin, or proteoglycan or non-proteoglycan polysaccharides of the extracellular matrix, which would support cell attachment. In one aspect, the second medium is a serum-free medium. In another aspect, the second medium contains serum at a concentration lower than the serum concentration in the first medium.

[0224] In some aspects, the pH of the growth medium can be between about 6.5 and about 7.5, between about 6.5 and about 7.4, between about 6.5 and about 7.3, between about 6.5 and about 7.2, between about 6.5 and about 7.1, between about 6.5 and about 7.0, between about 6.5 and about 6.9, between about 6.5 and about 6.8, between about 6.5 and about 6.7, between about 6.6 and about 7.5, between about 6.6 and about 7.4, between about 6.6 and about 7.3, between about 6.6 and about 7.2, between about 6.6 and about 7.1, between about 6.6 and about 7.0, between about 6.6 and about 6.9, between about 6.6 and about 6.8, between about 6.7 and about 7.5, between about 6.7 and about 7.4, between about 6.7 and about 7.3, between about 6.7 and about 7.2, between about 6.7 and about 7.1, between about 6.7 and about 7.0, between about 6.7 and about 6.9, between about 6.8 and about 7.5, between about 6.8 and about 7.4, between about 6.8 and about 7.3, between about 6.8 and about 7.2, between about 6.8 and about 7.1, between about 6.8 and about 7.0, between about 6.9 and about 7.5, between about 6.9 and about 7.4, between about 6.9 and about 7.3, between about 6.9 and about 7.2, between about 6.9 and about 7.1, between about 7.0 and about 7.5, between about 7.0 and about 7.4, between about 7.0 and about 7.3, between about 7.0 and about 7.2, between about 7.1 and about 7.5, between about 7.1 and about 7.4, between about 7.1 and about 7.3, between about 7.2 and about 7.5, between about 7.2 and about 7.4, or between about 7.3 and about 7.5.

[0225] In some aspects, the cells can be cultured at a temperature of from 32°C to about 39°C, from about 32°C to about 37°C, between about 32°C and about 37.5°C, between about 34°C and about 37°C, between about 35°C and about 37°C, between about 35.5°C and about 37.5°C, between about 36°C and about 37°C, or about 36.5°C. In some aspects, the cells can be incubated at a temperature of about 37°C from the start to the end of the culture period. In some aspects, the temperature can be changed or can vary slightly during the culture period, for example, by the hour or by the day. In some aspects, the temperature can be changed or shifted (e.g., increased or decreased) about one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, fourteen, or fifteen days after the start of the culture period or at any point in time during the culture period. In some aspects, the temperature can be shifted up by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0°C. In some aspects, the temperature can be shifted down by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10°C.

[0226] In some aspects, cell culture can be carried out in an atmosphere containing from about 1% to about 15% CO2. In some aspects, the cells can be cultured in an atmosphere containing about 14% CO2, 12% CO2, 10% CO2, 8% CO2, 6% CO2, 5% CO2, 4% CO2, 3% CO2, 2% CO2, or about 1% CO2.

[0227] In some aspects, cell culture can be carried out by maintaining the dissolved oxygen (dO2) in the cell culture at between about 3% and about 55%, between about 3% and about 50%, between about 3% and about 45%, between about 3% and about 40%, between about 3% and about 35%, between about 3% and about 30%, between about 3% and about 25%, between about 3% and about 20%, between about 3% and about 15%, between about 5% and about 55%, between about 5% and about 50%, between about 5% and about 45%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% and about 25%, between about 5% and about 20%, between about 5% and about 15%, between about 5% and about 10%, between about 10% and about 55%, between about 10% and about 50%, between about 10% and about 45%, between about 10% and about 40%, between about 10% and about 35%, between about 10% and about 30%, between about 10% and about 25%, between about 10% and about 20%, between about 15% and about 55%, between about 15% and about 50%, between about 15% and about 45%, between about 15% and about 40%, between about 15% and about 35%, between about 15% and about 30%, between about 15% and about 25%, between about 15% and about 20%, between about 20% and about 55%, between about 20% and about 50%, between about 20% and about 45%, between about 20% and about 40%, between about 20% and about 35%, between about 20% and about 30%, between about 20% and about 25%, between about 25% and about 55%, between about 25% and about 50%, between about 25% and about 45%, between about 25% and about 40%, between about 25% and about 35%, between about 25% and about 30%, between about 30% and about 55%, between about 30% and about 50%, between about 30% and about 45%, between about 30% and about 40%, between about 30% and about 35%, between about 35% and about 55%, between about 35% and about 50%, between about 35% and about 45%, between about 35% and about 40%, between about 40% and about 55%, between about 40% and about 50%, between about 40% and about 45%, between about 45% and about 55%, between about 45% and about 50%, or between about 50% and about 55%.

[0228] In some aspects, the pH of a cell culture can be maintained at a specific pH value by adding a base solution (such as an alkaline base solution). The pH of the cell culture can be maintained at the following pH values: between about 6.5 and about 7.5, between about 6.5 and about 7.4, between about 6.5 and about 7.3, between about 6.5 and about 7.2, between about 6.5 and about 7.1, between about 6.5 and about 7.0, between about 6.5 and about 6.9, between about 6.5 and about 6.8, between about 6.5 and about 6.7, between about 6.6 and about 7.5, between about 6.6 and about 7.4, between about 6.6 and about 7.3, between about 6.6 and about 7.2, between about 6.6 and about 7.1, between about 6.6 and about 7.0, between about 6.6 and about 6.9, between about 6.6 and about 6.8, between about 6.7 and about 7.5, between about 6.7 and about 7.4, between about 6.7 and about 7.3, between about 6.7 and about 7.2, between about 6.7 and about 7.1, between about 6.7 and about 7.0, between about 6.7 and about 6.9, between about 6.8 and about 7.5, between about 6.8 and about 7.4, between about 6.8 and about 7.3, between about 6.8 and about 7.2, between about 6.8 and about 7.1, between about 6.8 and about 7.0, between about 6.9 and about 7.5, between about 6.9 and about 7.4, between about 6.9 and about 7.3, between about 6.9 and about 7.2, between about 6.9 and about 7.1, between about 7.0 and about 7.5, between about 7.0 and about 7.4, between about 7.0 and about 7.3, between about 7.0 and about 7.2, between about 7.1 and about 7.5, between about 7.1 and about 7.4, between about 7.1 and about 7.3, between about 7.2 and about 7.5, between about 7.2 and about 7.4, or between about 7.3 and about 7.5.

[0229] In some aspects, cell culture under suspension conditions can be carried out in any type of cell culture flask suitable for stable or mixed / shaking suspension cell expansion, such as a T-flask, roller bottle, spinner flask, or shake flask, or a combination thereof. In some aspects, the N-1 container is a shake flask.

[0230] In some aspects, the suspension conditions may include some form of agitation. In some aspects, the agitation can be rotational agitation. In some aspects, the frequency of agitation can be from about 25 RPM to about 500 RPM, between about 25 RPM and about 480 RPM, between about 25 RPM and about 460 RPM, between about 25 RPM and about 440 RPM, between about 25 RPM and about 420 RPM, between about 25 RPM and about 400 RPM, between about 25 RPM and about 380 RPM, between about 25 RPM and about 360 RPM, between about 25 RPM and about 340 RPM, between about 25 RPM and about 320 RPM, between about 25 RPM and about 300 RPM, between about 25 RPM and about 280 RPM, between about 25 RPM and about 260 RPM, between about 25 RPM and about 240 RPM, between about 25 RPM and about 220 RPM, between about 25 RPM and about 200 RPM, between about 25 RPM and about 180 RPM, between about 25 RPM and about 160 RPM, between about 25 RPM and about 140 RPM, between about 25 RPM and about 120 RPM, between about 25 RPM and about 100 RPM, between about 25 RPM and about 80 RPM, between about 25 RPM and about 60 RPM, between about 25 RPM and about 40 RPM, between about 25 RPM and about 35 RPM, between about 25 RPM and about 30 RPM, from about 50 RPM to about 500 RPM, between about 50 RPM and about 480 RPM, between about 50 RPM and about 460 RPM, between about 50 RPM and about 440 RPM, between about 50 RPM and about 420 RPM, between about 50 RPM and about 400 RPM, between about 50 RPM and about 380 RPM, between about 50 RPM and about 360 RPM, between about 50 RPM and about 340 RPM, between about 50 RPM and about 320 RPM, between about 50 RPM and about 300 RPM, between about 50 RPM and about 280 RPM, between about 50 RPM and about 260 RPM, between about 50 RPM and about 240 RPM, between about 50 RPM and about 220 RPM, between about 50 RPM and about 200 RPM, between about 50 RPM and about 180 RPM, between about 50 RPM and about 160 RPM, between about 50 RPM and about 140 RPM, between about 50 RPM and about 120 RPM, between about 50 RPM and about 100 RPM, between about 50 RPM and about 80 RPM, between about 50 RPM and about 60 RPM, from about 75 RPM to about 500 RPM, between about 75 RPM and about 480 RPM, between about 75 RPM and about 460 RPM, between about 75 RPM and about 440 RPM,Between approximately 75 RPM and approximately 420 RPM, between approximately 75 RPM and approximately 400 RPM, between approximately 75 RPM and approximately 380 RPM, between approximately 75 RPM and approximately 360 RPM, between approximately 75 RPM and approximately 340 RPM, between approximately 75 RPM and approximately 320 RPM, between approximately 75 RPM and approximately 300 RPM, between approximately 75 RPM and approximately 280 RPM, between approximately 75 RPM and approximately 260 RPM, between approximately 75 RPM and approximately 240 RPM, between approximately 75 RPM and approximately 220 RPM, between approximately 75 RPM and approximately 200 RPM, between approximately 75 RPM and approximately 180 RPM, between approximately 75 RPM and approximately 160 RPM, between approximately 75 RPM and approximately 140 RPM, between approximately 75 RPM and approximately 120 RPM, between approximately 75 RPM and approximately 100 RPM, between approximately 75 RPM and approximately 80 RPM, between approximately 100 RPM and approximately 500 RPM, between approximately 100 RPM and approximately 480 RPM, between approximately 100 RPM and approximately 460 RPM, between approximately 100 RPM and approximately 440 RPM, between approximately 100 RPM and approximately 420 RPM, between approximately 100 RPM and approximately 400 RPM, between approximately 100 RPM and approximately 380 RPM, between approximately 100 RPM and approximately 360 RPM, between approximately 100 RPM and approximately 340 RPM, between approximately 100 RPM and approximately 320 RPM, between approximately 100 RPM and approximately 300 RPM, between approximately 100 RPM and approximately 280 RPM, between approximately 100 RPM and approximately 260 RPM, between approximately 100 RPM and approximately 240 RPM, between approximately 100 RPM and approximately 220 RPM, between approximately 100 RPM and approximately 200 RPM, between approximately 100 RPM and approximately 180 RPM, between approximately 100 RPM and approximately 160 RPM, between approximately 100 RPM and approximately 140 RPM, between approximately 100 RPM and approximately 120 RPM, between approximately 150 RPM and approximately 500 RPM, between approximately 150 RPM and approximately 480 RPM, between approximately 150 RPM and approximately 460 RPM, between approximately 150 RPM and approximately 440 RPM, between approximately 150 RPM and approximately 420 RPM, between approximately 150 RPM and approximately 400 RPM, between approximately 150 RPM and approximately 380 RPM, between approximately 150 RPM and approximately 360 RPM, between approximately 150 RPM and approximately 340 RPM, between approximately 150 RPM and approximately 320 RPM, between approximately 150 RPM and approximately 300 RPM, between approximately 150 RPM and approximately 280 RPM, between approximately 150 RPM and approximately 260 RPM, between approximately 150 RPM and approximately 240 RPM, between approximately 150 RPM and approximately 220 RPMBetween about 150 RPM and about 200 RPM, between about 150 RPM and about 180 RPM, between about 150 RPM and about 160 RPM, between about 200 RPM and about 500 RPM, between about 200 RPM and 480 RPM, between about 200 RPM and about 460 RPM, between about 200 RPM and about 440 RPM, between about 200 RPM and about 420 RPM, between about 200 RPM and about 400 RPM, between about 200 RPM and about 380 RPM, between about 200 RPM and about 360 RPM, between about 200 RPM and about 340 RPM, between about 200 RPM and about 320 RPM, between about 200 RPM and about 300 RPM, between about 200 RPM and about 280 RPM, between about 200 RPM and about 260 RPM, between about 200 RPM and about 240 RPM, between about 200 RPM and about 220 RPM, between about 240 RPM and about 500 RPM, between about 240 RPM and about 480 RPM, between about 240 RPM and about 460 RPM, between about 240 RPM and about 440 RPM, between about 240 RPM and about 420 RPM, between about 240 RPM and about 400 RPM, between about 240 RPM and about 380 RPM, between about 240 RPM and about 360 RPM, between about 240 RPM and about 340 RPM, between about 240 RPM and about 320 RPM, between about 240 RPM and about 300 RPM, between about 240 RPM and about 280 RPM, between about 240 RPM and about 260 RPM, between about 260 RPM and about 500 RPM, between about 260 RPM and about 480 RPM, between about 260 RPM and about 460 RPM, between about 260 RPM and about 440 RPM, between about 260 RPM and about 420 RPM, between about 260 RPM and about 400 RPM, between about 260 RPM and about 380 RPM, between about 260 RPM and about 360 RPM, between about 260 RPM and about 340 RPM, between about 260 RPM and about 320 RPM, between about 260 RPM and about 300 RPM, between about 260 RPM and about 280 RPM, between about 280 RPM and about 500 RPM, between about 280 RPM and about 480 RPM, between about 280 RPM and about 460 RPM, between about 280 RPM and about 440 RPM, between about 280 RPM and about 420 RPM, between about 280 RPM and about 400 RPM, between about 280 RPM and about 380 RPM, between about 280 RPM and about 360 RPM, between about 280 RPM and about 340 RPM,Between about 280 RPM and about 320 RPM, between about 280 RPM and about 280 RPM, between about 300 RPM and about 500 RPM, between about 380 RPM and about 480 RPM, between about 380 RPM and about 460 RPM, between about 380 RPM and about 440 RPM, between about 380 RPM and about 420 RPM, between about 380 RPM and about 400 RPM, between about 400 RPM and about 500 RPM, between about 400 RPM and about 480 RPM, between about 400 RPM and about 460 RPM, between about 400 RPM and about 440 RPM, or between about 400 RPM and about 420 RPM. Stirring can be continuous or periodic.,

[0231] In some aspects, passage of the cells under suspension conditions is no more than two times.,

[0232] In some aspects, the cells are cultured in suspension culture for about 24 to about 96 hours. In some aspects, the cells are cultured in suspension culture for about 36 to about 84 hours. In some aspects, the cells are cultured in suspension culture for about 48 to about 72 hours. In some aspects, the cells are cultured in suspension culture for about 54 to about 66 hours. In some aspects, the cells are cultured in suspension culture for about 24, about 30, about 36, about 42, about 48, about 54, about 60, about 66, about 72, about 78, about 84, about 90 or about 96 hours.,

[0233] In some aspects of the present disclosure, the cells are adherent cells. In some aspects, the adherent cells are HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells or COS cells. In some aspects, the adherent cells are human cells. In some aspects, the adherent cells are HeLa or HEK-293 cells. In some aspects, the adherent cells are HEK-293 cells.,

[0234] In some aspects, the adherent cells are not suspension-adapted. In some aspects, culturing the cells under suspension conditions does not change the adherent-dependence of the cells. In some aspects, the method does not change the cells to produce a new cell line. The methods described herein do not change the genomic or transcriptomic profile of the cells. The methods described herein do not change the phenotype of the cells.,

[0235] In some aspects, the cells are passaged multiple times under adherent conditions in a growth medium supplemented with serum prior to inoculation into the N-1 vessel. In some aspects, the cells are cultured in N-2, N-3, N-4, N-5, N-6, N-7, N-8, N-9 or N-10 vessels prior to inoculation into the N-1 vessel. In some aspects, the cells are cultured in N-3 and N-2 vessels. In some aspects, the cells are cultured in N-4, N-3 and N-2 vessels.

[0236] In some aspects, the bioreactor is an adherent bioreactor. In some aspects, the adherent cells are purified from a culture produced in the adherent bioreactor.

[0237] In some aspects, the bioreactor comprises at least one, more preferably a plurality of carriers, to which the expanded cells are intended to adhere, which may be floating or fixed in the bioreactor. Preferably, the carriers can be made of, for example, polyethylene terephthalate, polystyrene, polyester, polypropylene, DEAE-dextran, collagen, glass, alginate or acrylamide. In some aspects, the bioreactor can be a bioreactor containing bead-type microcarriers (e.g. beads of a brand, available from GE Healthcare Inc., a subsidiary of General Electric Corp.) or matrix-type carriers (e.g. Fibra-Cell TM slices of a brand, available from Eppendorf Corp.). In some aspects, the bioreactor uses polyester fiber carriers, such as those used in nano or 500 bioreactors (available from Advanced Technology Materials Inc. (Brussels, Belgium) and Pall Corp. (Fall River, Mass)).

[0238] In some aspects, the third medium in the bioreactor comprises at least one factor that promotes cell adhesion. In some aspects, the at least one factor that promotes cell adhesion is selected from FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof. In some aspects, the at least one factor that promotes cell adhesion can be added to the third medium just before, during or after inoculating the suspension cells into the bioreactor.

[0239] In some aspects, the growth medium comprises DMEM and about 10 wt% FBS. In some aspects, the growth medium comprises about 2 wt% to about 20 wt% FBS. In some aspects, the growth medium comprises about 3 wt% to about 19 wt% FBS. In some aspects, the growth medium comprises about 4 wt% to about 18 wt% FBS. In some aspects, the growth medium comprises about 5 wt% to about 17 wt% FBS. In some aspects, the growth medium comprises about 6 wt% to about 16 wt% FBS. In some aspects, the growth medium comprises about 7 wt% to about 15 wt% FBS. In some aspects, the growth medium comprises about 8 wt% to about 14 wt% FBS. In some aspects, the growth medium comprises about 9 wt% to about 13 wt% FBS. In some aspects, the growth medium comprises about 10 wt% to about 12 wt% FBS. In some aspects, the growth medium comprises about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt% or about 20 wt% FBS.

[0240] In some aspects, the suspension-expanded cells from step (d) can be directly inoculated into the bioreactor. In some aspects, the number of cells inoculated into the bioreactor varies based on the size of the bioreactor. In some aspects, a 4 m2 bioreactor (e.g., a nano-bioreactor) is used. In some aspects, the 4 m2 bioreactor is inoculated with about 1×10 8 to 1×10 9 cells. In some aspects, the 4 m2 bioreactor is inoculated with about 3×10 8 to 7×10 8 cells. In some aspects, the 4 m2 bioreactor is inoculated with about 4×10 8 to 6×10 8 cells. In some aspects, the 4 m2 bioreactor is inoculated with about 5×10 8 cells. In some aspects, an equivalent cell density is used for bioreactors of other sizes.

[0241] In some aspects, the methods of the present disclosure may further include culturing the cells in the bioreactor. In some aspects, the cell culture includes batch culture. In some aspects, the cell culture includes fed-batch culture. In some aspects, the cell culture comprises perfusion culture.

[0242] Fed-batch culture involves incrementally (periodically) or continuously adding a feed medium to an initial cell culture without substantially or significantly removing growth medium from the cell culture. The cell culture in fed-batch culture can be placed in a bioreactor (e.g., a production bioreactor such as a 10,000 L production bioreactor). In some aspects, the feed medium can be the same as the growth medium. The feed medium can be in liquid or dry powder form. In some aspects, the feed medium is a concentrated form of the growth medium and / or added in dry powder form. In some aspects, both a first liquid feed medium and a different second liquid feed medium can be added (e.g., continuously) to the growth medium. In some aspects, adding the first liquid feed medium to the culture and adding the second liquid feed medium to the culture can be initiated at approximately the same time. In some aspects, the total volume of the first liquid feed medium and the second liquid feed medium added to the culture over the entire culture period can be approximately the same.

[0243] When the feed medium is added continuously, the addition rate of the feed medium can remain constant or increase (e.g., increase steadily) during the culture period. The continuous addition of the feed medium can start at a specific time point during the culture period (e.g., when the cells reach a target viable cell density, such as about 1×10 6 cells / mL, about 1.1×10 6 cells / mL, about 1.2×10 6 cells / mL, about 1.3×10 6 cells / mL, about 1.4×10 6 cells / mL, about 1.5×10 6 cells / mL, about 1.6×10 6 cells / mL, about 1.7×10 6 cells / mL, about 1.8×10 6 cells / mL, about 1.9×10 6 cells / mL or about 2.0×10 6 cells / mL of viable cell density). In some aspects, the continuous addition of the feed medium can start on the 2nd, 3rd, 4th, or 5th day of the culture period.

[0244] In some aspects, the incremental (periodic) addition of the feed medium can be initiated when the cells reach a target cell density (e.g., about 1×10 6 cells / mL, about 1.1×10 6 cells / mL, about 1.2×10 6 cells / mL, about 1.3×10 6 cells / mL, about 1.4×10 6 cells / mL, about 1.5×106 cells / ml, about 1.6×10 6 cells / ml, about 1.7×10 6 cells / ml, about 1.8×10 6 cells / ml, about 1.9×10 6 cells / ml or about 2.0×10 6 cells / ml). In some aspects, the fed-batch medium addition can be carried out at regular time intervals (e.g., daily, every other day, or every three days), or can be carried out when the cells reach a specific target cell density (e.g., the target cell density increase during the culture period). In some aspects, the amount of the fed-batch medium added can be gradually increased between the first incremental addition of the fed-batch medium and subsequent additions of the fed-batch medium. In some aspects, the volume of the liquid culture fed-batch medium added to the initial cell culture within any 24-hour period of the culture period can be a certain proportion of the initial volume of the bioreactor containing the culture or a certain proportion of the initial culture volume.

[0245] In some aspects, the (continuous or periodic) addition of the liquid fed-batch medium can be carried out at a time point between 6 hours and 7 days, about 6 hours and about 6 days, about 6 hours and about 5 days, about 6 hours and about 4 days, about 6 hours and about 3 days, about 6 hours and about 2 days, about 6 hours and about 1 day, about 12 hours and about 7 days, about 12 hours and about 6 days, about 12 hours and about 5 days, about 12 hours and about 4 days, about 12 hours and about 3 days, about 12 hours and about 2 days, about 1 day and about 7 days, about 1 day and about 6 days, about 1 day and about 5 days, about 1 day and about 4 days, about 1 day and about 3 days, about 1 day and about 2 days, about 2 days and about 7 days, about 2 days and about 6 days, about 2 days and about 5 days, about 2 days and about 4 days, about 2 days and about 3 days, about 3 days and about 7 days, about 3 days and about 6 days, about 3 days and about 5 days, about 3 days and about 4 days, about 4 days and about 7 days, about 4 days and about 6 days, about 4 days and about 5 days, about 5 days and about 7 days, or about 5 days and about 6 days after the start of the culture period.

[0246] In some aspects, the volume of liquid feed medium added to the initial cell culture over any 24-hour period (continuous or periodic) can be between 0.01× and about 0.3× of the bioreactor capacity. This ratio can be between about 0.01× and about 0.28× of the bioreactor capacity, between about 0.01× and about 0.26×, between about 0.01× and about 0.24×, between about 0.01× and about 0.22×, between about 0.01× and about 0.20×, between about 0.01× and about 0.18×, between about 0.01× and about 0.16×, between about 0.01× and about 0.14×, between about 0.01× and about 0.12×, between about 0.01× and about 0.10×, between about 0.01× and about 0.08×, between about 0.01× and about 0.06×, between about 0.01× and about 0.04×, between about 0.02× and about 0.3×, between about 0.02× and about 0.28×, between about 0.02× and about 0.26×, between about 0.02× and about 0.24×, between about 0.02× and about 0.22×, between about 0.02× and about 0.20×, between about 0.02× and about 0.18×, between about 0.02× and about 0.16×, between about 0.02× and about 0.14×, between about 0.02× and about 0.12×, between about 0.02× and about 0.10×, between about 0.02× and about 0.08×, between about 0.02× and about 0.06×, between about 0.02× and about 0.05×, between about 0.02× and about 0.04×, between about 0.02× and about 0.03×, between about 0.025× and about 0.3×, between about 0.025× and about 0.28×, between about 0.025× and about 0.26×, between about 0.025× and about 0.24×, between about 0.025× and about 0.22×, between about 0.025× and about 0.20×, between about 0.025× and about 0.18×, between about 0.025× and about 0.16×, between about 0.025× and about 0.14×, between about 0.025× and about 0.12×, between about 0.025× and about 0.10×, between about 0.025× and about 0.08×, between about 0.025× and about 0.06×, between about 0.025× and about 0.04×, between about 0.05× and about 0.3×, between about 0.05× and about 0.28×, between about 0.05× and about 0.26×, between about 0.05× and about 0.24×, between about 0.05× and about 0.22×, between about 0.05× and about 0.20×, between about 0.05× and about 0.18×, between about 0.05× and about 0.16×, between about 0.05× and about 0.14×, between about 0.05× and about 0.12×, between about 0.05× and about 0.10×, between about 0.1× and about 0.3×, between about 0.1× and about 0.28×, between about 0.1× and about 0.Between about 0.1× and about 0.24×, between about 0.1× and about 0.22×, between about 0.1× and about 0.20×, between about 0.1× and about 0.18×, between about 0.1× and about 0.16×, between about 0.1× and about 0.14×, between about 0.1× and about 0.1×, between about 0.15× and about 0.3×, between about 0.15× and about 0.2×, between about 0.2× and about 0.3×, or between about 0.25× and about 0.3×.

[0247] In some aspects, the volume of the liquid feed medium added to the initial cell culture during any 24-hour period (continuous or periodic) during cultivation can be between 0.02× and about 1.0×, between about 0.02× and about 0.9×, between about 0.02× and about 0.8×, between about 0.02× and about 0.7×, between about 0.02× and about 0.6×, between about 0.02× and about 0.5×, between about 0.02× and about 0.4×, between about 0.02× and about 0.3×, between about 0.02× and about 0.2×, between about 0.02× and about 0.1×, between about 0.02× and about 0.08×, between about 0.02× and about 0.06×, between about 0.02× and about 0.05×, between about 0.02× and about 0.04×, between about 0.02× and about 0.03×, between about 0.05× and about 1.0×, between about 0.05× and about 0.8×, between about 0.05× and about 0.7×, between about 0.05× and about 0.6×, between about 0.05× and about 0.5×, between about 0.05× and about 0.4×, between about 0.05× and about 0.3×, between about 0.05× and about 0.2×, between about 0.05× and about 0.1×, between about 0.1× and about 1.0×, between about 0.1× and about 0.9×, between about 0.1× and about 0.8×, between about 0.1× and about 0.7×, between about 0.1× and about 0.6×, between about 0.1× and about 0.5×, between about 0.1× and about 0.4×, between about 0.1× and about 0.3×, between about 0.1× and about 0.2×, between about 0.2× and about 1.0×, between about 0.2× and about 0.9×, between about 0.2× and about 0.8×, between about 0.2× and about 0.7×, between about 0.2× and about 0.6×, between about 0.2× and about 0.5×, or between about 0.2× and about 0.4× of the initial cell culture volume.

[0248] In some aspects, the total amount of feed medium added during the entire cultivation period (continuously or periodically) can be between about 1% and about 40% of the initial culture volume (e.g., between about 1% and about 35%, between about 1% and about 30%, between about 1% and about 25%, between about 1% and about 20%, between about 1% and about 15%, between about 1% and about 10%, between about 1% and about 5%, between about 1% and about 4%, between about 2% and about 40%, between about 2% and about 35%, between about 2% and about 30%, between about 2% and about 25%, between about 2% and about 20%, between about 2% and about 15%, between about 2% and about 10%, between about 2% and about 5%, between about 3% and about 40%, between about 3% and about 35%, between about 3% and about 30%, between about 3% and about 25%, between about 3% and about 20%, between about 3% and about 15%, between about 3% and about 10%, between about 3% and about 5%, between about 4% and about 40%, between about 4% and about 35%, between about 4% and about 30%, between about 4% and about 25%, between about 4% and about 20%, between about 4% and about 15%, between about 4% and about 10%, between about 4% and about 8%, between about 5% and about 40%, between about 5% and about 35%, between about 5% and about 30%, between about 5% and about 25%, between about 5% and about 20%, between about 5% and about 15%, between about 5% and about 10%, between about 10% and about 40%, between about 10% and about 35%, between about 10% and about 30%, between about 10% and about 25%, between about 10% and about 20%, between about 10% and about 15%, between about 15% and about 40%, between about 15% and about 35%, between about 15% and about 30%, between about 15% and about 25%, between about 15% and about 20%, between about 20% and about 40%, between about 20% and about 35%, between about 20% and about 30%, between about 20% and about 25%, between about 25% and about 40%, between about 25% and about 35%, between about 25% and about 30%, between about 30% and about 40%, between about 30% and about 35%, or between about 35% and about 40%).

[0249] In some aspects, two different feed media are added during fed-batch cultivation (continuously or incrementally). In some aspects, the amounts or volumes of the first and second feed media added can be substantially the same or can be different. In some aspects, the first feed medium can be in liquid form and the second feed medium can be in solid form. In some aspects, the first and second feed media can be liquid feed media.

[0250] Perfusion culture involves removing a first volume of growth medium from a bioreactor and adding a second volume of a second growth medium to the production bioreactor, where the first volume and the second volume are substantially equal. Cells are retained in the bioreactor by a cell retention device or by techniques such as cell sedimentation in a settling cone. In some aspects, the removal and addition of the growth medium can be performed simultaneously, sequentially, or in some combination of both. In some aspects, the removal and addition can be performed continuously, such as at a rate of removing and replacing a volume that is between 0.1% and 800%, between 1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and 400%, between 1% and 350%, between 1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and 200%, between 5% and 150%, between 10% and 50%, between 15% and 40%, between 8% and 80%, or between 4% and 30% of the bioreactor capacity.

[0251] In some aspects, the first volume of the first growth medium removed and the second volume of the second growth medium added can remain substantially the same within each 24-hour period. In some aspects, the rate (volume per unit time) of removing the first volume of the first growth medium and the rate (volume per unit time) of adding the second volume of the second growth medium can vary and depend on the conditions of a particular cell culture system. In some aspects, the rate (volume per unit time) of removing the first volume of the first growth medium and the rate (volume per unit time) of adding the second volume of the second growth medium can be substantially the same or can be different.

[0252] In some aspects, the volume of removal and addition can vary by gradually increasing within each 24-hour period. In some aspects, the volume of the first growth medium removed and the volume of the second growth medium added within each 24-hour period can increase during the culture period. In some aspects, the volume can increase by an amount between 0.5% and about 20% of the bioreactor capacity within a 24-hour period. In some aspects, the volume can increase during the culture period to an amount that is about 25% to about 150% of the bioreactor capacity or the volume of the first liquid medium within a 24-hour period.

[0253] In some aspects, after the first 48 to 96 hours of the culture period, within each 24-hour period, the first volume of the first growth medium removed and the second volume of the second growth medium added are about 10% to about 95%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 85% to about 95%, about 60% to about 80%, or about 70% of the volume of the first growth medium.

[0254] In some aspects, the first growth medium and the second growth medium can be the same type of medium. In some aspects, the first growth medium and the second growth medium can be different. In some aspects, the second liquid medium can be more concentrated with respect to one or more medium components.

[0255] In some aspects, the first volume of the first growth medium can be removed by using any automated system. In some aspects, alternating tangential flow filtration can be used. In some aspects, the first volume of the first growth medium can be removed by having the first volume of the first growth medium permeate or flow by gravity through a sterile membrane having a molecular weight cut-off that excludes the cells. In some aspects, the first volume of the first growth medium can be removed by stopping or significantly reducing the agitation rate for at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 50 minutes, or 1 hour and removing or aspirating the first volume of the growth medium from the top of the production bioreactor.

[0256] In some aspects, the second volume of the second liquid medium can be added to the first liquid medium by a pump. In some aspects, the second liquid medium can be added manually to the first liquid medium, such as by directly pipetting or injecting the second volume of the second liquid medium onto the first liquid medium, or added in an automated manner.

[0257] In some aspects, the method further includes contacting the cells with a first polynucleotide sequence. In some aspects, the method further includes transfecting the cells with a polynucleotide sequence. In some aspects, the polynucleotide sequence is a plasmid. In some aspects, the plasmid encodes a capsid of a recombinant viral particle selected from: AAV, lentivirus, herpesvirus, polyomavirus, and vaccinia virus. In some aspects, the cells are transfected before being inoculated into the bioreactor. In some aspects, the cells are transfected after being inoculated into the bioreactor. In some aspects, the cells are contacted with or transfected with a second polynucleotide and contain a nucleic acid encoding a transgene. In some aspects, the cells are cultured under conditions for producing a viral vector. In some aspects, the method further includes isolating the produced viral vector.

[0258] In some aspects, the polynucleotide is a viral vector. In some aspects, the viral vectors are adenovirus and adeno-associated virus (AAV) vectors. These vectors infect a wide range of dividing and non-dividing cell types, including synoviocytes and hepatocytes. The episomal nature of adenovirus and AAV vectors after cell entry makes these vectors suitable for therapeutic applications (Russell, J. Gen. Virol. 81: 2573-2604 (2000)); Goncalves, Virol J. 2(1):43 2005)), as shown above. AAV vectors can result in very stable long-term expression of the transgene (up to 9 years in dogs (Niemeyer et al., Blood 113(4):797-806 (2009)), up to 2 years in humans (Nathwani et al., N Engl J Med. 365(25): 2357-2365 (2011); Simonelli et al., Mol Ther. 18(3):643-50 (2010), electronic edition (Epub), December 1, 2009)). In some aspects, adenovirus vectors are modified to reduce the host response, as reviewed by Russell (2000, ibid.). Methods of using AAV vectors for gene therapy are described in: Wang et al., 2005, J GeneMed. March 9 (electronic edition before print); Mandel et al., Curr Opin Mol Ther. 6:482-90 (2004); Martin et al., Eye 18:1049-55 (2004); Nathwani et al., N Engl J Med. 22; 365:2357-65 (2011); and Apparailly et al., Hum Gene Ther. 16(4):426-34 (2005).

[0259] In some aspects, the first polynucleotide sequence comprises one or more of the following: inverted terminal repeats, a nucleic acid encoding at least one AAV replication protein, a nucleic acid encoding at least one AAV packaging protein, a nucleic acid encoding at least one AAV structural capsid protein, or a combination thereof.

[0260] In some aspects, the cells are cultured under conditions for producing recombinant virus particles. In some aspects, the method further comprises isolating the produced recombinant virus particles.

[0261] In some aspects, the viral vector comprises a transgene operably linked to appropriate regulatory sequences. The term "regulatory sequences" includes promoters, enhancers and other expression control elements (such as polyadenylation signals) that control the transcription or translation of a protein. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology, Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). In some aspects, the regulatory sequences may comprise a promoter sequence. In some aspects, the promoter sequence may be the cytomegalovirus (CMV) immediate early promoter, a viral long terminal repeat promoter (LTR) (such as the promoter from Moloney murine leukemia virus (MMLV), Rous sarcoma virus or HTLV-1), the simian virus 40 (SV 40) early promoter or the herpes simplex virus thymidine kinase promoter.

[0262] In some aspects, the viral vector comprises another nucleotide sequence encoding another polypeptide. In some aspects, the another polypeptide may be a (selectable) marker polypeptide that allows the identification, selection and / or screening of cells containing the viral vector. In some aspects, the marker polypeptide may be a fluorescent protein GFP and selectable marker genes such as HSV thymidine kinase (for HAT medium selection), bacterial hygromycin B phosphotransferase (for hygromycin B selection), Tn5 aminoglycoside phosphotransferase (for G418 selection) and dihydrofolate reductase (DHFR) (for methotrexate selection), CD20, low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are provided in Sambrook and Russel (2001), "Molecular Cloning: A Laboratory Manual" (3rd Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.

[0263] Methods for treating muscular dystrophy (e.g., DMD)

[0264] Some aspects of the present invention relate to a method for producing a viral vector (such as the rAAV described herein), which comprises: amplifying cells according to any of the inoculation chain amplification methods described herein; inoculating a growth medium in a bioreactor with the cells; transfecting the cells with a polynucleotide sequence encoding viral particles; and culturing the cells in the bioreactor under conditions for producing viral particles. In some aspects, the production of viral vectors is disclosed in U.S. Application No. 63 / 123,602, which is hereby incorporated by reference in its entirety.

[0265] Methods for introducing exogenous nucleic acids into host cells are well known in the art and vary with the host cells used. Techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polyethyleneimine-mediated transfection, polybrene-mediated transfection, protoplast fusion, electroporation, viral or phage infection, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Transfection can be transient or stable.

[0266] In some aspects, the polynucleotide sequence is a plasmid. In some aspects, the plasmid encodes viral particles from AAV.

[0267] In some aspects, the polynucleotide sequence is a viral vector. In some aspects, the viral vector encodes viral particles. In preferred aspects, the viral particles are from AAV. In some aspects, the rAAV contains the nucleic acid sequence of SEQ ID NO:9. In some aspects, the present disclosure provides rAAV particles containing the nucleic acid sequence of SEQ ID NO:9. In some aspects, the present disclosure provides rAAV containing nucleotides 55 - 5021 of SEQ ID NO:3. In some aspects, the present disclosure provides rAAV particles containing nucleotides 55 - 5021 of SEQ ID NO:3. In some aspects, the rAAV contains nucleotides 1 - 4988 of SEQ ID NO:8. In some aspects, the present disclosure provides rAAV particles containing nucleotides 1 - 4977 of SEQ ID NO:8.

[0268] In some aspects, the viral vector is an AAV vector. In some aspects, the AAV vector can comprise a recombinant AAV vector (rAAV). As used herein, an "rAAV vector" refers to a recombinant vector comprising a portion of the AAV genome encapsulated in a protein shell containing a capsid protein derived from a serotype of AAV as described herein. In some aspects, the AAV vector can comprise inverted terminal repeats (ITRs) derived from an adeno-associated virus serotype (such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVRH10, AAV11, AAV12, and others).

[0269] Typically, the vector genome requires the use of flanking 5' and 3' ITR sequences to allow efficient packaging of the vector genome into the rAAV capsid. In some aspects, the rAAV genome present in the rAAV vector comprises at least the nucleotide sequence of the inverted terminal repeat region (ITR) of a serotype of AAV or a nucleotide sequence substantially identical thereto, and a nucleic acid sequence encoding a transgene under the control of a suitable regulatory element (e.g., a promoter), wherein the regulatory element and the modified nucleic acid sequence are inserted between the two ITRs.

[0270] The complete genomes of several AAV serotypes and the corresponding ITRs have been sequenced (Chiorini et al., J. of Virology 73: 1309-1319 (1999)). They can be prepared by chemical synthesis as known in the art, e.g., using an oligonucleotide synthesizer such as those provided by Applied Biosystems Inc. (Fosters, Calif., USA), or by cloning or preparation by standard molecular biology techniques. The ITRs can be cloned from the AAV viral genome or excised from a vector containing the AAV ITR. The ITR nucleotide sequence can be ligated to the nucleotide sequence encoding one or more therapeutic proteins at either end using standard molecular biology techniques, or the wild-type AAV sequence between the ITRs can be replaced with the desired nucleotide sequence.

[0271] In some aspects, the viral capsid component of the packaged viral vector can be a parvovirus capsid, such as an AAV Cap and / or a chimeric capsid. Examples of suitable parvovirus capsid components are capsid components from the family Parvoviridae, such as autonomous parvoviruses or the genus Dependovirus. For example, the viral capsid can be an AAV capsid (e.g., an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVRH8, AAV9, AAV10, AAVRH10, AAV11 or AAV12 capsid; those skilled in the art will appreciate that there may be other as yet unidentified variants performing the same or similar functions), or can comprise components from two or more AAV capsids. The complete components of the AAV Cap protein include VP1, VP2, and VP3. The ORF containing the nucleotide sequence encoding the AAV VP capsid protein can include less than all of the AAV Cap protein components, or can provide all of the AAV Cap protein components.

[0272] In some aspects, one or more of the AAV Cap proteins can be chimeric proteins, including AAV Cap amino acid sequences from two or more viruses, preferably two or more AAVs. For example, a chimeric viral capsid can include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. In some aspects, the rAAV genome present in an rAAV vector does not contain any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. In some aspects, the rAAV genome can also contain a marker or reporter gene, such as a gene encoding an antibiotic resistance gene, a fluorescent protein (e.g., gfp), or a gene encoding a product that can be detected and / or selected chemically, enzymatically, or otherwise known in the art (e.g., lacZ, aph, etc.).

[0273] In some aspects, the rAAV genome present in the rAAV vector can also contain a promoter sequence that is operably linked to a nucleotide sequence encoding a transgene.

[0274] In some aspects, a suitable 3' untranslated sequence can also be operably linked to the modified nucleic acid sequence encoding the transgene. A suitable 3' untranslated region can be a region naturally associated with the nucleotide sequence or can be derived from a different gene, such as the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal and enhancer sequence).

[0275] Unless otherwise noted, methods known to those skilled in the art can be used to construct recombinant parvovirus and AAV (rAAV) constructs, packaging vectors expressing parvovirus Rep and / or Cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor, N.Y., 1989); AUSUBEL et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

[0276] In some aspects, a suitable 3' untranslated sequence can also be operably linked to the nucleic acid sequence encoding the transgene. A suitable 3' untranslated region can be a region naturally associated with the nucleotide sequence or can be derived from a different gene, such as the bovine growth hormone 3' untranslated region (e.g., bGH polyadenylation signal, SV40 polyadenylation signal, SV40 polyadenylation signal and enhancer sequence).

[0277] In some aspects, additional nucleotide sequences can be operably linked to a nucleic acid sequence encoding a transgene, such as nucleotide sequences encoding signal sequences, nuclear localization signals, expression enhancers, and the like.

[0278] Unless otherwise stated, methods known to those skilled in the art can be used to construct lentiviral constructs, vectors, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor, N.Y., 1989); AUSUBEL et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley Sons, Inc., New York).

[0279] In some aspects, the methods according to the present disclosure include transfecting cells with a transgenic plasmid comprising an rAAVrh74.MHCK7.minidystrophin construct, a plasmid comprising an AAV rep gene and an AAV cap gene, and an adenovirus helper plasmid. In some aspects, the transgenic plasmid comprising the rAAVrh74.MHCK7.minidystrophin construct comprises: the nucleic acid sequence of SEQ ID NO:9; nucleotides 55-5021 of SEQ ID NO:3; or nucleotides 1-4977 of SEQ ID NO:8. In some aspects, the plasmid comprising the AAV rep gene and the AAV cap gene comprises an AAV2 rep gene and an rAAVrh74 cap gene. In some aspects, the adenovirus helper plasmid comprises adenovirus 5 E2A, E4 ORF6, and VA RNA genes.

[0280] In some aspects, the method further includes isolating the resulting viral particles. The viral vector replicates within the cell and thus amplifies and produces viral particles. Viral infection causes lysis of the transfected cells. Thus, the lytic characteristics of viral vectors, such as AAV, allow for two different modes of viral particle production and isolation. The first mode is to collect the viral particles by lysing the cells with an exogenous factor before cell lysis. The second mode is to collect the viral particles from the supernatant after the cells are almost completely lysed by the produced virus.

[0281] Methods for active cell lysis are known to those skilled in the art. In some aspects, cells can be lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high-pressure extrusion, detergent lysis, or a combination of the above.

[0282] In some aspects, cells can be lysed using at least one detergent. In some aspects, the detergents can include anionic, cationic, zwitterionic, and nonionic detergents. In some aspects, the concentration of the detergent can be about 0.1%-5% (w / w). In some aspects, the detergent can be Triton X-100.

[0283] In some aspects, nucleases can be used to remove contaminating nucleic acids, i.e., native nucleic acids from transfected cells. In some aspects, the nuclease can be or any other DNase and / or RNase commonly used in the art.

[0284] Methods for collecting or isolating viral vectors from transfected cells have been widely disclosed in WO 2005 / 080556, which is incorporated herein by reference in its entirety.

[0285] In some aspects, the time for collecting or isolating the viral vector is between about 24 and 120 hours after transfection, between about 36 and 108 hours after transfection, between about 48 and about 96 hours after transfection, between about 60 and about 84 hours after transfection. In some aspects, the time for collecting or isolating the vector is about 72 hours after transfection.

[0286] In some aspects, the isolated viral particles can be further purified. In some aspects, the purification of viral particles can be carried out in several steps including clarification, ultrafiltration, diafiltration, or separation using chromatography. Such methods are described in WO 2005 / 080556, which is incorporated herein by reference in its entirety. In some aspects, clarification can be carried out by removing cell debris and other impurities from the cell lysate through a filtration step. In some aspects, ultrafiltration is used to concentrate the viral solution. In some aspects, diafiltration, buffer exchange, or ultrafiltration can be used to remove and exchange salts, sugars, etc. Those skilled in the art know how to find the optimal conditions for various purification steps.

[0287] In some aspects, purification can be achieved by density gradient centrifugation. In some aspects, at least one chromatography step is employed. In some aspects, the viral vector can be purified by anion exchange chromatography, size exclusion chromatography, or a combination thereof.

[0288] Glucocorticoid

[0289] The present disclosure provides a method for treating a human subject in need of treatment for muscular dystrophy, which includes the step of administering recombinant adeno-associated virus (rAAV) rAAV.MHCK7.minidystrophin, wherein the rAAV is administered via a systemic administration route at about 5.0×10 12 vg / kg to about 1.0×1015 administered at a dose of vg / kg, and wherein the rAAV is produced in mammalian adherent cells, and wherein the adherent cells are cultured under suspension conditions in an N-1 container. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the rAAV is administered by the systemic administration route at a dose of about 1.33×10 14 vg / kg. In some aspects, the rAAV is administered by intravenous (IV) infusion at a dose of about 1.33×10 14 vg / kg. In some aspects, the rAAV is administered by the systemic route as a single infusion. In some aspects, the rAAV is administered by the systemic route as multiple infusions. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy. The terms "human subject" and "human patient" are used interchangeably herein.

[0290] The present disclosure also provides a method of treating muscular dystrophy in a human subject in need thereof, comprising administering to the human subject a composition comprising the rAAV described herein. In some aspects, the composition is prepared by the method described herein. In some aspects, the rAAV is used by the systemic administration route and at a dose of about 5.0×10 12 vg / kg to about 1.0×10 15 vg / kg. In some aspects, the systemic administration route is the intravenous route, and the administration dose of the rAAV is about 2×10 14 vg / kg. In some aspects, the systemic administration route is the intravenous route, and the administration dose of the rAAV is about 1.33×10 14 vg / kg.

[0291] In some aspects, the rAAV is administered to a human subject weighing less than 70 kg. In some aspects, the rAAV is administered to a human subject weighing more than 70 kg. In some aspects, the rAAV is administered by the intravenous route as a single dose of about 1.33×10 14 vg / kg to a human subject weighing less than 70 kg. In some aspects, the rAAV is administered by the intravenous route as a total fixed dose of about 9.31×10 15 vg to a human subject weighing more than 70 kg.

[0292] In some aspects, the dose of the rAAV is administered at a concentration of about 10 mL / kg. In some aspects, the rAAV is administered by injection, infusion or implantation. In some aspects, the rAAV is administered by infusion within about 1 hour. In some aspects, the rAAV is administered by the intravenous route via a peripheral limb vein.

[0293] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0294] In some aspects, the level of micro-dystrophin gene expression in the cells of the subject increases after administration of rAAV as compared to the level of micro-dystrophin gene expression before administration of rAAV. In some aspects, the level of micro-dystrophin gene expression in the muscle cells of the subject increases after administration of rAAV as compared to the level of micro-dystrophin gene expression before administration of rAAV. In some aspects, the level of micro-dystrophin gene expression in the cardiomyocytes of the subject increases after administration of rAAV as compared to the level of micro-dystrophin gene expression before administration of rAAV.

[0295] In some aspects, the expression of the micro-dystrophin gene in the cells is detected by measuring the level of micro-dystrophin in the western blot of the muscle biopsied before and after administration of rAAV. In some aspects, the expression after administration of rAAV is at least 55.4% as compared to that before administration.

[0296] In some aspects, the average percentage of micro-dystrophin-positive fibers in the muscle tissue of the subject increases after administration of rAAV as compared to the number of micro-dystrophin-positive fibers before administration of rAAV. In some aspects, the average percentage of micro-dystrophin-positive fibers is at least 70.5% and the average intensity is at least 116.9%, as detected by immunofluorescence (IF) in muscle biopsies before and after administration of rAAV. In some aspects, the micro-dystrophin transduction counted by the vector genome is at least 3.87 average vector genome copies per cell nucleus.

[0297] In some aspects, the average percentage of centralized nuclei-positive fibers in the muscle tissue of the subject increases after administration of rAAV as compared to the number of centralized nuclei-positive fibers before administration of rAAV.

[0298] In some aspects, the average percentage of micro-dystrophin-positive fibers in the cardiac tissue of the subject increases after administration of rAAV as compared to the number of micro-dystrophin-positive fibers before administration of rAAV.

[0299] Pre - infusion glucocorticoid

[0300] In some aspects, prior to rAAV treatment, subjects to be treated with rAAV as described herein receive a stable weekly (e.g., daily or every weekend, rather than 10 doses / 10 withdrawals) dose equivalent of oral glucocorticoids for at least 12 weeks. In some aspects, after rAAV administration, the glucocorticoid dose remains unchanged (except for modifications to account for weight changes). In some aspects, subjects to be treated with rAAV as described herein are non-ambulatory subjects and the subjects do not receive glucocorticoids. In some aspects, subjects to be treated with rAAV as described herein may not require long-term use of steroids to treat their DMD.

[0301] Post - infusion glucocorticoid

[0302] In some aspects, prior to treatment with rAAV as described herein, subjects to be treated with rAAV as described herein are also treated with an oral glucocorticoid (prednisone or prednisolone) at 1 mg / kg / day for immunosuppression in addition to the baseline stable oral glucocorticoids for DMD that the subjects receive.

[0303] In some aspects, subjects to be treated with rAAV as described herein do not receive steroids for DMD and will begin receiving an oral glucocorticoid (prednisone or prednisolone) at 1.5 mg / kg / day for immunosuppression 1 week prior to rAAV infusion.

[0304] Genotyping of the DMD gene in a subject

[0305] In some aspects, subjects to be treated with rAAV as described herein maintain their baseline stable oral glucocorticoid dose for DMD and additionally take an oral glucocorticoid (prednisone or prednisolone) at 1 mg / kg / day. In some aspects, the subjects additionally take an oral glucocorticoid at 1 mg / kg / day for the first 60 days after rAAV infusion.

[0306] In some aspects, subjects to be treated with rAAV as described herein have not been previously treated with glucocorticoids, begin receiving an oral glucocorticoid at 1.5 mg / kg / day prior to rAAV treatment and continue to receive a dose of 1.5 mg / kg / day of an oral glucocorticoid for immunosuppression for the first 60 days after rAAV infusion.

[0307] In some aspects, if the GGT level is confirmed to be ≥150 U / L after infusion or there are other clinically significant liver function abnormalities, the oral glucocorticoid dose is adjusted. In some aspects, if the subject is receiving an additional 1 mg / kg of steroid for immunosuppression in addition to their DMD steroid, this dose can be increased to 2 mg / kg of additional steroid for immunosuppression in addition to their DMD steroid for administration. In some aspects, if the subject is receiving a fixed dose of 60 mg / day, this dose can be increased to 120 mg / day. In some aspects, if the subject is receiving 1.5 mg / kg / day for immunosuppression, this dose can be increased to 2.5 mg / kg / day.

[0308] In some aspects, if the subject experiences a severe or dramatic elevation of liver biochemistry (including GGT, bilirubin, and ALT relative to baseline), or the elevation does not respond to an increase in oral steroids, the subject will receive an intravenous (IV) bolus of steroid.

[0309] Figure 1

[0310] In some aspects, at least one mutation in the DMD gene of the subject has been genotyped. In some aspects, the human dystrophin (DMD) gene of the subject is genotyped before treatment (i.e., before administration of the rAAV described herein). In some aspects, at least one mutation in exons 18 - 79 of the subject's DMD gene is genotyped. In some aspects, at least one mutation is a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the absence of expression of human dystrophin. In some aspects, detection of at least one of these mutations in the DMD gene indicates that the composition described herein can be administered to the subject.

[0311] In some aspects, genotyping of the subject's DMD gene identifies that rAAV gene therapy as described herein should be contraindicated. For example, a subject with a deletion that completely encompasses exons 9 - 13 of the DMD gene should be contraindicated for rAAV gene therapy. In another example, a subject with a deletion in exon 8 and / or 9 of the DMD gene (i.e., any deletion) should be contraindicated for rAAV gene therapy.

[0312] In some aspects, at least one mutation in the DMD gene is a mutation in exons 1 - 17, in-frame deletion, in-frame duplication, variant of unknown significance (VUS), or a mutation that is completely contained within exon 45, thereby identifying the subject as not suitable for administration of the composition described herein.

[0313] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles, the AAV viral particles encapsulating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion that completely encompasses exons 9-13 of the DMD gene.

[0314] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles, the AAV viral particles encapsulating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion in exon 8 and / or exon 9 of the DMD gene.

[0315] In some aspects, the DMD gene of the subject is genotyped prior to treatment.

[0316] In some aspects, the AAV viral particles are rh74 serotype AAV viral particles.

[0317] In some aspects, the rAAV vector is administered as a composition comprising: a) rh74 serotype AAV viral particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rh74 serotype AAV viral particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rh74 serotype AAV viral particles encapsulating nucleotides 1-4977 of SEQ ID NO:8.

[0318] The present disclosure also provides a method of treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) administering to the subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin, provided that genotyping does not identify a deletion that completely encompasses exons 9-13 of the DMD gene; wherein the composition comprises: a) rAAV particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsulating nucleotides 1-4977 of SEQ ID NO:8.

[0319] The present disclosure also provides a method for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) administering to the subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, provided that the genotyping does not identify a deletion in exon 8 and / or 9 of the DMD gene; wherein the composition comprises: a) rAAV particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsulating nucleotides 1-4977 of SEQ ID NO:8.

[0320] In some aspects, the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin administered to the subject is prepared by any of the methods described herein.

[0321] The present disclosure also provides the use of the composition described herein for treating muscular dystrophy in a human subject in need thereof. In some aspects, the present disclosure also provides the use of the composition described herein in the preparation of a medicament for treating muscular dystrophy.

[0322] In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy.

[0323] For example, the administration dose of rAAV is about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 5.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 13 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15vg / kg, or 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or approximately 1.0×10 13 vg / kg to 1.0×10 14 vg / kg, or approximately 1.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or approximately 1.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or approximately 1.0×10 13 vg / kg to approximately 5.0×10 13 vg / kg, or approximately 1.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.0×10 13 vg / kg to approximately 5.0×10 14 vg / kg, or approximately 1.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or 1.0×10 13 vg / kg to approximately 1.0×10 15 vg / kg, or 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or approximately 5.0×10 13 vg / kg to 1.0×10 14 vg / kg, or approximately 5.0×10 13 vg / kg to approximately 2.0×10 14 vg / kg, or approximately 5.0×10 13 vg / kg to approximately 1.0×10 14 vg / kg, or approximately 5.0×10 13 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 5.0×10 13 vg / kg to approximately 5.0×10 14 vg / kg, or approximately 5.0×10 13 vg / kg to approximately 6.0×10 14 vg / kg, or 5.0×10 13 vg / kg to approximately 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to approximately 6.0×10 14 vg / kg, or 1.0×10 14 vg / kg to approximately 5.0×10 14vg / kg, or 1.0×10 14 vg / kg to approximately 4.0×10 14 vg / kg, or 1.0×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.0×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or 1.0×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 6.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 5.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 4.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 1.0×10 15 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or 1.5×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or approximately 1.5×10 14 vg / kg to 6.0×1014 vg / kg, or about 1.5×10 14 vg / kg to 5.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to 4.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.25×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.0×10 14 vg / kg, or 1.75×10 14 vg / kg to about 1.0×10 15 vg / kg, or about 1.75×10 14 vg / kg to 6.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to 5.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to 4.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.25×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.75×10 14vg / kg to about 2.5×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.25×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to 1.0×10 15 vg / kg, or about 2.0×10 14 vg / kg to 6.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to 5.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.25×10 14 vg / kg.

[0324] In some aspects, the human subject is between about 2 years and less than 3 years old. In some aspects, the human subject is about 2 years old, about 2.25 years old, about 2.5 years old, or about 2.75 years old.

[0325] In some aspects, the human subject is 2 to 3 years old.

[0326] In some aspects, the human subject is 2 years old. In some aspects, the human subject is 3 years old. In some aspects, the human subject is between about 4 years and less than 8 years old. In some aspects, the human subject is about 4 years old, about 4.25 years old, about 4.5 years old, about 4.75 years old, about 5 years old, about 5.25 years old, about 5.5 years old, about 5.75 years old, about 6 years old, about 6.25 years old, about 6.5 years old, about 6.75 years old, about 7 years old, about 7.25 years old, about 7.5 years old, or about 7.75 years old.

[0327] In some aspects, the human subject is 4 to 5 years old.

[0328] In some aspects, the human subject is between about 8 years old and less than 18 years old. In some aspects, the human subject is about 8 years old, about 8.25 years old, about 8.5 years old, about 8.75 years old, about 9 years old, about 9.25 years old, about 9.5 years old, about 9.75 years old, about 10 years old, about 10.25 years old, about 10.5 years old, about 10.75 years old, about 11 years old, about 11.25 years old, about 11.5 years old, about 11.75 years old, about 12 years old, about 12.25 years old, about 12.5 years old, about 12.75 years old, about 13 years old, about 13.25 years old, about 13.5 years old, about 13.75 years old, about 14 years old, about 14.5 years old, about 14.75 years old, about 15 years old, about 15.25 years old, about 15.5 years old, about 15.75 years old, about 16 years old, about 16.25 years old, about 16.5 years old, about 16.75 years old, about 17 years old, about 17.25 years old, about 17.5 years old or about 17.75 years old.

[0329] In one aspect, the method of the present disclosure includes systemic administration of rAAV, wherein the systemic administration route is the intravenous route, and the administration dose of rAAV is about 2.0×10 14 vg / kg. In some aspects, the method of the present disclosure includes systemic administration of rAAV, wherein the systemic administration route is the intravenous route, and the administration dose of rAAV is about 1.33×10 14 vg / kg. In another aspect, the method of the present disclosure includes systemic administration of rAAV, wherein the systemic administration route is the intravenous route, and the administration dose of rAAV is about 5.0×10 12 vg / kg, or about 6.0×10 12 vg / kg, or about 7.0×10 12 vg / kg, or about 8.0×10 12 vg / kg, or about 9.0×10 12 vg / kg, or about 1.0×10 13 vg / kg, or about 1.25×10 13 vg / kg, or about 1.5×10 13 vg / kg, or about 1.75×10 13 vg / kg, or about 2.25×10 13 vg / kg, or about 2.5×10 13 vg / kg, or about 2.75×10 13 vg / kg, or about 3.0×10 13 vg / kg, or about 3.25×10 13 vg / kg, or about 3.5×10 13 vg / kg, or about 3.75×10 13 vg / kg, or about 4.0×10 13vg / kg, or about 5.0×10 13 vg / kg, or about 6.0×10 13 vg / kg, or about 7.0×10 13 vg / kg, or about 8.0×10 13 vg / kg, or about 9.0×10 13 vg / kg, or about 1.0×10 14 vg / kg, or about 1.33×10 14 vg / kg, or about 1.25×10 14 vg / kg, or about 1.5×10 14 vg / kg, or about 1.75×10 14 vg / kg, or about 2.25×10 14 vg / kg, or about 2.5×10 14 vg / kg, or about 2.75×10 14 vg / kg, or about 3.0×10 14 vg / kg, or about 3.25×10 14 vg / kg, or about 3.5×10 14 vg / kg, or about 3.75×10 14 vg / kg, or about 4.0×10 14 vg / kg, or about 5.0×10 14 vg / kg, or about 6.0×10 14 vg / kg, or about 1×10 15 vg / kg. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin or AAVrh74.MCK.minidystrophin. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0330] In any of the methods of the present disclosure, the dose of rAAV can be administered at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg, or 10 mL / kg to about 20 mL / kg. In a particular aspect, the dose of rAAV is administered at about 10 mL / kg. In one aspect, rAAV is AAVrh74.MHCK7.minidystrophin or AAVrh74.MCK.minidystrophin. In one aspect, rAAV is AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, nucleotides 56 - 5022 of SEQ ID NO:6, or SEQ ID NO:9. In one aspect, rAAV is AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0331] In any of the methods of the present disclosure, the dose of rAAV can be administered by injection, infusion, or implantation. For example, the dose of rAAV is administered by infusion within about 1 hour. Additionally, the dose of rAAV is administered by the intravenous route via a peripheral limb vein (such as a peripheral arm vein or a peripheral leg vein). Alternatively, the infusion can be administered within about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours. In one aspect, rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, AAVrh74.MHCK7.minidystrophin is AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, nucleotides 56 - 5022 of SEQ ID NO:6, or SEQ ID NO:9. In one aspect, rAAV is AAVrh74.MCK.minidystrophin. In one aspect, AAVrh74.MCK.minidystrophin is AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0332] rAAV administered by any of the methods of the present disclosure may comprise the human micro-dystrophin nucleotide sequence of SEQ ID NO:1, the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7. In addition, rAAV administered by any of the methods of the present disclosure comprises the human micro-dystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7. For example, the rAAV may comprise the AAVrh74.MHCK7.micro-dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MHCK7.micro-dystrophin. In one aspect, the AAVrh74.MHCK7.micro-dystrophin is the AAVrh74.MHCK7.micro-dystrophin of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0333] In one aspect, the rAAV is AAVrh74.MCK.micro-dystrophin. In one aspect, the AAVrh74.MCK.micro-dystrophin is the AAVrh74.MCK.micro-dystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0334] In any of the methods of the present disclosure, the administered rAAV is of the AAVrh74 serotype rAAV.

[0335] In some aspects, the methods of the present disclosure treat Duchenne muscular dystrophy or Becker muscular dystrophy. An exemplary aspect is a method of treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, comprising the step of administering a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.micro-dystrophin, wherein the administration route is intravenous infusion and the administration dose of rAAV is about 2×10 14vg / kg, and wherein the rAAV vector comprises the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct of nucleotides 55 - 5021 of SEQ ID NO:9 or SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of SEQ ID NO:9, or nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0336] In any method of treating muscular dystrophy, after administration of the rAAV, the level of minidystrophin gene expression in the cells of the subject increases. In any method of treating muscular dystrophy, after administration of the rAAV, the level of minidystrophin gene expression in the muscle cells of the subject increases. In any method of treating muscular dystrophy, after administration of the rAAV, the level of minidystrophin gene expression in the cardiomyocytes of the subject increases. The expression of the minidystrophin gene in the cells (such as muscle cells or cardiomyocytes) is detected by measuring the level of minidystrophin by Western blot of a muscle biopsy before and after administration of the rAAV. Specifically, compared to the level of minidystrophin before administration of the rAAV, the level of minidystrophin after administration of the rAAV increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90%. For example, compared to the level of minidystrophin before administration of the rAAV, the level of minidystrophin after administration of the rAAV increases by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84% or at least about 85%.

[0337] In addition, expression of the mini - dystrophin gene in the cells is detected by measuring the mini - dystrophin levels by immunohistochemistry in muscle biopsies before and after administration of rAAV. The level of mini - dystrophin after administration of rAAV 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% compared to the level of mini - dystrophin before administration of rAAV. For example, the level of mini - dystrophin after administration of rAAV 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% compared to the level of mini - dystrophin before administration of rAAV.

[0338] In any of the methods of treating muscular dystrophy, the serum CK level of the subject after administration of rAAV is decreased compared to the serum CK level before administration of rAAV. For example, compared to the serum CK level before administration of rAAV, the serum CK level of the subject 60 days after administration of rAAV is decreased by about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%. Specifically, in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level before administration of rAAV, the serum CK level of the subject 60 days after administration of rAAV is decreased by about 87%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level before administration of rAAV, the serum CK level of the subject 60 days after administration of rAAV is decreased by about 72%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level before administration of rAAV, the serum CK level of the subject 60 days after administration of rAAV is decreased by about 73%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level before administration of rAAV, the serum CK level of the subject 60 days after administration of rAAV is decreased by about 78%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level before administration of rAAV, the serum CK level of the subject 60 days after administration of rAAV is decreased by about 95%. In any of the methods of treating muscular dystrophy, the number of micro-dystrophin positive fibers in the muscle tissue of the subject is increased compared to the number of micro-dystrophin positive fibers before administration of rAAV. In any of the methods of treating muscular dystrophy described herein, the number of micro-dystrophin positive fibers in the myocardial tissue of the subject is increased compared to the number of micro-dystrophin positive fibers before administration of rAAV. For example, the number of micro-dystrophin positive fibers is detected by measuring the micro-dystrophin level by Western blot or immunohistochemistry in muscle biopsies (including myocardial biopsies) before and after administration of rAAV.

[0339] In any of the methods for treating muscular dystrophy as described herein, administration of rAAV upregulates the expression of DAPC proteins such as α-dystroglycan or β-dystroglycan. For example, the level of α-dystroglycan in a subject increases after administration of rAAV compared to the level of α-dystroglycan before administration of rAAV. In addition, the level of β-dystroglycan in a subject increases after administration of rAAV compared to the level of β-dystroglycan before administration of rAAV. The level of α-dystroglycan or β-dystroglycan is detected by measuring the α-dystroglycan or β-dystroglycan protein level in a muscle biopsy by Western blot or immunohistochemistry before and after administration of rAAV.

[0340] In any of the methods for treating muscular dystrophy, disease progression in the subject is delayed after administration of rAAV, as measured by any of the following: 6-minute walk test, time to rise, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment (NSAA), 10-meter timed test, 100-meter timed test, hand held dynamometry (HHD), Timed Up and Go, Bayley-III score, and / or Bayley-IV scale for gross and fine motor skills.

[0341] For example, in any of the methods, the NSAA score of a subject at least 270 days after administration of rAAV is improved by at least 1.5, 2.0, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5 points compared to the NSAA score before administration of rAAV. In addition, in any of the methods, the time to rise of a subject at least 270 days after administration of rAAV is improved by at least about 0.8 seconds compared to the time to rise before administration of rAAV. In addition, in any of the methods, the time for ascending 4 steps of a subject at least 270 days after administration of rAAV is improved by at least about 1.2 seconds compared to the time for ascending 4 steps before administration of rAAV. In addition, in any of the methods, the 100m timed test of a subject at least 270 days after administration of rAAV is improved by at least about 7 seconds compared to the 100m timed test before administration of rAAV.

[0342] In another aspect, the present disclosure provides a method of expressing a mini-dystrophin gene in a patient's cells, comprising administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.mini-dystrophin construct of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, expression of the mini-dystrophin gene in the patient's cells is detected by measuring the mini-dystrophin level by Western blot or immunohistochemistry in a muscle biopsy before or after administration of the AAVrh74.MHCK7.mini-dystrophin construct. In addition, expression of the mini-dystrophin gene in the patient is measured by detecting an increase in the number of vector genomes per nucleus, wherein there is approximately 50% mini-dystrophin expression with 1 vector genome per nucleus, and more than 1 copy per nucleus correlates with the mini-dystrophin expression level. For example, the cells have 1.2 vector copies / nucleus, or 1.3 vector copies / nucleus, or 1.4 vector copies / nucleus, or 1.5 vector copies / nucleus, or 1.6 vector copies / nucleus, or 1.7 vector copies / nucleus, or 1.8 vector copies / nucleus, or 1.9 vector copies / nucleus.

[0343] In some aspects, the present disclosure provides a method of reducing serum CK levels in a patient in need thereof, the method comprising administering to the patient an AAVrh74.MHCK7.minidystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, compared to the serum CK level prior to administration of rAAV, the serum CK level of the patient 60 days after administration of rAAV is reduced by at least about 65% to about 90%, or about 65% to about 95%, or about 75% to about 90%, or about 80% to about 90%, or about 85% to about 95%, or about 87% to about 95%, or about 87% to about 90%. Specifically, compared to the serum CK level prior to administration of rAAV, the serum CK level of the patient 60 days after administration of rAAV is reduced by about 87%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level prior to administration of rAAV, the serum CK level of the patient 60 days after administration of rAAV is reduced by about 72%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level prior to administration of rAAV, the serum CK level of the patient 60 days after administration of rAAV is reduced by about 73%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level prior to administration of rAAV, the serum CK level of the patient 60 days after administration of rAAV is reduced by about 78%, or in any of the methods of treating muscular dystrophy of the present disclosure, compared to the serum CK level prior to administration of rAAV, the serum CK level of the patient 60 days after administration of rAAV is reduced by about 95%.

[0344] The present disclosure also provides methods for increasing the number of mini-dystrophin positive fibers in a patient's muscle tissue, which include administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.mini-dystrophin construct of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In some aspects, the present disclosure provides methods for increasing the number of mini-dystrophin positive fibers in a patient's cardiac muscle tissue, which include administering to the patient the 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 of the AAVrh74.MHCK7.mini-dystrophin construct. For example, the number of mini-dystrophin positive fibers is detected by measuring the mini-dystrophin level by Western blot or immunohistochemistry on a muscle biopsy (including a cardiac muscle biopsy) before or after administration of rAAV. In addition, the expression of the mini-dystrophin gene in a patient is measured by detecting an increase in the number of vector genomes per cell nucleus, wherein there is approximately 50% mini-dystrophin expression for 1 vector genome per cell nucleus, and more than 1 copy per cell nucleus is consistent with the mini-dystrophin expression level. For example, the cells have 1.2 vector copies / nucleus, or 1.3 vector copies / nucleus, or 1.4 vector copies / nucleus, or 1.5 vector copies / nucleus, or 1.6 vector copies / nucleus, or 1.7 vector copies / nucleus, or 1.8 vector copies / nucleus, or 1.9 vector copies / nucleus.

[0345] In another aspect, the present disclosure provides methods for increasing α-sarcoglycan expression in a patient in need thereof, which include administering to the patient the nucleotide sequence of the AAVrh74.MHCK7.mini-dystrophin construct of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, the level of α-sarcoglycan is detected by measuring the α-sarcoglycan protein level by Western blot or immunohistochemistry on a muscle biopsy before and after administration of rAAV.

[0346] In addition, the present disclosure provides methods for increasing β - dystroglycan expression in patients in need thereof, which include administering to the patient the nucleotide sequence of the AAVrh74.MHCK7. micro - dystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. For example, the level of β - dystroglycan is detected by measuring the β - dystroglycan protein level in muscle biopsies before and after administration of rAAV by Western blot or immunohistochemistry.

[0347] The present disclosure also provides methods for treating patients with Duchenne muscular dystrophy or Becker muscular dystrophy, which include administering to the patient the nucleotide sequence of the AAVrh74.MHCK7. micro - dystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6, such that the disease progression of the patient is delayed, as measured by any of the following: 6 - minute walk test, time to rise, walk up 4 steps, walk up and down 4 steps, North Star Ambulatory Assessment (NSAA), 10 - meter timed test, 100 - meter timed test, hand - held dynamometry (HHD), timed up - and - go test, Bayley Scales of Infant and Toddler Development, Third Edition (Bayley - III) score, and / or Bayley Scales of Infant Development, Fourth Edition (Bayley - IV).

[0348] For example, in any of the methods, compared to the NSAA score before administration of rAAV, the NSAA score of the subject at least 270 days after administration of rAAV has an improvement of at least 1.5 points, 2.0 points, 2.5 points, 2.6 points, 2.7 points, 2.8 points, 2.9 points, 3.0 points, 3.1 points, 3.2 points, 3.3 points, 3.4 points, 3.5 points, 4.0 points, 4.5 points, 5.0 points, 5.5 points, 6.0 points, 6.1 points, 6.2 points, 6.3 points, 6.4 points, or 6.5 points. In addition, in any of the methods, compared to the time to rise before administration of rAAV, the time to rise of the subject at least 270 days after administration of rAAV has an improvement of at least about 0.8 seconds. In addition, in any of the methods, compared to the time for the 4 - step walk - up test before administration of rAAV, the time for the 4 - step walk - up test of the subject at least 270 days after administration of rAAV has an improvement of at least about 1.2 seconds. In addition, in any of the methods, compared to the 100 - m timed test before administration of rAAV, the 100 - m timed test of the subject at least 270 days after administration of rAAV has an improvement of at least about 7 seconds.

[0349] "Fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues after injury, said tissues including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas. The deposited ECM components include fibronectin and collagen, such as collagen 1, collagen 2, or collagen 3.

[0350] The present disclosure also provides methods for reducing or preventing fibrosis in a subject with muscular dystrophy, which include administering a therapeutically effective amount of an rAAV comprising the human mini - dystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or an rAAV vector comprising the AAVrh74.MHCK7.mini - dystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MHCK7.mini - dystrophin. In one aspect, AAVrh74.MHCK7.mini - dystrophin is AAVrh74.MHCK7.mini - dystrophin of nucleotides 55 - 5021 of SEQ ID NO:3. In another aspect, AAVrh74.MHCK7.mini - dystrophin is AAVrh74.MHCK7.mini - dystrophin of SEQ ID NO:9. In another aspect, AAVrh74.MHCK7.mini - dystrophin is AAVrh74.MHCK7.mini - dystrophin of nucleotides 1 - 4977 of SEQ ID NO:8 or nucleotides 56 - 5066 of SEQ ID NO:6. In another aspect, the rAAV is AAVrh74.MCK.mini - dystrophin. In one aspect, AAVrh74.MCK.mini - dystrophin is AAVrh74.MCK.mini - dystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0351] In another aspect, the present disclosure provides a method for preventing fibrosis in a subject in need thereof, which comprises administering a therapeutically effective amount of the human minidystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or an rAAV vector comprising the AAVrh74.MHCK7.minidystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. For example, any of the rAAVs of the present disclosure can be administered to a subject with muscular dystrophy to prevent fibrosis, for example, administering the rAAV expressing human minidystrophin of the present disclosure before fibrosis is observed in the subject. In addition, the rAAV expressing the human minidystrophin gene of the present disclosure can be administered to a subject at risk of developing fibrosis, such as those subjects with or diagnosed with muscular dystrophy (e.g., DMD). The rAAVs of the present disclosure can be administered to subjects with muscular dystrophy to prevent new fibrosis in these subjects.

[0352] The present disclosure contemplates administering the rAAV before fibrosis is observed in the subject. In addition, the rAAV can be administered to a subject at risk of developing fibrosis, such as those subjects with or diagnosed with muscular dystrophy (e.g., DMD). The rAAV can be administered to a subject with muscular dystrophy who has already developed fibrosis to prevent new fibrosis in these subjects.

[0353] The present disclosure also provides a method for increasing muscle strength and / or muscle mass in a subject with muscular dystrophy, which comprises administering a therapeutically effective amount of the human minidystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or an rAAV vector comprising the AAVrh74.MHCK7.minidystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0354] The present disclosure contemplates administering the rAAV vector to a subject diagnosed with DMD before fibrosis is observed in the subject or before muscle strength decreases or before muscle mass decreases.

[0355] The present disclosure also contemplates administering to a subject with muscular dystrophy who has developed fibrosis the human minidystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or an rAAV comprising the AAVrh74.MHCK7.minidystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6, to prevent new fibrosis or reduce fibrosis in these subjects. The present disclosure also provides administering to a subject with muscular dystrophy who has reduced muscle strength or reduced muscle mass the human minidystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:7; or an rAAV vector comprising the AAVrh74.MHCK7.minidystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6, to protect muscle from further damage.

[0356] The present disclosure also provides methods of treating cardiomyopathy in a human subject with muscular dystrophy (such as DMD), comprising administering to the human subject any one of the compositions described herein (such as delandistrogenemoxeparvovec). In some aspects, the methods are for improving cardiac function in a subject with DMD. In some aspects, the methods are for reducing fibrosis of the myocardium. In some aspects, the methods are for reducing central nucleation of the myocardium. In some aspects, cardiac function is improved by, for example, an increase in ejection fraction (EF), an increase in fractional shortening (FS), an increase in cardiac output, a decrease in left ventricular end - diastolic diameter (LVIDd), a decrease in left ventricular end - systolic diameter (LVESD), a decrease in systolic volume, an increase in left ventricular anterior wall systolic thickness, an increase in left ventricular posterior wall systolic thickness, an increase in peak height of sarcomere length, an increase in sarcomere length shortening rate, a decrease in the time to reach 90% of the sarcomere baseline length, Ca2 + transient peak height increase, a decrease in the time to reach 90% of the Ca2 + transient baseline, and / or maintaining or reducing serum troponin blood levels. In any of the methods of the present disclosure, the subject may have muscular dystrophy, such as DMD, or any other dystrophin - related muscular dystrophy.

[0357] In other aspects of any of the methods of the present disclosure described herein, the serum CK level of the subject after administration of rAAV is reduced by a percentage level selected from the following compared to the serum CK level before administration of rAAV:

[0358] a) at least 78% reduction at 90 days, 180 days, or 270 days after administration;

[0359] b) at least 46%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, or 85% reduction at 270 days after administration;

[0360] c) at least 72%, 73%, 74%, or 95% reduction at 180 days after administration;

[0361] d) at least 87%, 88%, 93%, or 95% reduction at 90 days after administration;

[0362] e) at least 70% reduction at 270 days after administration;

[0363] f) 70% to 95% reduction at 90 days, 180 days, or 270 days after administration;

[0364] g) at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% reduction at 90 days, 180 days, or 270 days after administration; and

[0365] h) at least 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% reduction at 90 days, 180 days, or 270 days after administration.

[0366] In another aspect, the present disclosure provides a composition for treating muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.minidystrophin, wherein the composition is formulated for systemic administration, and the dose of rAAV is about 1×10 14 vg / kg to about 4×10 14 vg / kg. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55-5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0367] For example, the composition of the present disclosure comprises the following dose of rAAV: about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 5.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 13 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to 1.0×10 14vg / kg, or about 1.0×10 13 vg / kg to about 2.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 13 vg / kg, or about 1.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 5.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 5.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15vg / kg, or 1.0×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.0×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or 1.0×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 6.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 5.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 4.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to 1.0×10 15 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.0×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.5×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 2.0×10 14 vg / kg, or 1.25×10 14 vg / kg to approximately 3.75×10 14 vg / kg, or approximately 1.25×10 14 vg / kg to approximately 3.5×10 14 vg / kg, or 1.5×10 14 vg / kg to approximately 1.0×10 15 vg / kg, or approximately 1.5×10 14 vg / kg to 6.0×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to 5.0×10 14 vg / kg, or approximately 1.5×10 14 vg / kg to 4.0×1014 vg / kg, or about 1.5×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.25×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.0×10 14 vg / kg, or 1.75×10 14 vg / kg to about 1.0×10 15 vg / kg, or about 1.75×10 14 vg / kg to 6.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to 5.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to 4.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.25×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.25×10 14 vg / kg, or about 1.75×1014 vg / kg to about 2.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to 1.0×10 15 vg / kg, or about 2.0×10 14 vg / kg to 6.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to 5.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.25×10 14 vg / kg. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0368] In one aspect, the compositions of the present disclosure are formulated for intravenous administration and contain about 2.0×10 14 vg / kg dose of rAAV. In one aspect, the compositions of the present disclosure are formulated for intravenous administration and contain about 1.33×10 14 vg / kg dose of rAAV. In another aspect, the compositions of the present disclosure are formulated for intravenous administration and contain the following doses of rAAV: about 5.0×10 12 vg / kg, or about 6.0×10 12 vg / kg, or about 7.0×10 12 vg / kg, or about 8.0×10 12 vg / kg, or about 9.0×10 12vg / 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 14 vg / kg, or approximately 1×10 15 。

[0369] In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In another aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0370] 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 a particular aspect, the composition comprises a dose of rAAV delivered at about 10 mL / kg. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In another aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0371] The compositions of the present disclosure are formulated for administration by injection, infusion, or implantation. For example, the composition is formulated for administration by infusion within about one hour. Additionally, the compositions of the present disclosure are formulated for intravenous administration via a peripheral limb vein (such as a peripheral arm vein or a peripheral leg vein). Alternatively, the infusion can be administered within about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0372] Any composition of the present disclosure comprises an rAAV or an rAAV vector, wherein the rAAV comprises the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 or SEQ ID NO:7; the rAAV vector comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0373] Specifically, the compositions of the present disclosure are for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present disclosure provides a composition for the treatment of Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the composition comprises a dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin, wherein the composition is formulated for administration by intravenous infusion over about one hour, and the administration dose of the rAAV is about 2×10 14 vg / kg, and wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0374] In another aspect, the present disclosure also provides a composition comprising an rAAV for reducing fibrosis in a subject in need thereof. In addition, the present disclosure provides a composition comprising an rAAV vector for preventing fibrosis in a subject with muscular dystrophy.

[0375] The present disclosure also provides a composition comprising an rAAV for increasing muscle strength and / or muscle mass in a subject with muscular dystrophy. In another aspect, the present disclosure provides a composition comprising any of the rAAVs of the present disclosure for the treatment of muscular dystrophy.

[0376] In other aspects of any of the compositions of the present disclosure, after administering the composition to a human subject in need of treatment for muscular dystrophy, the serum CK level of the subject is reduced by a percentage level selected from the following compared to the serum CK level before administering the composition:

[0377] a) at least 78% reduction at 90 days, 180 days, or 270 days after administration;

[0378] b) at least 46%, 55%, 70% or 85% reduction 270 days after administration;

[0379] c) at least 72%, 73%, 74% or 95% reduction 180 days after administration;

[0380] d) at least 87%, 99%, 93% or 95% reduction 90 days after administration;

[0381] e) at least 70% reduction 270 days after administration;

[0382] f) 70% to 95% reduction at 90 days, 180 days or 270 days after administration;

[0383] 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% reduction at 90 days, 180 days or 270 days after administration; and

[0384] 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% reduction at 90 days, 180 days or 270 days after administration.

[0385] In another aspect, the present disclosure provides the use of a certain dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.minidystrophin in the preparation of a medicament for treating muscular dystrophy in a human subject in need thereof, wherein the medicament is formulated for systemic administration and the dose of rAAV is about 1×10 14 vg / kg to about 4×10 14vg / kg. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0386] For example, the medicament comprises the following dose of rAAV: about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 12 vg / kg to about 5.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 2.0×10 13 vg / kg, or about 5.0×10 12 vg / kg to about 1.0×10 13 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 2.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 13vg / kg, or about 1.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 5.0×10 14 vg / kg, or about 1.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 2.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 1.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 3.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 5.0×10 14 vg / kg, or about 5.0×10 13 vg / kg to about 6.0×10 14 vg / kg, or 5.0×10 13 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to about 6.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 5.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or 1.0×10 14 vg / kg to about 1.0×10 15 vg / kg, or 1.0×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.0×10 14 vg / kg to about 2.5×10 14 vg / kg, or 1.0×10 14 vg / kg to about 2.0×10 14vg / kg, or about 1.25×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to 6.0×10 14 vg / kg, or about 1.25×10 14 vg / kg to 5.0×10 14 vg / kg, or about 1.25×10 14 vg / kg to 4.0×10 14 vg / kg, or about 1.25×10 14 vg / kg to 1.0×10 15 vg / kg, or about 1.25×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 2.0×10 14 vg / kg, or 1.25×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.25×10 14 vg / kg to about 3.5×10 14 vg / kg, or 1.5×10 14 vg / kg to about 1.0×10 15 vg / kg, or about 1.5×10 14 vg / kg to 6.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to 5.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to 4.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.5×10 14vg / kg to about 3.25×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.5×10 14 vg / kg to about 2.0×10 14 vg / kg, or 1.75×10 14 vg / kg to about 1.0×10 15 vg / kg, or about 1.75×10 14 vg / kg to 6.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to 5.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to 4.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.5×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.25×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 3.0×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.75×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.5×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.25×10 14 vg / kg, or about 1.75×10 14 vg / kg to about 2.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to 1.0×10 15 vg / kg, or about 2.0×10 14 vg / kg to 6.0×10 14vg / kg, or about 2.0×10 14 vg / kg to 5.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 4.0×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.75×10 14 vg / kg, or about 2.0×10 14 vg / kg to about 3.5×10 14 vg / kg or about 2.0×10 14 vg / kg to about 3.25×10 14 vg / kg. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55 - 5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56 - 4820 of SEQ ID NO:5.

[0387] In one aspect, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, wherein the systemic administration route is the intravenous route, and the administration dose of rAAV is about 2.0×10 14 vg / kg. In another aspect, the medicament of the present disclosure is formulated for systemic administration of a dose of rAAV, wherein the systemic administration route is the intravenous route, and the dose of rAAV is about 5.0×10 12 vg / kg, or about 6.0×10 12 vg / kg, or about 7.0×10 12 vg / kg, or about 8.0×10 12 vg / kg, or about 9.0×10 12 vg / kg, or about 1.0×10 13 vg / kg, or about 1.25×10 13 vg / kg, or about 1.5×10 13 vg / kg, or about 1.75×10 13 vg / kg, or about 2.25×10 13 vg / kg, or about 2.5×10 13vg / 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 1.33×10 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.

[0388] In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55-5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0389] In any use of the present disclosure, a certain dose of rAAV comprised in the medicament is present at about 5 mL / kg to about 15 mL / kg, or about 8 mL / kg to about 12 mL / kg, or 8 mL / kg to about 10 mL / kg, or 5 mL / kg to about 10 mL / kg, or about 10 mL / kg to 12 mL / kg, or about 10 mL / kg to 15 mL / kg or 10 mL / kg to about 20 mL / kg. In a particular aspect, the dose of rAAV is present at about 10 mL / kg. In one aspect, the rAAV is AAVrh74.MHCK7.minidystrophin. In one aspect, the AAVrh74.MHCK7.minidystrophin is the AAVrh74.MHCK7.minidystrophin of nucleotides 55-5021 of SEQ ID NO:9, SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8 or nucleotides 56-5022 of SEQ ID NO:6. In one aspect, the rAAV is AAVrh74.MCK.minidystrophin. In one aspect, the AAVrh74.MCK.minidystrophin is the AAVrh74.MCK.minidystrophin of nucleotides 56-4820 of SEQ ID NO:5.

[0390] In any use of the present disclosure, the medicament is formulated for administration by injection, infusion or implantation. For example, the medicament is formulated for administration by infusion within about one hour. Additionally, the medicament 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 within about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours or about 3 hours.

[0391] In any use of the present disclosure, the drug comprises: 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.

[0392] A specific use of the present disclosure is for the preparation of a drug for treating Duchenne muscular dystrophy or Becker muscular dystrophy. For example, the present disclosure provides the use of a certain dose of recombinant adeno-associated virus (rAAV) rAAV.MHCK7.microdystrophin for the preparation of a drug for treating Duchenne muscular dystrophy or Becker muscular dystrophy in a human subject in need thereof, wherein the drug is formulated for administration by intravenous infusion over about one hour, and the administration dose of the rAAV is about 2×10 14 vg / kg, and wherein the rAAV comprises the AAVrh74.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6.

[0393] In another aspect, the present disclosure provides the use of rAAV for the preparation of a drug for reducing fibrosis in a subject in need thereof. For example, the subject in need may have muscular dystrophy, such as DMD or any other dystrophin-related muscular dystrophy.

[0394] In another aspect, the present disclosure provides the use of rAAV for the preparation of a drug for preventing fibrosis in a subject with muscular dystrophy.

[0395] In addition, the present disclosure provides the use of rAAV for the preparation of a drug for increasing muscle strength and / or muscle mass in a subject with muscular dystrophy.

[0396] The present disclosure also provides the use of rAAV for the preparation of a drug for treating muscular dystrophy.

[0397] The present disclosure provides 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 treating 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 treating muscular dystrophy.

[0398] In other aspects of any of the uses of the present disclosure, after administering the rAAV to the subject, the serum CK level of the subject is reduced by a percentage level selected from the following compared to the serum CK level before administering the rAAV:

[0399] a) at least 78% reduction at 90 days, 180 days, or 270 days after administration;

[0400] b) at least 46%, 55%, 70%, or 95% reduction at 270 days after administration;

[0401] c) at least 72%, 73%, 74%, or 95% reduction at 180 days after administration;

[0402] d) at least 87%, 99%, 93%, or 95% reduction at 90 days after administration;

[0403] e) at least 70% reduction at 270 days after administration;

[0404] f) 70% to 95% reduction at 90 days, 180 days, or 270 days after administration;

[0405] 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% reduction at 90 days, 180 days, or 270 days after administration; and

[0406] 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% reduction at 90 days, 180 days, or 270 days after administration.

[0407] In any of the uses of a composition or a medicament for treating muscular dystrophy for treating muscular dystrophy, after administering the composition or the medicament, the level of expression of the micro-dystrophin gene in the cells of the subject increases. The expression of the micro-dystrophin gene in the cells is detected by measuring the level of micro-dystrophin by Western blot of a muscle biopsy before and after administering the composition or the medicament. Specifically, compared with the level of micro-dystrophin before administering the composition or the medicament, the level of micro-dystrophin after administering the composition or the medicament increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90%. For example, compared with the level of micro-dystrophin before administering the composition or the medicament, the level of micro-dystrophin after administering the composition increases by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84% or at least about 85%.

[0408] In addition, the expression of the micro-dystrophin gene in the cells is detected by measuring the level of micro-dystrophin by immunohistochemistry in a muscle biopsy before and after administering the composition or the medicament. Compared with the level of micro-dystrophin before administering the composition or the medicament, the level of micro-dystrophin after administering rAAV increases by at least about 70% to at least about 80%, or at least about 70% to at least about 90%, or at least about 80% to at least about 90%. For example, compared with the level of micro-dystrophin before administering the composition or the medicament, the level of micro-dystrophin after administering the composition or the medicament increases by at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84% or at least about 85%.

[0409] In any of the compositions for treating muscular dystrophy, the serum CK level of the subject is reduced after administration of the rAAV compared to the serum CK level before administration of the composition or drug. For example, compared to the serum CK level before administration of the composition or drug, the serum CK level of 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% 60 days after administration of the composition or drug. Specifically, in any of the compositions for treating muscular dystrophy of the present disclosure, the serum CK level of the subject is reduced by about 87% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or in any of the uses of the composition or drug for treating muscular dystrophy of the present disclosure for treating muscular dystrophy, the serum CK level of the subject is reduced by about 72% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or in any of the compositions for treating muscular dystrophy of the present disclosure, the serum CK level of the subject is reduced by about 73% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug, or in any of the uses of the composition or drug for treating muscular dystrophy of the present disclosure for treating muscular dystrophy, the serum CK level of the subject is reduced by about 78% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition, or in any of the uses of the composition or drug for treating muscular dystrophy of the present disclosure for treating muscular dystrophy, the serum CK level of the subject is reduced by about 95% 60 days after administration of the composition or drug compared to the serum CK level before administration of the composition or drug. In any of the uses of the composition or drug for treating muscular dystrophy for treating muscular dystrophy, the number of micro-dystrophin positive fibers in the muscle tissue of the subject is increased compared to the number of micro-dystrophin positive fibers before administration of the composition or drug. For example, the number of micro-dystrophin positive fibers is detected by measuring the micro-dystrophin level by Western blot or immunohistochemistry on muscle biopsies before and after administration of the composition or drug.

[0410] In any of the uses of a composition or a medicament for treating muscular dystrophy for treating muscular dystrophy, administration of the composition or the medicament upregulates the expression of DAPC proteins, such as α - dystroglycan or β - dystroglycan. For example, compared with the level of α - dystroglycan before administration of the composition or the medicament, the level of α - dystroglycan in a subject increases after administration of the composition or the medicament. In addition, compared with the level of β - dystroglycan before administration of the composition or the medicament, the level of β - dystroglycan in a subject increases after administration of the composition or the medicament. The level of α - dystroglycan or β - dystroglycan is detected by measuring the level of α - dystroglycan or β - dystroglycan protein in a muscle biopsy by Western blot or immunohistochemistry before and after administration of the composition or the medicament.

[0411] In any of the uses of a composition or a medicament for treating muscular dystrophy for treating muscular dystrophy, the disease progression in a subject is delayed after administration of the composition or the medicament, which is measured by any of the following: 6 - minute walk test, time to rise, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment (NSAA), 10 - meter timed test, 100 - meter timed test, hand - held dynamometry (HHD), Timed Up and Go test, Bayley Scales of Infant and Toddler Development, Third Edition (Bayley - III) score, and / or Bayley Scales of Infant Development, Fourth Edition (Bayley - IV).

[0412] For example, after administration of any composition for treating muscular dystrophy or in the use of a medicament for treating muscular dystrophy, compared with the NSAA score before administration of rAAV, the NSAA score of a subject is improved by at least 1.5 points, 2.0 points, 2.5 points, 2.6 points, 2.7 points, 2.8 points, 2.9 points, 3.0 points, 3.1 points, 3.2 points, 3.3 points, 3.4 points, 3.5 points, 4.0 points, 4.5 points, 5.0 points, 5.5 points, 6.0 points, 6.1 points, 6.2 points, 6.3 points, 6.4 points or 6.5 points at least 270 days after administration of the composition or the medicament. In addition, in any of the methods, compared with the time to rise before administration of the composition or the medicament, the time to rise of a subject is improved by at least about 0.8 seconds at least 270 days after administration of the composition or the medicament. In addition, in any of the methods or uses of the present disclosure, compared with the time for the 4 - step ascent test before administration of the composition or the medicament, the time for the 4 - step ascent test of a subject is improved by at least about 1.2 seconds at least 270 days after administration of the composition or the medicament. In addition, in any of the methods or uses of the present disclosure, compared with the 100 - m timed test before administration of the composition or the medicament, the 100 - m timed test of a subject is improved by at least about 7 seconds at least 270 days after administration of the composition or the medicament.

[0413] In another aspect, the present disclosure provides a composition for expressing the mini - dystrophin gene in patient cells, comprising the nucleotide sequence of the AAVrh74.MHCK7.mini - dystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. In another aspect, the present disclosure provides the use of a certain dose of the nucleotide sequence of the AAVrh74.MHCK7.mini - dystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6 for the preparation of a medicament for expressing the mini - dystrophin gene in patient cells. For example, the expression of the mini - dystrophin gene in patient cells is detected by measuring the mini - dystrophin level by Western blot or immunohistochemistry in muscle biopsies before and after administration of the rAAV.MHCK7.mini - dystrophin construct. In addition, the expression of the mini - dystrophin gene in a patient is measured by detecting an increase in the number of vector genomes per cell nucleus, wherein about 50% mini - dystrophin expression is present with 1 vector genome per cell nucleus, and more than 1 copy per cell nucleus correlates with the mini - dystrophin expression level. For example, the cells have 1.2 vector copies / nucleus, or 1.3 vector copies / nucleus, or 1.4 vector copies / nucleus, or 1.5 vector copies / nucleus, or 1.6 vector copies / nucleus, or 1.7 vector copies / nucleus, or 1.8 vector copies / nucleus, or 1.9 vector copies / nucleus.

[0414] In another aspect, the present disclosure provides a composition for reducing serum CK levels in a patient in need thereof, the composition comprising the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. Additionally, the present disclosure provides the use of a certain dose of the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for reducing serum CK levels in a patient in need thereof. For example, compared to the serum CK level before using the composition or medicament, the serum CK level of 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% 60 days after administration of the composition or medicament. Specifically, the serum CK level of the subject is reduced by about 87% 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 72% 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 73% 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 78% 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament, or is reduced by about 95% 60 days after administration of the composition or medicament compared to the serum CK level before administration of the composition or medicament.

[0415] The present disclosure also provides a composition for increasing the number of mini-dystrophin positive fibers in a patient's muscle tissue, which comprises the nucleotide sequence of the AAVrh74.MHCK7.mini-dystrophin construct of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6. In addition, the present disclosure provides the use of a certain dose of the nucleotide sequence of the AAVrh74.MHCK7.mini-dystrophin construct of SEQ ID NO:9, nucleotides 55-5021 of SEQ ID NO:3, nucleotides 1-4977 of SEQ ID NO:8, or nucleotides 56-5022 of SEQ ID NO:6 for the preparation of a medicament for increasing the number of mini-dystrophin positive fibers in a patient's muscle tissue. For example, the number of mini-dystrophin positive fibers is detected by measuring the mini-dystrophin level by Western blot or immunohistochemistry on a muscle biopsy before and after administration of the composition or medicament. In addition, the expression of the mini-dystrophin gene in a patient is measured by detecting an increase in the number of vector genomes per cell nucleus, wherein there is approximately 50% mini-dystrophin expression for 1 vector genome per cell nucleus, and more than 1 copy per cell nucleus is consistent with the mini-dystrophin expression level. For example, the cell has 1.2 vector copies / nucleus, or 1.3 vector copies / nucleus, or 1.4 vector copies / nucleus, or 1.5 vector copies / nucleus, or 1.6 vector copies / nucleus, or 1.7 vector copies / nucleus per cell nucleus, or 1.8 vector copies / nucleus per cell nucleus, or 1.9 vector copies / nucleus per cell nucleus.

[0416] In another aspect, the present disclosure provides a composition for increasing α - dystroglycan expression in a patient in need thereof, comprising the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. The present disclosure also provides the use of a certain dose of the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6 for the preparation of a medicament for increasing α - dystroglycan expression in a patient in need thereof. For example, the level of α - dystroglycan is detected by measuring the α - dystroglycan protein level in a muscle biopsy by Western blot or immunohistochemistry before and after administration of the composition or medicament.

[0417] In addition, the present disclosure provides a composition for increasing β - dystroglycan expression in a patient in need thereof, comprising the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6. The present disclosure also provides the use of the nucleotide sequence of the AAVrh74.MHCK7.minidystrophin construct of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6 for the preparation of a medicament for increasing β - dystroglycan expression in a patient in need thereof. For example, the level of β - dystroglycan is detected by measuring the β - dystroglycan protein level in a muscle biopsy by Western blot or immunohistochemistry before and after administration of the composition or medicament.

[0418] The present disclosure also provides the use of a nucleotide sequence of an AAVrh74.MHCK7.minidystrophin construct of a certain dose of SEQ ID NO:9, nucleotides 55 - 5021 of SEQ ID NO:3, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6 for the preparation of a medicament for treating a patient suffering from Duchenne muscular dystrophy or Becker muscular dystrophy, such that administration of the medicament will delay the disease progression of the patient, as measured by any one of the following: 6-minute walk test, time to rise, ascending 4 steps, ascending and descending 4 steps, North Star Ambulatory Assessment (NSAA), 10-meter timed test, 100-meter timed test, hand-held dynamometry (HHD), timed up and go test, Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III) score, and / or Bayley Scales of Infant Development, Fourth Edition (Bayley-IV).

[0419] For example, compared to the NSAA score before administration of the composition or medicament, the NSAA score of the subject at least 270 days after administration of the composition or medicament has an improvement of at least 1.5 points, 2.0 points, 2.5 points, 2.6 points, 2.7 points, 2.8 points, 2.9 points, 3.0 points, 3.1 points, 3.2 points, 3.3 points, 3.4 points, 3.5 points, 4.0 points, 4.5 points, 5.0 points, 5.5 points, 6.0 points, 6.1 points, 6.2 points, 6.3 points, 6.4 points, or 6.5 points. In addition, compared to the time to rise before administration of the composition or medicament, the time to rise of the subject at least 270 days after administration of the composition or medicament has an improvement of at least about 0.8 seconds. In addition, compared to the time for ascending 4 steps test before administration of the composition or medicament, the time for ascending 4 steps test of the subject at least 270 days after administration of the composition or medicament has an improvement of at least about 1.2 seconds. In addition, compared to the 100m timed test before administration of the composition or medicament, the 100m timed test of the subject at least 270 days after administration of the composition or medicament has an improvement of at least about 7 seconds.

[0420] In any of the methods for treating muscular dystrophy in a human subject described herein, the human subject can or cannot walk. In some aspects, the human subject can walk. In another aspect, the ambulatory human subject is ≥8 years old to <18 years old. In some aspects, the human subject cannot walk. In some aspects, the human subject is ≥2 years old to <3 years old. In some aspects, the human subject is ≥2 years old to <3 years old and can walk.

[0421] In some aspects, a human subject being treated for muscular dystrophy according to any of the methods described herein is between 4 and 5 years old (i.e., the subject is at least 4 years old up to and including 6 years old. In other words, the subject is at least 4 years old and less than 6 years old). In some aspects, the human subject between 4 and 5 years old is ambulatory.

[0422] In some aspects, a human subject being treated for muscular dystrophy according to any of the methods described herein is between 2 and 3 years old (i.e., the subject is at least 2 years old up to and including 3 years old. In other words, the subject is at least 2 years old and less than 3 years old). In some aspects, the human subject between 2 and 3 years old is ambulatory.

[0423] In some aspects, the human subject is non-ambulatory. In some aspects, the human subject has been non-ambulatory for at least 9 months. In some aspects, the human subject has a stable FVC of less than 40% and / or requires nocturnal ventilatory support.

[0424] It should be understood that the detailed description section, and not the summary of the invention and abstract sections, is intended to be used to interpret the claims. The summary of the invention and abstract sections may set forth one or more, but not all, exemplary aspects of the disclosure as contemplated by the inventors, and as such, are not intended to limit the disclosure and the appended claims in any way.

[0425] The following examples are provided by way of illustration and not limitation. The numerical ranges include all integer values within the ranges and include the lowest and highest integers.

[0426] Examples

[0427] Example 1

[0428] A) Generation of the AAVrh74.MHCK7.minidystrophin construct

[0429] The AAVrh74.MHCK7.minidystrophin plasmid contains a human minidystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 1)。The mini-dystrophin construct is characterized by an in-frame rod deletion (R4-R23), while hinges 1, 2, and 4 and the cysteine-rich domain are retained, resulting in a 138 kDa protein. The expression of mini-dystrophin (3579 bp) is driven by the MHCK7 promoter (792 bp). The plasmid was constructed from the rAAV.MCK.mini-dystrophin plasmid by removing the MCK promoter and inserting the MHCK7 promoter. After the core promoter, there is a 53 bp endogenous murine MCK exon 1 (untranslated) for efficient transcriptional initiation, followed by the SV40 late 16S / 19S splice signal (150 bp) and a small 5'UTR (61 bp). The intron and 5'UTR are derived from the plasmid pCMVβ (Clontech). The mini-dystrophin cassette has a consensus Kozak immediately before the ATG start, and a small 53 bp synthetic poly A signal for mRNA termination. The human mini-dystrophin cassette contains the (R4-R23 / Δ71-78) domain as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)). The complementary DNA was codon-optimized for human use and synthesized by GenScript (Piscataway, NJ) (Mol Ther 18, 109-117 (2010)). The only viral sequences included in this vector are the AAV2 inverted terminal repeats, which are required for viral DNA replication and packaging. The mini-dystrophin cassette has a small 53 bp synthetic poly A signal for mRNA termination.

[0430] Previous studies have used the MHCK7 promoter (Salva et al., Mol Ther 15, 320-329 (2007)) and AAVrh74, which enables expression in skeletal, diaphragm, and cardiac muscles (Sondergaard et al., Annals of Clinical and Transl Neurology 2, 256-270 (2015)) to verify cardiac expression. Figure 14 The sequence of the construct was packaged into AAVrh.74 viral particles. The molecular cloning of serotype AAVrh.74 was cloned from rhesus monkey lymph nodes and described in Rodino-Klapac et al., Journal of Translational Medicine 5, 45 (2007).

[0431] Table 1 shows the molecular characteristics of the plasmid AAVrh74.MHCK7 mini-dystrophin (SEQ ID NO: 3).

[0432] Table 1. Molecular characteristics of plasmid rAAV.MHCK7.mini-dystrophin

[0433]

[0434] B) Generation of the AAVrh74.MHCK7.micro-dystrophin construct and the plasmid encoding the kanamycin (Kan) resistance gene from the plasmid encoding the kanamycin (Kan) resistance gene

[0435] Cloning of MHCK7.μDys.KAN was completed by isolating the MHCK7.μDys fragment and the kanamycin backbone from the MHCK7.μDys.AMP plasmid and annealing and ligating them using the NEBuilder cloning workflow. The MHCK7.μDys fragment was isolated via restriction enzyme digestion with SnaBI. The digestion was carried out at 37 °C for 1 h in a total reaction volume of 50 μL in 1× CutSmart buffer (NEB) and 1 μL SnaBI. The resulting fragments were separated using a 1% agarose gel, electrophoresed at 105 V for 1.5 h. The band corresponding to the MHCK7.μDys insert was excised and purified using a gel purification kit (Macherey-Nagel). The resulting fragment had a DNA concentration of 10 ng / μL. The Kan backbone fragment was isolated by digesting with XbaI restriction enzyme for 1 h in a 50 μL reaction in the presence of 1× CutSmart buffer (NEB) and 1 μL XbaI at 37 °C. The resulting fragments were separated using a 1% agarose gel, electrophoresed at 105 V for 1.5 h. The band corresponding to the Kan backbone was excised and purified via a gel purification kit (Macherey-Nagel). The resulting fragment had a DNA concentration of 8.1 ng / μL. The two fragments were annealed and ligated using the NEBuilder cloning workflow, which is capable of joining two fragments with overlapping sequences. The NEBuilder cloning reaction was carried out at 50 °C for 15 min in a total reaction volume of 20 μL using a 1:1 ratio of MHCK7.μDys to the kanamycin backbone in 1× NEBuilder HiFi DNA Assembly premix according to the manufacturer's protocol. The resulting clone was transformed into competent cells by adding 2.5 μL of the cloning product to the cells, followed by incubation on ice for 30 min, then at 42 °C for 30 s and on ice for an additional 5 min. In stable competent Escherichia coli (E. coli) (C3040). After transformation, 950 μL of outgrowth media was added to the cells and they were grown at 30 °C for 1.5 hours with shaking at 225 rpm. After recovery, 450 μL of these cells were spread on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 30 °C in a dry incubator. Colonies were picked from this plate and grown overnight in LB containing 50 μg / mL kanamycin. DNA was isolated from 3 mL of this culture using the Spin Miniprep kit (Qiagen). The cloned product was confirmed using this DNA. The cloned product was confirmed by restriction enzyme digestion with PmeI, MscI, and SmaI followed by gel electrophoresis. In addition, the cloned product was confirmed by sequencing. The resulting plasmid is shown in SEQ ID NO:8 and is shown in Figure 15 and Figure 13 As described above, the sequence of the construct corresponding to nucleotides 1 - 4977 of SEQ ID NO:9 and SEQ ID NO:8 was packaged into AAVrh.74 virions. Eligibility criteria of the construct

[0436] Example 2

[0437] Systemic gene delivery clinical trial for Duchenne muscular dystrophy

[0438] This was a single - dose controlled trial using rAAVrh74.MHCK7.micro - dystrophin with nucleotides 55 - 5021 of SEQ ID NO:3 in DMD subjects. Group A included 6 subjects aged 3 months to 3 years, and Group B included 6 subjects aged 4 years to 7 years. All subjects received the micro - dystrophin vector intravenously (2×10 14 vg / kg, 10 mL / kg). rAAVrh74.MHCK7.micro - dystrophin was formulated in a buffer containing 20 mM Tris (pH 8.0), 1 mM magnesium chloride (MgCl2), 200 mM sodium chloride (NaCl), and 0.001% poloxamer 188.

[0439] In this study, the rAAVrh74.MHCK7.micro - dystrophin was infused via the peripheral arm vein so that it could reach all muscles in the body. Six DMD subjects aged 3 months to 3 years in Group A and six DMD subjects aged 4 years to 7 years in Group B were recruited. All subjects received the micro - dystrophin vector intravenously (2×10 14vg / kg, 10 mL / kg). Quantitative PCR was used, with the Prism 7500 Taqman detector system (PE Applied Biosystems), and primers for the MHCK7 promoter, to assay the titer of the encapsulated vector genome in the administered dose relative to a supercoiled DNA plasmid standard (Pozsgai et al., Mol. Ther. 25(4):855 - 869 (2017)).

[0440] Subjects received a 1 - hour infusion in the Pediatric Intensive Care Unit (PICU) of Nationwide Children's Hospital. Muscle biopsies were performed at the screening visit prior to gene therapy. At 90 days post - delivery, subjects underwent a second muscle biopsy to determine whether the gene could replace the missing dystrophin. After gene transfer, any treatment side effects in the patients were carefully monitored. This monitoring included blood and urine tests, as well as physical examinations, during the screening visit and on days 0, 1, 7, 14, 30, 60, 90, and 180, and at 9 months, 12 months, 18 months, 24 months, 30 months, and 36 months to ensure no side effects caused by gene injection.

[0441] Subjects in Group A (n = 6) were between 3 months and 3 years of age and received the rAAVrh74.MHCK7.minidystrophin vector (2×10 14 vg / kg, 10 mL / kg) intravenously. One day prior to gene transfer, subjects in Group A started and maintained prednisone or deflazacort at 1 mg / kg for 30 days while monitoring the immune response. If negative on day 30, steroids were discontinued within 1 week. If the T - cell response to AAV or minidystrophin > 125 SFC / 106 PBMC, steroids were maintained until the level dropped below the threshold.

[0442] Subjects in Group B (n = 6) were between 4 and 7 years of age and received the rAAVrh74.MHCK7.minidystrophin vector (2×10 14 vg / kg, 10 mL / kg) intravenously. These subjects maintained a stable dose of corticosteroids throughout the trial, but the dose could be increased briefly if the T - cell response to AAV or minidystrophin > 125 SFC / 106 PBMC.

[0443] Minidystrophin gene expression

[0444] The inclusion criteria for this study were as follows:

[0445] · Recruitment age: Group A: 3 months to 7 years old; Group B: between 4 - 7 years old, including the endpoints.

[0446] · Molecular characterization of the DMD gene with frameshift (deletion or duplication) or premature termination codon mutations between exons 18 to 58.

[0447] · CK elevation > 1000 U / L.

[0448] · Subjects in Group A: Gross motor skills below the mean at Bayley - III motor assessment, defined as a scale score ≤ 9.

[0449] · Group B: Below the mean at the 100 - meter timed test, defined as < 80% predicted value.

[0450] · Males of any race.

[0451] · Able to cooperate with the motor assessment test.

[0452] · Subjects in Group A: Not previously treated with corticosteroids.

[0453] · Subjects in Group B: Oral corticosteroids at a stable dose equivalent for at least 12 weeks before screening and expected to remain unchanged throughout the study (except for modifications to accommodate weight changes).

[0454] The exclusion criteria for this study are as follows:

[0455] · Active viral infection based on clinical observations.

[0456] · Signs of cardiomyopathy, including an echocardiogram ejection fraction below 40%.

[0457] · Serological evidence of HIV infection, or hepatitis B or C infection.

[0458] · Diagnosed with (or receiving treatment for) an autoimmune disease.

[0459] · Abnormal laboratory values considered to be of clinical significance.

[0460] · Concurrent diseases or need for long - term drug treatment that the PI believes would pose an unnecessary risk to gene transfer.

[0461] · Subject's AAVrh74 or AAV8 antibody titer > 1:400 as measured by ELISA immunoassay.

[0462] · Medical conditions or special circumstances that the researchers believe may compromise the subject's ability to comply with the tests or procedures required by the protocol, or may compromise the subject's health, safety, or clinical interpretability.

[0463] · Have had a serious infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks before the gene transfer visit (recruitment may be postponed).

[0464] · Have received any investigational drug (other than corticosteroids) or exon skipping drug (including ExonDys ) within the last 6 months before screening for this study, whether experimental or otherwise.

[0465] · Have undergone any type of gene therapy, cell-based therapy (e.g., stem cell transplantation), or CRISPR / Cas9 therapy.

[0466] · Family members do not want to disclose the patient's study participation to the attending physician and other healthcare providers.

[0467] Outcome Measures

[0468] The primary outcome measure is safety based on the number of participants who experience adverse events (Time frame: 3 years). Adverse effects are monitored and scored for severity and relevance to the study drug.

[0469] The secondary outcome measures are as follows:

[0470] Bayley-III Gross Motor Subscale score (Time frame: Screening, Day 30 to 3 years): The gross motor scale score measured by motor development. At each follow-up visit from Day 30 to 3 years, Group A is scored using the Bayley-III Gross Motor Subscale. At screening, any subject aged 43 - 47 months (including endpoints) has a scale score calculated compared to the standardized data of a 42-month-old child. The Bayley-III provides standardized data for children aged 1 - 42 months.

[0471] Physical therapy assessment - 100-meter Timed Test (100m) (Time frame: Screening, Day 30 to 3 years): The 100m is the primary motor outcome for Group B. For Group A, the 100-meter Timed Test is an exploratory outcome initiated when the child is 3 years old.

[0472] Physical therapy assessment - North Star Ambulatory Assessment (NSAA) (Time frame: Screening, Day 30 to 3 years): The North Star Ambulatory Assessment (NSAA) is an exploratory outcome initiated when the child in Group A is 4 years old and is an exploratory outcome for Group B. The NSAA measures the walking quality of young boys with Duchenne muscular dystrophy.

[0473] Physical therapy assessment - Timed Up and Go Test (TUG) modified for children (Time frame: Screening, Day 30 to 3 years): Exploratory outcomes for Group B include the Timed Up and Go Test (TUG) modified for children.

[0474] Physical therapy assessment - ascending and descending 4 steps (time range: screening, day 30 to 3 years): Exploratory results in Group B included ascending and descending 4 steps.

[0475] Physical therapy assessment - hand-held dynamometry (HHD) (time range: screening, day 30 to 3 years): Exploratory results in Group B included hand-held dynamometry (HHD) for knee extensors and knee flexors, as well as elbow flexors and elbow extensors.

[0476] Quantification of mini-dystrophin gene expression by immunofluorescence (time range: screening, day 90): Mini-dystrophin gene expression levels were quantified and compared by immunofluorescence in muscle biopsies before and after treatment.

[0477] Quantification of mini-dystrophin gene expression by Western blot (time range: screening, day 90): Mini-dystrophin gene expression levels were quantified and compared by Western blot analysis in muscle biopsies before and after treatment.

[0478] Reduction of CK after gene therapy (time range: 3 years): CK levels in circulating blood decreased.

[0479] Cardiac magnetic resonance imaging (at 1 year).

[0480] Figure 2

[0481] Analyze and quantify the change in mini-dystrophin expression from baseline via immunofluorescence (IF) fiber intensity. As Figure 7 shown, Subject 1 (5 years old) showed 78% mini-dystrophin expression in the muscle fibers of the gastrocnemius muscle biopsy after administration of the rAAVrh74.MHCK7.mini-dystrophin; Subject 2 (4 years old) showed 73.5% mini-dystrophin expression in the muscle fibers of the gastrocnemius muscle biopsy after administration of the rAAVrh74.MHCK7.mini-dystrophin; Subject 3 (6 years old) showed 77.0% mini-dystrophin expression in the muscle fibers of the gastrocnemius muscle biopsy after administration of the rAAVrh74.MHCK7.mini-dystrophin. Subject 4 (4 years old) showed 96.2% mini-dystrophin expression in the muscle fibers of the gastrocnemius muscle biopsy after administration of the rAAVrh74.MHCK7.mini-dystrophin. As measured by immunohistochemistry, all patients showed stable expression of the transduced mini-dystrophin, which was completely restricted to the muscle sarcolemma ( Subject ).

[0482] Table 2

[0483] Average intensity Percentage of minidystrophin - positive fibers Mean value 1 82.0％ 78.0％ 2 59.0％ 73.5％ 3 83.0％ 77.0％ 4 160.0％ 96.2％ Figure 8A 96.0％ 81.2％

[0484] The change in microdystrophin gene expression from baseline to day 60 was also evaluated by quantifying microdystrophin expression, which was measured by Western blot of biopsy muscle tissue. As Figure 8B and Figure 8C shown, Western blot analysis detected protein expression of microdystrophin in Subject 1 (5 years old), Subject 2 (4 years old), and Subject 3 (6 years old). Subject Western blot analysis was provided to detect protein expression of microdystrophin in Subject 4 (4 years old). All post-treatment biopsies showed stable levels of microdystrophin, as measured by Western blot, where using Method 1, the average value of Subjects 1 - 4 was 74.3 compared to the normal value, and according to Method 2 adjusted for adipose and fibrotic tissue, the average value of Subjects 1 - 4 was 95.8% compared to the normal value.

[0485] For each subject, the copy number of the vector genome per nucleus of muscle fibers was measured. As shown in Table 3, after administration of rAAVrh74.MHCK7.microdystrophin, for each subject, the copy number of the vector genome per nucleus was greater than 1. One copy of the vector indicates approximately 50% microdystrophin gene expression. An average of 1.6 vector copies per nucleus was measured in Subjects 1 - 3, consistent with the observed high levels of microdystrophin expression. When including the value of Subject 4, the average vector copies per microgram of DNA > 10 5 , with an average of 3.3 vector copies per nucleus.

[0486] Table 3

[0487] Vector copies / μg DNA Copies per cell nucleus Figure 9A 1 <![CDATA[>10 5 > 1.7 2 <![CDATA[>10 5 > 1.3 3 <![CDATA[>10 5 > 1.9

[0488] The protein levels of α - sarcoglycan and β - sarcoglycan in muscle biopsy tissues were measured by immunohistochemistry before and after administration of rAAVrh74.MHCK7.microdystrophin. Administration of rAAVrh74.MHCK7 also upregulated the DAPC proteins in the subjects. As shown in Figure 9, in Subject 1 ( Figure 9B ), Subject 2 ( Figure 9C ), and Subject 3 ( Circulating serum CK level ), the expression of α - sarcoglycan and β - sarcoglycan in muscle biopsy tissues increased compared to the levels of these proteins in muscle biopsies before administration of rAAVrh74.MHCK7.

[0489] Figure 10

[0490] Blood samples were obtained every 30 days after intravenous infusion of the rAAVrh74.MHCK7.minidystrophin vector (2×10 14 vg / kg, 10 mL / kg). CK levels were measured at each visit and compared to the baseline levels obtained prior to administration of rAAVrh74.MHCK7.minidystrophin (visit day 0). The baseline serum CK levels (units / liter) are provided in Table 4 below. As Figure 10 shown, at 2 months after administration of rAAVrh74.MHCK7.minidystrophin, the level of circulating serum CK was reduced by approximately 87%. At 2 months after treatment, all subjects showed a significant reduction in serum creatine kinase (CK) levels, with an average CK reduction of more than 87% (n = 3). CK is an enzyme associated with muscle damage, and DMD patients all have elevated CK levels. In fact, significantly elevated CK is commonly used as an initial diagnostic tool for DMD and is then confirmed by genetic testing.

[0491] Table 4 and Figure 11 provide the CK levels for each subject. Subject Provide the change in average CK levels over time and show that the average CK levels decreased significantly over time after administration of rAAVrh74.MHCK7.minidystrophin. The average baseline CK level of 27,064 U / L (average of Table 4) decreased by approximately 63% to reach an average of 9,982 U / L (average, day 270, Table 5).

[0492] Table 4

[0493] Age (years) CK level at baseline, units per liter (U / L) Subject 1 5 20691 2 4 23414 3 6 34942 4 4 29210

[0494] Table 5: Change in CK Levels from Baseline to Day 270

[0495] Baseline Day 30 Day 60 Day 90 Day 180 Day 270 Day 360 Efficacy assessment 1 - 2984 2444 18476 6317 - 2 23414 10427 4283 41920 6209 10494 - 3 34942 10430 2966 2546 9650 18855 6410 4 29210 7215 908 1382 2580 4262 -

[0496] Figure 12

[0497] In addition to minidystrophin and CK levels, efficacy was measured by the following functional tests: time to get up from the floor, climb 4 steps, North Star Ambulatory Assessment (NSAA), get up time test, 4-Stair Up Test, 10-meter timed test (10m), and 100-meter timed test (100m). Data are provided in Tables 6 and 7 below, and this data shows consistent and sustained improvement at 9 months after administration of rAAVrh74.MHCK7.minidystrophin. Safety assessment Improvement in NSAA over time is also provided.

[0498] Table 6: NSAA changes from baseline to day 270

[0499]

[0500] Table 7: Changes from baseline to day 270

[0501]

[0502] Efficacy assessment

[0503] No serious adverse events (SAEs) were observed in this study. Three subjects had elevated gamma-glutamyl transferase (GGT), which resolved within a week with increased steroids and returned to baseline levels. There were no other clinically significant laboratory findings. Patients typically experienced transient nausea during the first week of treatment, concurrent with an increase in steroid dose. This was not associated with elevated liver enzymes or any other abnormalities.

[0504] Example 3

[0505] Randomized, double-blind, placebo-controlled, systemic gene delivery Phase I / IIa clinical trial

[0506] This was a randomized, double-blind, single-dose trial of rAAVrh74.MHCK7. micro-dystrophin in DMD subjects. This study included 24 subjects aged 4 to 7 years. Subjects were randomly assigned to the treatment or placebo group at the time of recruitment. Twelve subjects received the rAAVrh74.MHCK7. micro-dystrophin vector (2×10 14 vg / kg, approximately 10 mL / kg) intravenously, and 13 subjects received 10 mL / kg of placebo (lactated Ringer's solution). Placebo subjects continued with treatment, which was given in the same manner as the 12 previously treated subjects one year after the last treated subject was dosed. Subjects received an infusion of rAAV with micro-dystrophin or lactated Ringer's solution over approximately 1 hour. Muscle biopsies of the gastrocnemius muscle were performed before and after treatment (90 days).

[0507] The primary objective of this study was to evaluate the safety of intravenous administration of rAAVrh74.MHCK7.minidystrophin to DMD subjects via the peripheral limb veins. Safety endpoints were evaluated by changes in hematology, serum chemistry, urinalysis, immune responses to rAAVrh74 and minidystrophin, and reported medical history and symptom observations. Dystrophin gene expression was used as a primary outcome measure together with safety and was quantified using validated immunofluorescence and immunoblot assays. Reduction of CK after gene therapy was used as a secondary outcome. Efficacy was measured by the following functional tests: time to rise from sitting, ascending 4 steps, North Star Ambulatory Assessment (NSAA), 10-meter timed test (10m), 100-meter timed test (100m). Exploratory measures included hand-held dynamometry (HHD) for knee extensors and flexors and elbow flexors and extensors.

[0508] The inclusion criteria for this study were as follows:

[0509] · Enrollment age: between 4 - 7 years, inclusive of endpoints.

[0510] · Molecular characterization of the DMD gene with a frameshift (deletion or duplication) or premature stop codon mutation between exons 18 to 58.

[0511] · Indication of symptomatic muscular dystrophy: CK elevation > 1000 U / L and < percentage of mean predicted time on the 100-meter walk test.

[0512] · Males of any race were eligible.

[0513] · Able to cooperate with the motor assessment tests.

[0514] · Oral stable dose equivalent of corticosteroids for at least 12 weeks prior to screening and expected to remain unchanged throughout the study (except for possible modification to accommodate weight changes).

[0515] The exclusion criteria for this study were as follows:

[0516] · Active viral infection based on clinical observations.

[0517] · Signs of cardiomyopathy, including an echocardiographic ejection fraction < 40%.

[0518] · Serological evidence of HIV infection, or hepatitis B or C infection.

[0519] · Diagnosis of (or being treated for) an autoimmune disease.

[0520] · Abnormal laboratory values considered clinically significant (GGT > 3X ULN, bilirubin ≥ 3.0

[0521] mg / dL, creatinine ≥ 1.8 mg / dL, Hgb < 8 or > 18 g / Dl; WBC > 18,500 cells / cmm), platelets ≤ 50,000.

[0522] · Concomitant diseases or need for long-term drug therapy that the PI believes pose an unnecessary risk to gene transfer.

[0523] · Subject's AAVrh74 or AAV8 antibody titer > 1:400 as measured by ELISA immunoassay. If the endpoint titer is positive at screening, the test can be repeated before exclusion.

[0524] · Have a medical condition or special circumstance that the researchers believe may impair the subject's ability to comply with the tests or procedures required by the protocol, or may impair the subject's health, safety, or clinical interpretability.

[0525] · Have had a severe infection (e.g., pneumonia, pyelonephritis, or meningitis) within 4 weeks prior to the gene transfer visit (recruitment may be postponed).

[0526] · Received any investigational drug (except corticosteroids) or exon skipping drug (including ExonDys ) within the last 6 months prior to screening in this study, whether experimental or otherwise.

[0527] · Have undergone any type of gene therapy, cell-based therapy (e.g., stem cell transplantation), or CRISPR / Cas9 therapy.

[0528] · Family members do not want to disclose the subject's study participation to the attending physician and other healthcare providers.

[0529] Efficacy analysis

[0530] Dystrophin gene expression is used as a primary outcome measure together with safety. Quantification is performed using validated immunofluorescence and immunoblot assays. The reduction of CK after gene therapy is used as a secondary outcome. In addition, efficacy is measured by the following functional tests: time to rise from the floor, climb 4 steps, North Star Ambulatory Assessment (NSAA), 10-meter timed test (10m), 100-meter timed test (100m). Exploratory measures include hand-held dynamometry (HHD) for knee extensors and flexors, and elbow flexors and extensors.

[0531] Muscle biopsies were performed under ultrasound guidance to quantify transgene expression and compare expression at baseline and day 90. Biopsies were performed in the same muscle as the initial biopsy but on the opposite leg. One year after dosing all subjects, placebo crossover subjects restarted the study schedule at Visit 1. Placebo subjects did not have the following at the second baseline screen: cardiac MRI and muscle biopsy. Placebo subjects had a muscle biopsy at day 90 (total of 3 muscle biopsies). Cryosections were stained for dystrophin using indirect immunofluorescence (IF). Whole slide scans were performed and microdystrophin intensity and percentage of positive fibers were quantified using a validated image scanning and MuscleMapTM analysis algorithm. Muscle morphometry was performed blinded and included fiber size histograms. Blinded frozen muscle biopsy shavings were used for quantitative protein analysis of microdystrophin using a validated Western blot method.

[0532] Microdystrophin expression was quantified using a percutaneous muscle biopsy of the gastrocnemius muscle (unless the PI considered this procedure contraindicated for a particular subject, in which case the PI would select an alternative muscle for biopsy).

[0533] Figure 5

[0534] The primary efficacy endpoint was the change in the amount of microdystrophin expression from baseline to day 90 as measured by Western blot of muscle tissue obtained by biopsy. Treatment group differences in the primary efficacy endpoint were evaluated using an analysis of covariance (ANCOVA) model with treatment as the fixed factor and baseline value as the covariate. A Wilcoxon rank-sum test was performed as a supportive analysis. Changes in microdystrophin expression from baseline were analyzed in a similar manner via immunofluorescence (IF) fiber intensity.

[0535] Supportive efficacy endpoints included changes from baseline to each pre-specified assessment regarding time to rise from the floor, climb 4 steps, NSAA, 10-meter timed test (10m), 100-meter timed test (100m), and CK. Exploratory measures included HHD for knee extensors and flexors and elbow flexors and extensors. Treatment group differences were evaluated using an ANCOVA model with treatment as the fixed factor and baseline value as the covariate. A Wilcoxon rank-sum test was performed as a supportive analysis.

[0536] Example 4

[0537] Alternatively, the assays and studies described in Examples 2 and 3 above were performed using the rAAVrh74.MHCK7.minidystrophin construct shown in SEQ ID NO:9, nucleotides 1 - 4977 of SEQ ID NO:8, or nucleotides 56 - 5022 of SEQ ID NO:6.

[0538] Example 5

[0539] Generation of the pAAV.MCK.minidystrophin construct

[0540] The pAAV.MCK.minidystrophin plasmid was constructed by inserting the MCK expression cassette driving the codon - optimized human minidystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski, R. J. et al., J. Virol. 61(10):3096 - 3101(1987)). Muscle - specific regulatory elements were included in the construct to drive muscle - specific gene expression. The regulatory element contains the murine MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). After the core promoter, the construct contains 53 bp of endogenous murine MCK exon 1 (untranslated) for efficient transcriptional initiation, followed by the SV40 late 16S / 19S splice signal (97 bp) and a small 5'UTR (61 bp). The intron and 5'UTR are derived from the plasmid pCMVβ (Clontech). The minidystrophin cassette has a consensus Kozak 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 minidystrophin cassette contains the (R4 - R23 / Δ71 - 78) domain.

[0541] The pAAV.MCK.minidystrophin plasmid contains the human minidystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 19A ). This sequence was packaged into AAVrh.74 viral particles. The molecular clone of serotype AAVrh.74 was cloned from rhesus monkey lymph nodes as described by Rodino - Klapac et al., J Transl. Med. 5:45(2007).

[0542] Example 6

[0543] Generation of rAAV using hybrid - primer chain amplification

[0544] The rAAV constructs described herein can be generated using the following method.

[0545] The HEK-293 cells were passaged four times under adherent conditions. Before the second-to-last amplification culture, the cells were collected and centrifuged to wash away the serum (at 300 g for 5 minutes), and then resuspended in serum-free growth medium (EXPI293) in a suspension shake flask at an inoculation density of 0.5 + E6 cells / mL. Subsequently, they were allowed to grow and the number was amplified for 48 - 72 hours. Then, according to the number of viable cells required for bioreactor inoculation, the suspended cells were collected and inoculated into a shake flask or a WAVE bag. At the end of 72 hours, the concentration of viable cells was measured using a cell counting device. Then, the necessary volume containing the preferred total viable cells was added to an adherent bioreactor containing DMEM and 10% FBS. Considering the added volume of the serum-free suspension culture, FBS was appropriately supplemented so that the final FBS concentration was maintained at 10%.

[0546] As shown in Figure 18, the cell viability in the inoculation chain system and the adherent system was similar. Regarding the viable cell density (VCD), in the hybrid inoculation chain system, the VCD after the 6th passage was higher than that after the 1st passage ( Figure 19B ), which was comparable to that in the adherent system ( Characteristics ).

[0547] After inoculating the adherent bioreactor , the adherent culture was transiently transfected with a transgenic plasmid carrying the mini-dystrophin construct described herein, including, for example, the construct shown in SEQ ID NO:9 contained in the transgenic plasmid of SEQ ID NO:8. In addition to the transgenic plasmid, a rep / cap plasmid (AAV2 rep / rh74cap) and a helper plasmid were also included. After the required growth period, rAAV particles were collected by cell lysis and column chromatography.

[0548] In some aspects, rAAV is produced by a suspension inoculation method, which includes:

[0549] (a) culturing cells in a first growth medium containing serum in an N-2 container;

[0550] (b) removing the cells from the first medium;

[0551] (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing a lower concentration of serum than the first medium;

[0552] (d) culturing the cells in suspension in the N-1 container; and

[0553] (e) inoculating the bioreactor with the cells from step (d) in a third medium.

[0554] In some aspects, the suspension inoculation method further includes:

[0555] (f) Transfecting the cells with a transgenic plasmid containing the rAAVrh74.MHCK7.minidystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

[0556] In some aspects, the transgenic plasmid containing the rAAVrh74.MHCK7.minidystrophin construct contains: the nucleic acid sequence of SEQ ID NO:9; nucleotides 55 - 5021 of SEQ ID NO:3; or nucleotides 1 - 4977 of SEQ ID NO:8. In some aspects, the plasmid containing the AAV rep gene and the AAV cap gene contains the AAV2 rep gene and the rAAVrh74 cap gene.

[0557] In some aspects, the adenovirus helper plasmid contains the adenovirus 5 E2A, E4 ORF6, and VA RNA genes.

[0558] In some aspects, the suspension inoculation method further includes: (g) lysing the cells. In some aspects, the cells are lysed by freeze - thaw, solid shearing, hypertonic and / or hypotonic lysis, liquid shearing, sonication, high - pressure extrusion, detergent lysis, or a combination thereof.

[0559] In some aspects, the suspension inoculation method further includes: (h) purifying the rAAV by at least one column chromatography step. In some aspects, the at least one column chromatography step includes anion - exchange chromatography, size - exclusion chromatography, or a combination thereof.

[0560] Example 7

[0561] An open - label, systemic gene delivery study using commercially representative materials to evaluate the safety and expression of the rAAVrh74.MHCK7.minidystrophin construct in subjects with Duchenne muscular dystrophy

[0562] This is a Phase 1b open-label study of a commercially representative material using the rAAVrh74.MHCK7.minidystrophin construct, conducted in boys with Duchenne muscular dystrophy. Initially, 20 patients were recruited, and Example 7 provides data for the first 11 patients <8 years of age (e.g., 2 patients 4 to 5 years of age; 9 patients 6 to 7 years of age) (Group 1; 20 ambulant male DMD subjects aged ≥4 years to <8 years) (Table 7). The study was later expanded to include Group 2 (approximately 6 ambulant male DMD subjects aged ≥8 years to <18 years) and Group 3 (approximately 6 non-ambulant male DMD subjects), as further described in Example 8.

[0563] Table 8: Baseline demographics: First 11 patients in Group 1

[0564] 4 - 5 year - old group (N = 2) 6 - 7 year - old group (N = 9) Total (N = 11) Age (years); mean value (SD) Gender, male; n (%) 5.5(0.6) 6.8(0.7) 6.6(0.9) Race, Caucasian; n (%) 2(100.0) 9(100.0) 11(100.0) Years since DMD diagnosis; mean value (SD) 1(50.0) 6(66.7) 7(63.6.) Dosing weight (kg); mean value (SD) 2.3(0.7) 2.8(1.8) 2.7(1.7) NSAA at baseline; mean value (SD) 24.5(0.3) 23.4(4.4) 23.6(4.0) <![CDATA[Body mass index (kg / m 2 ); mean (SD)]]> 20.2(2.3) 17.5(2.9) 18.0(2.9) The inclusion criteria for the studies applicable to Examples 7 and 8 are as follows: 21.5(4.9) 22.6(3.2) 22.4(3.3)

[0565] The primary objective was to evaluate minidystrophin expression of the rAAVrh74.MHCK7.minidystrophin construct (e.g., commercially representative material) at 12 weeks post-infusion (Part 1), measured by Western blot of biopsied muscle tissue, where the corresponding endpoint was the change in minidystrophin expression level measured by Western blot from baseline to 12 weeks (Part 1). The secondary objectives were: (1) to evaluate minidystrophin protein expression by immunofluorescence (IF) fiber intensity at 12 weeks; (2) to evaluate minidystrophin expression by IF percentage of dystrophin-positive fibers (PDPF) at 12 weeks; and (3) to evaluate safety.

[0566] All

[0567] Subjects must meet The exclusion criteria for this study are as follows: the following criteria to be eligible for this study:

[0568] 1. Only Group 1 (ambulant, <8 years): Male at birth, ambulant, and at screening ≥4 to <8 years of age, and NSAA score >17 and ≤26 at the screening visit.

[0569] 2. Only Group 2 (ambulant, ≥8 years): Male at birth, ambulant, and at screening ≥8 to <18 years of age, and NSAA score ≥15 and ≤26 at the screening visit.

[0570] 3. Only Group 3 (non-ambulatory): Male at birth and unable to walk for at least 9 months, with an NSAA walking score of "0" and unable to perform the 10MWR at screening visit, and a PUL enrollment item score ≥ 2. The onset of loss of walking ability was defined as the age at which the participant or caregiver reported continuous use of a wheelchair, approximated to the nearest month.

[0571] 4. Prior to screening, a definitive diagnosis of DMD was made based on clinical examination findings documentation and confirmatory genetic testing previously performed using clinical diagnostic genetic tests.

[0572] 5. Have signs of symptomatic muscular dystrophy:

[0573] · CK elevation > 1000 U / L, and

[0574] · Only Groups 1 and 2 (ambulatory): Below 95% predicted time at 100MWR.

[0575] 6. Be able to cooperate with the motor assessment tests.

[0576] 7. Prior to screening, receive an oral stable weekly dose equivalent of corticosteroids for at least 12 weeks, and the dose is expected to remain constant during the first year of the study (except for modifications to accommodate weight changes).

[0577] 8. As measured by ELISA, the AAVrh74 antibody titer ≤ 1:400 (i.e., not elevated).

[0578] 9. Sexually active subjects must agree to use condoms throughout the duration of the study, and female sexual partners must also use medically acceptable contraception methods (e.g., oral contraceptives).

[0579] 10. Parents or legal guardians or subjects ≥ 18 years old are able to understand and comply with the study visit schedule and all other protocol requirements.

[0580] 11. Willing to provide an informed assent or consent form (if applicable), and parents or legal guardians or subjects ≥ 18 years old are willing to provide written informed consent for the subject's participation in the study.

[0581] Any one

[0582] Subjects who meet the following criteria Efficacy assessment: will be excluded from the study:

[0583] 1. Left ventricular ejection fraction on screening ECHO < 40% or have clinical signs and / or symptoms of cardiomyopathy.

[0584] 2. Only in Groups 2 and 3 (ambulatory, ≥8 years old and non-ambulatory): Forced vital capacity (FVC) < 50% of predicted value and / or need for nocturnal respiratory support at screening.

[0585] 3. Had major surgery within 3 months prior to Day 1 or is scheduled for surgery at any time during the study.

[0586] 4. Have any other major genetic disease other than DMD.

[0587] 5. Have serological evidence of current, chronic, or active human immunodeficiency virus, hepatitis C, or hepatitis B infection.

[0588] 6. Diagnosed with an autoimmune disease.

[0589] 7. Have a concomitant disease or require long-term drug treatment that the investigator believes poses an undue risk to gene transfer.

[0590] 8. Medical conditions or special circumstances that the investigator believes may impair the subject's ability to comply with protocol-required tests or procedures, or may compromise the subject's health, safety, or clinical interpretability.

[0591] 9. Had a symptomatic infection (e.g., upper respiratory tract infection, pneumonia, pyelonephritis, meningitis) within 4 weeks prior to Day 1.

[0592] 10. Exhibit cognitive retardation or impairment that the investigator believes may affect motor development.

[0593] 11. Treated with any of the following therapies within the specified time frames:

[0594] · At any time:

[0595] - Gene therapy,

[0596] - Cell-based therapy (e.g., stem cell transplantation),

[0597] - CRISPR / Cas9 or any other form of gene editing,

[0598] · Within 12 weeks from Day 1:

[0599] - Use of human growth factors or vamorolone,

[0600] · Within 6 months from Day 1:

[0601] - Any investigational drug,

[0602] - Only in Group 1: Any therapeutic agent designed to increase dystrophin expression (e.g., TranslarnaTM , EXONDYS 51, VYONDYS 53, VILTEPSO TM ). Note: Subjects in Groups 2 and 3 are expected to discontinue these therapeutic agents prior to Day 1. Therapeutic agents aimed at increasing dystrophin expression may be resumed and / or initiated after Week 72.

[0603] 12. Have received a live virus vaccine within 4 weeks of the Day 1 visit, or an inactivated vaccine within 2 weeks of the Day 1 visit, or are expected to receive vaccination within the first 3 months after Day 1.

[0604] 13. Have abnormal laboratory values considered to be of clinical significance, including but not limited to:

[0605] · Gamma-glutamyl transferase (GGT) > 2 x upper limit of normal (ULN),

[0606] · Total bilirubin > ULN. Note that elevations in total bilirubin considered to be due to Gilbert's syndrome are not excluded.

[0607] · White blood cell count > 18,500 / μl,

[0608] · Platelets ≤ 150,000 / μL.

[0609] 14. The subject or family does not want to disclose the subject's study participation to the general practitioner / attending physician and other healthcare providers.

[0610] The investigators believe that the subject is unlikely to comply with the study protocol. All subjects received the rAAVrh74.MHCK7.minidystrophin construct (1.33 x 10 14 vg / kg) (e.g., commercially representative material) by a single intravenous (IV) infusion.

[0611] Figure 20

[0612] Muscle biopsies for evaluation of minidystrophin expression were collected from all subjects at baseline and Week 12. Muscle biopsies were obtained using ...

Claims

1. A method for producing recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7. micro-dystrophin in adherent mammalian cells by suspension seeding, comprising: (a) culturing the cells in a first growth medium containing serum in an N-2 container; (b) removing the cells from the first medium; (c) inoculating the cells from step (b) into a second medium in an N-1 container, the second medium being serum-free or containing a lower concentration of serum than the first medium; (d) culturing the cells in suspension in the N-1 container; and (e) inoculating the third medium in a bioreactor with the cells from step (d).

2. The method according to claim 1, wherein the rAAV contains the human micro-dystrophin nucleotide sequence of SEQ ID NO:

1.

3. The method according to claim 2, wherein the rAAV contains the MHCK7 promoter sequence of SEQ ID NO:

7.

4. The method according to any one of claims 1 to 3, wherein the rAAV contains the human micro-dystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:

7.

5. The method according to any one of claims 1 to 4, wherein the suspension seeding method further comprises: (f) transfecting the adherent cells with a transgenic plasmid containing the rAAVrh74.MHCK7. micro-dystrophin construct, a plasmid containing the AAV rep gene and the AAV cap gene, and an adenovirus helper plasmid.

6. The method according to claim 5, wherein the transgenic plasmid containing the rAAVrh74.MHCK7. micro-dystrophin construct contains: the nucleic acid sequence of SEQ ID NO:9; nucleotides 55 - 5021 of SEQ ID NO:3; or nucleotides 1 - 4977 of SEQ ID NO:

8.

7. The method according to claim 5 or 6, wherein the plasmid containing the AAV rep gene and the AAV cap gene contains the AAV2 rep gene and the rAAVrh74 cap gene.

8. The method according to any one of claims 5 to 7, wherein the adenovirus helper plasmid contains the adenovirus 5E2A, E4ORF6, and VA RNA genes.

9. The method according to any one of claims 1 to 8, wherein the suspension seeding method further comprises: (g) lysing the adherent cells.

10. The method according to claim 9, wherein the adherent cells are lysed by freeze-thaw, solid shearing, hypertonic and / or hypotonic lysis, liquid shearing, sonication, high-pressure extrusion, detergent lysis, or a combination thereof.

11. The method according to any one of claims 1 to 10, wherein the suspension seeding method further comprises: (h) purifying the rAAV by at least one column chromatography step.

12. The method according to claim 11, wherein the at least one column chromatography step comprises anion exchange chromatography, size exclusion chromatography, or a combination thereof.

13. The method according to any one of claims 1 to 12, wherein the suspension seeding method further comprises culturing the cells in the first growth medium in an N-3 container.

14. The method according to claim 13, wherein the suspension seeding method further comprises culturing the cells in the first growth medium in an N-4 container.

15. The method according to any one of claims 1 to 14, wherein the bioreactor is an adherent bioreactor.

16. The method according to claim 15, wherein the rAAV is purified from the culture produced in the adherent bioreactor.

17. The method according to claim 15 or 16, wherein the third medium in the bioreactor comprises at least one factor promoting cell adhesion.

18. The method according to claim 17, wherein the at least one factor promoting cell adhesion is selected from serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix, and combinations thereof.

19. The method according to claim 17 or 18, wherein the third medium in the bioreactor comprises DMEM and 10% FBS.

20. The method according to any one of claims 1 to 19, wherein the adherent cells are cultured under suspension conditions for about 48 - 72 hours.

21. The method according to any one of claims 1 to 20, wherein the N-1 container is a suspension shake flask.

22. The method according to any one of claims 1 to 21, wherein the adherent cells are selected from HeLa cells, CHO cells, HEK-293 cells, VERO cells, BHK cells, MDCK cells, MDBK cells, and COS cells.

23. The method according to claim 22, wherein the adherent cells are HeLa cells or HEK-293 cells.

24. The method according to claim 23, wherein the adherent cells are HEK-293 cells.

25. The method according to any one of claims 1 to 24, wherein the adherent cells are not suspension-adapted.

26. The method according to any one of claims 1 to 25, wherein culturing the cells under suspension conditions does not change the cell's adhesion dependence.

27. The method according to any one of claims 1 to 26, wherein the culturing does not change the cells to produce a new cell line.

28. A composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin, wherein the rAAV is produced by the method according to any one of claims 1 to 27.

29. The composition according to claim 28, wherein the composition comprises: a) rAAV particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsulating nucleotides 55 - 5021 of SEQ ID NO:3; and / or c) rAAV particles encapsulating nucleotides 1 - 4977 of SEQ ID NO:

8.

30. A method for treating muscular dystrophy in a human subject in need thereof, comprising administering the composition according to claim 29 to the human subject.

31. The method according to claim 30, wherein the systemic administration route is used and the rAAV is administered at a dose of about 5.0×10 12 vg / kg to about 1.0×10 15 vg / kg.

32. The method according to claim 31, wherein the systemic administration route is an intravenous route, and the administration dose of the rAAV is about 2×10 14 vg / kg.

33. The method according to any one of claims 30 to 32, wherein the rAAV of the dose is administered at a concentration of about 10 mL / kg.

34. The method according to any one of claims 30 to 33, wherein the rAAV is administered by injection, infusion or implantation.

35. The method according to claim 34, wherein the rAAV is administered by infusion within about one hour.

36. The method according to any one of claims 30 to 35, wherein the rAAV is administered via a peripheral limb vein by an intravenous route.

37. The method according to any one of claims 30 to 36, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

38. The method according to claim 37, wherein the muscular dystrophy is Duchenne muscular dystrophy.

39. The method according to any one of claims 30 to 38, wherein the level of micro - dystrophin gene expression in the cells of the subject increases after administering the rAAV as compared to the level of micro - dystrophin gene expression before administering the rAAV.

40. The method according to claim 39, wherein the expression of the micro - dystrophin gene in the cells is detected by measuring the level of micro - dystrophin by Western blot of a muscle biopsy before and after administering the rAAV.

41. The method according to claim 40, wherein the expression after administering the rAAV is at least 55.4% as compared to before administration.

42. The method according to any one of claims 30 to 41, wherein the average percentage of micro - dystrophin - positive fibers in the muscle tissue of the subject increases after administering the rAAV as compared to the number of micro - dystrophin - positive fibers before administering the rAAV.

43. The method according to claim 42, wherein the average percentage of micro - dystrophin - positive fibers is at least 70.5% and the average intensity is at least 116.9%, as detected by immunofluorescence (IF) in muscle biopsies before and after administering the rAAV.

44. The method according to any one of claims 30 to 43, wherein the micro - dystrophin transduction counted by vector genome is at least 3.87 average vector genome copies per cell nucleus.

45. The method according to any one of claims 30 to 44, wherein at least one mutation in the human dystrophin (DMD) gene of the subject has been genotyped.

46. The method according to claim 45, wherein the at least one mutation is a frameshift deletion, frameshift duplication, premature termination, or other pathogenic variant that results in the absence of expression of the human dystrophin.

47. Use of the composition according to claim 28 or 29 for the treatment of muscular dystrophy in a human subject in need thereof.

48. Use of the composition according to claim 28 or 29 in the preparation of a medicament for the treatment of muscular dystrophy.

49. The use according to claim 47 or 48, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

50. The use according to claim 49, wherein the muscular dystrophy is Duchenne muscular dystrophy.

51. A method for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles, the AAV viral particles encapsulating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion of exons 9-13 of the DMD gene in its entirety.

52. A method for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising AAV viral particles, the AAV viral particles encapsulating an expression cassette comprising a human micro-dystrophin transgene, provided that the subject does not have a deletion of exon 8 and / or exon 9 of the human dystrophin (DMD) gene.

53. The method according to claim 51 or 52, wherein the DMD gene of the subject is genotyped prior to treatment.

54. The method according to any one of claims 51 to 53, wherein the AAV viral particles are rh74 serotype AAV viral particles.

55. The method according to claim 54, wherein the rAAV vector is administered as a composition, the composition comprising: a) rh74 serotype AAV viral particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rh74 serotype AAV viral particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rh74 serotype AAV viral particles encapsulating nucleotides 1-4977 of SEQ ID NO:

8.

56. A method for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) if genotyping does not identify a deletion of exons 9-13 of the DMD gene in its entirety, administering to the subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.micro-dystrophin; wherein the composition comprises: a) rAAV particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsulating nucleotides 55-5021 of SEQ ID NO:3; and / or c) rAAV particles encapsulating nucleotides 1 - 4977 of SEQ ID NO:

8.

57. A method for treating Duchenne muscular dystrophy in a human subject in need thereof, comprising: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) if the genotyping does not identify a deletion of exon 8 and / or 9 of the DMD gene, administering to the subject a composition comprising recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin; wherein the composition comprises: a) rAAV particles encapsulating the nucleic acid sequence of SEQ ID NO:9; b) rAAV particles encapsulating nucleotides 55 - 5021 of SEQ ID NO:3; and / or c) rAAV particles encapsulating nucleotides 1 - 4977 of SEQ ID NO:

8.

58. The method according to any one of claims 30 - 46 or 51 - 57, wherein the human subject is ambulant.

59. The method according to any one of claims 30 - 46 or 51 - 57, wherein the human subject is non-ambulant.

60. The method according to any one of claims 30 - 46 or 51 - 59, wherein the human subject is 2 to 3 years old.

61. The method according to any one of claims 30 - 46 or 51 - 59, wherein the human subject is 4 to 5 years old.

62. The method according to claim 59, wherein the human subject has been non-ambulant for at least 9 months.

63. The method according to claim 62, wherein the human subject has a stable forced vital capacity (FVC) of less than 40% of predicted value and / or requires nocturnal ventilator support.

64. The method according to any one of claims 56 - 63, wherein the recombinant adeno-associated virus (rAAV) rAAVrh74.MHCK7.minidystrophin is made by the method according to any one of claims 1 and 7 - 27.

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