Methods of treating muscular dystrophy

Abstract

The present disclosure relates to methods of treating muscular dystrophy in a subject in need thereof. In certain aspects, the methods comprise administering to the subject an AAV vector, such as an AAVrh74 vector, and an enzyme that cleaves IgG.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 357,029, filed on June 30, 2022, and U.S. Provisional Application No. 63 / 382,047, filed on November 2, 2022. The entire teachings of the above applications are incorporated by reference in their entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The contents of the electronically submitted sequence listing (Name: 8183WO00_SequenceListing.XML; Size: 91 kilobytes; Creation Date: June 28, 2023) submitted with this application are incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure provides methods for treating muscular dystrophy in a human subject in need thereof. Background Art

[0006] Muscular dystrophy (MD) is a rare but highly debilitating class of genetic disorders. For example, Duchenne muscular dystrophy (DMD) is caused by a defect in the expression of the protein dystrophin. The gene encoding the protein contains seventy-nine exons distributed over more than two million nucleotides of DNA. Any exon mutation that changes the reading frame of the exon, or introduces a stop codon, or is characterized by the entire removal of one or more frame exons or duplication of one or more exons may disrupt the production of functional dystrophin, thereby producing MD.

[0007] Gene therapy provides a lasting means of treating a variety of diseases, and adeno-associated virus (AAV) is one of the most commonly studied gene therapy vectors. AAV is a protein shell that surrounds and protects a small single-stranded DNA genome of approximately 4.8 kilobases (kb). Naso et al., BioDrugs, 31(4): 317-334 (2017). AAV belongs to the parvovirus family and relies on co-infection with other viruses (mainly adenoviruses) for replication. Same as above. Its single-stranded genome contains three genes: Rep (replication), Cap (capsid) and aap (assembly). Same as above. These coding sequences are flanked by reverse terminal repeats (ITRs) required for genome replication and packaging. Same as above. The two cis-acting AAV ITRs are approximately 145 nucleotides in length, with an interrupted palindromic sequence that can fold into a T-shaped hairpin structure that acts as a primer during DNA replication initiation.

[0008] Although early results in animal models suggest that AAV gene therapy may be a viable approach for treating DMD and BMD, it was found that the AAV copy number decreased over time, which was associated with reduced efficacy. One suspected reason for this decline in the AAV copy number is the body's immune response to the AAV viral particles. After the first AAV therapy, human subjects will typically produce neutralizing antibodies that promote the destruction of foreign AAV vectors. Therefore, the AAV copy number decreases over time, and subsequent therapies using the same vector may result in a complete lack of response. Therefore, there is still a need for improved treatment methods for treating MD. Summary of the invention

[0009] Certain aspects of the present disclosure relate to a method of treating a subject suffering from muscular dystrophy, the method comprising administering to the subject a recombinant AAVrh74 viral vector and an enzyme that cleaves immunoglobulin G (IgG).

[0010] In some aspects, the recombinant AAVrh74 viral vector and the IgG-cleaving enzyme are administered to the subject simultaneously.

[0011] In some aspects, the recombinant AAVrh74 viral vector and the enzyme that cleaves IgG are administered to the subject sequentially.

[0012] In some aspects, the subject is determined to have anti-AAVrh74 antibodies.

[0013] In some aspects, the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

[0014] In some aspects, the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

[0015] In some aspects, the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

[0016] In some aspects, the subject has an anti-AAVrh74 antibody titer greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

[0017] In some aspects, the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

[0018] In some aspects, the recombinant AAVrh74 viral vector is administered after the enzyme that cleaves IgG.

[0019] In some aspects, the subject is administered the recombinant AAVrh74 viral vector no greater than 54 hours after the administration of the enzyme that cleaves IgG.

[0020] In some aspects, the method further comprises administering to the subject a second dose of the IgG-cleaving enzyme prior to administering the recombinant AAVrh74 viral vector if the subject is determined to have anti-AAVrh74 antibodies after administration of the first dose of the IgG-cleaving enzyme.

[0021] In some aspects, the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

[0022] In some aspects, the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

[0023] In some aspects, the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

[0024] In some aspects, the subject has an anti-AAVrh74 antibody titer greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

[0025] In some aspects, the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

[0026] In some aspects, the recombinant AAVrh74 viral vector is administered after the second dose of the IgG-cleaving enzyme.

[0027] In some aspects, the subject is administered the recombinant AAVrh74 viral vector no greater than 54 hours after the administration of the second dose of the IgG-cleaving enzyme.

[0028] In some aspects, the subject is administered the second dose of the enzyme no greater than 60 hours after the first dose of the IgG-cleaving enzyme.

[0029] In some aspects, the subject has an anti-AAVrh74 antibody titer of about 1:1600 to about 1:3200 in a total anti-AAVrh74 antibody ELISA assay.

[0030] In some aspects, the method further comprises administering to the subject a third dose of the IgG-cleaving enzyme prior to administering the recombinant AAVrh74 viral vector.

[0031] In some aspects, the recombinant AAVrh74 viral vector is administered after the third dose of the IgG-cleaving enzyme.

[0032] In some aspects, the subject is administered the recombinant AAVrh74 viral vector no greater than 54 hours after the administration of the third dose of the IgG-cleaving enzyme.

[0033] In some aspects, the subject is administered the third dose of the enzyme no greater than 60 hours after the second dose of the IgG-cleaving enzyme.

[0034] Further provided is a method of treating a subject suffering from muscular dystrophy, the method comprising administering to the subject a recombinant AAVrh74 viral vector, wherein the subject has previously been administered an enzyme that cleaves IgG.

[0035] In some aspects, the subject has been administered an enzyme that cleaves IgG no more than 54 hours prior to administration of the recombinant AAVrh74 viral vector.

[0036] A method of preparing a subject for gene therapy for muscular dystrophy is provided, the method comprising administering to the subject an enzyme that cleaves IgG prior to administering a recombinant AAVrh74 viral vector.

[0037] A method for removing anti-AAVrh74 antibodies in a subject is provided, the method comprising administering an enzyme that cleaves IgG, and measuring the titer of anti-rAAVrh74 antibodies in the subject after administering the enzyme that cleaves IgG.

[0038] In some aspects, the method further comprises measuring the titer of anti-rAAVrh74 antibodies in the subject prior to administering the IgG-cleaving enzyme.

[0039] In some aspects, the method further comprises administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is at or below 1:400.

[0040] In some aspects, if the anti-rAAVrh74 antibodies in the subject are above 1:400 in a total anti-AAVrh74 antibody ELISA assay, the recombinant AAVrh74 viral vector is not administered to the subject.

[0041] In some aspects, the method further comprises administering to the subject a second dose of the IgG-cleaving enzyme.

[0042] In some aspects, the method further comprises measuring the titer of anti-rAAVrh74 antibodies in the subject after the second dose of the IgG-cleaving enzyme.

[0043] In some aspects, the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

[0044] In some aspects, the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

[0045] In some aspects, the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

[0046] In some aspects, the subject has an anti-AAVrh74 antibody titer greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

[0047] In some aspects, the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

[0048] In some aspects, the method further comprises administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is less than 1:400.

[0049] In some aspects, the recombinant AAVrh74 viral vector is administered no greater than 54 hours after the second dose of the IgG-cleaving enzyme.

[0050] In some aspects, if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is higher than 1:400, the recombinant AAVrh74 viral vector is not administered to the subject.

[0051] In some aspects, the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy (LGMD).

[0052] In some aspects, the muscular dystrophy is DMD.

[0053] In some aspects, the IgG is human IgG.

[0054] In some aspects, the IgG-cleaving enzyme specifically targets and cleaves human IgG selected from IgG1, IgG2, IgG3, IgG4, and any combination thereof.

[0055] In some aspects, the IgG-cleaving enzyme specifically targets and cleaves human IgG1, human IgG2, human IgG3, and human IgG4.

[0056] In some aspects, the IgG is associated with the recombinant AAVrh74 viral vector.

[0057] In some aspects, the enzyme that cleaves immunoglobulin IgG comprises a protease.

[0058] In some aspects, the protease comprises a cysteine ​​protease or a thiol protease.

[0059] In some aspects, the enzyme that cleaves IgG inactivates the IgG.

[0060] In some aspects, the IgG-cleaving enzyme cleaves human IgG at the hinge region.

[0061] In some aspects, the protease is isolated or derived from a protease expressed by Streptococcus pyogenes, Streptococcus equi, or Mycoplasma canis.

[0062] In some aspects, the enzyme that cleaves IgG comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1 or 2.

[0063] In some aspects, the IgG-cleaving enzyme comprises the amino acid sequence set forth in any one of SEQ ID NOs: 3-18.

[0064] In some aspects, the enzyme that cleaves IgG comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0065] In some aspects, the enzyme is Imlifidase.

[0066] In some aspects, the subject has one or more IgG antibodies that specifically bind to a protein present in the recombinant AAVrh74 viral vector.

[0067] In some aspects, the subject has one or more IgG antibodies that specifically bind to a capsid protein of a recombinant AAVrh74 viral vector.

[0068] In some aspects, the one or more IgG antibodies are AAVrh74 neutralizing antibodies.

[0069] In some aspects, the one or more IgG antibodies are AAVrh74 non-neutralizing antibodies.

[0070] In some aspects, the one or more IgG antibodies are total AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

[0071] In some aspects, the subject has an anti-AAVrh74 antibody titer greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

[0072] In some aspects, the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

[0073] In some aspects, the subject has been previously administered a recombinant AAVrh74 viral vector.

[0074] In some aspects, the recombinant AAVrh74 viral vector comprises a gene cassette encoding a therapeutic molecule.

[0075] In some aspects, the therapeutic molecule comprises a polypeptide, an RNA molecule, or a DNA molecule.

[0076] In some aspects, the gene cassette encodes a therapeutic polypeptide selected from micro-dystrophin, beta sarcoglycan, alpha sarcoglycan, and any combination thereof.

[0077] In some aspects, the gene-cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19.

[0078] In some aspects, the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO:19.

[0079] In some aspects, the gene-cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21.

[0080] In some aspects, the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO:21.

[0081] In some aspects, the gene-cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:23.

[0082] In some aspects, the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO:23.

[0083] In some aspects, the recombinant AAVrh74 viral vector further comprises a promoter.

[0084] In some aspects, the promoter is a tissue-specific promoter.

[0085] In some aspects, the promoter is selected from the group consisting of a MHCK7 promoter and a tMCK promoter.

[0086] In some aspects, the promoter comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:25 or 26.

[0087] In some aspects, the promoter comprises the nucleic acid sequence shown in SEQ ID NO:25 or 26.

[0088] In some aspects, the recombinant AAVrh74 viral vector further comprises an intron.

[0089] In some aspects, the intron comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NOs:27-29.

[0090] In some aspects, the intron comprises the nucleic acid sequence shown in SEQ ID NOs: 27-29.

[0091] In some aspects, the recombinant AAVrh74 viral vector further comprises a 3'UTR poly (A) tail sequence.

[0092] In some aspects, the 3'UTR poly (A) tail sequence comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO:30.

[0093] In some aspects, the AAVrh74 viral vector comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a nucleic acid sequence selected from the nucleic acid sequences shown in SEQ ID NOs: 20, 22 and 24.

[0094] In some aspects, imrilase is administered at a dose of 0.25 mg / kg.

[0095] In some aspects, the recombinant AAVrh74 viral vector is delandistrogene moxeparvovec (or delandistrogene moxeparvovec-rokl), bidridistrogene xeboparvovec, or patidistrogene bexoparvovec.

[0096] In some aspects, the diandroestrogens moxpavovek, petriestrogens cospavovek, or patiestrogens becospavovek are present at about 1.33×10 14 vg / kg dose.

[0097] In some aspects, the administration of the imrilase is intravenous.

[0098] In some aspects, the administration of the diandroestrogen moxpavovec, petriestrogen cospavovec, or patiestrogen becospavovec is intravenous.

[0099] In some aspects, the recombinant AAVrh74 is the androestrogen moxepavik.

[0100] Further provided is a kit for treating muscular dystrophy in a subject in need thereof, the kit comprising (i) an enzyme that cleaves IgG, (ii) a recombinant AAVrh74 viral vector, and (iii) instructions for administering the enzyme and / or the recombinant AAVrh74 viral vector to the subject as described herein.

[0101] Provided is a use of a recombinant AAVrh74 viral vector for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof, wherein the subject has previously been administered an enzyme that cleaves IgG.

[0102] Provided is a use of a recombinant AAVrh74 viral vector in combination with an enzyme that cleaves IgG for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof.

[0103] In some aspects, the recombinant AAVrh74 viral vector and the enzyme are administered simultaneously to treat the muscular dystrophy in the subject in need thereof.

[0104] In some aspects, the recombinant AAVrh74 viral vector and the enzyme are administered sequentially.

[0105] In some aspects, the recombinant AAVrh74 viral vector is administered no later than 54 hours after the administration of the enzyme.

[0106] In some aspects, the muscular dystrophy is selected from DMD and LGMD.

[0107] In some aspects, the muscular dystrophy is DMD.

[0108] Also provided is a composition comprising a recombinant AAVrh74 viral vector for use in treating muscular dystrophy in a subject in need thereof, wherein the subject has previously been administered an enzyme that cleaves IgG.

[0109] Provided is a composition comprising a recombinant AAVrh74 viral vector in combination with an enzyme that cleaves IgG, the composition being useful for treating muscular dystrophy in a subject in need thereof.

[0110] In some aspects, the recombinant AAVrh74 viral vector and the enzyme are administered simultaneously to treat muscular dystrophy in a subject in need thereof.

[0111] In some aspects, the recombinant AAVrh74 viral vector and the enzyme are administered sequentially.

[0112] In some aspects, the recombinant AAVrh74 viral vector is administered no later than 54 hours after the administration of the enzyme.

[0113] In some aspects, the muscular dystrophy is selected from DMD and LGMD.

[0114] In some aspects, the muscular dystrophy is DMD.

[0115] Also provided is a method of removing anti-AAVrh74 antibodies to treat a subject in need thereof, the method comprising administering imrilase and measuring the titer of anti-rAAVrh74 antibodies in the subject after administration of imrilase.

[0116] In some aspects, the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

[0117] In some aspects, the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

[0118] In some aspects, the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

[0119] In some aspects, the subject has an anti-AAVrh74 antibody titer greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

[0120] In some aspects, the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

[0121] In some aspects, the method further comprises measuring the titer of anti-rAAVrh74 antibodies in the subject prior to administering imrilase.

[0122] In some aspects, the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

[0123] In some aspects, the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

[0124] In some aspects, the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

[0125] In some aspects, the subject has an anti-AAVrh74 antibody titer greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

[0126] In some aspects, the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

[0127] In some aspects, the method further comprises administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is at or below 1:400.

[0128] In some aspects, imrilase is administered at a dose of 0.25 mg / kg.

[0129] In some aspects, the recombinant AAVrh74 viral vector is diastolic estrogen moxpavovek, petreline estrogen cospovovek, or patiline estrogen becospovovek.

[0130] In some aspects, the recombinant AAVrh74 is the androestrogen moxepavik.

[0131] In some aspects, the diandroestrogens moxpavovek, petriestrogens cospavovek, or patiestrogens becospavovek are present at about 1.33×10 14 vg / kg dose.

[0132] In some aspects, the diandroestrogen moxpavovek is present at about 1.33×10 14 vg / kg dose.

[0133] In some aspects, the administration of the imrilase is intravenous.

[0134] In some aspects, the administration of the diandroestrogen moxpavovec, petriestrogen cospavovec, or patiestrogen becospavovec is intravenous.

[0135] In some aspects, if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is higher than 1:400, the recombinant AAVrh74 viral vector is not administered to the subject.

[0136] In some aspects, the method further comprises administering a second dose of imrilase to the subject.

[0137] In some aspects, the second dose of imrilase is 0.25 mg / kg.

[0138] In some aspects, the second dose of imrase is administered no greater than 60 hours after the first dose.

[0139] In some aspects, the method further comprises measuring the titer of anti-rAAVrh74 antibodies in the subject after the second dose of imrplase.

[0140] In some aspects, the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

[0141] In some aspects, the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

[0142] In some aspects, the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

[0143] In some aspects, the method further comprises administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is at or below 1:400.

[0144] In some aspects, the recombinant AAVrh74 viral vector is administered no greater than 54 hours after the second dose of imrase. BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 Schematic diagram showing the study design for imliase recipients only as described in Example 2.

[0146] Figure 2 A schematic diagram of the overall study design as described in Example 2 is shown.

[0147] Figure 3 Schematic diagram showing the study design for pre-infusion and imlirase infusion periods as described in Example 2.

[0148] Figure 4 Total antibody (TAb) responses to AAVrh74 in non-human primates as described in Example 1 are shown.

[0149] Figure 5 Total antibody (TAb) responses to AAVrh74 following imlimidase treatment in non-human primates of Groups 4, 5, and 6 as described in Example 1 are shown.

[0150] Fig. 6A Total antibody (TAb) responses to AAVrh74 following imlimidase treatment in non-human primates of Group 7 as described in Example 1 are shown. Figure 6B Total antibody (TAb) responses to AAVrh74 in nonhuman primates of Group 7 following re-administration of AAVrh74 as described in Example 1 are shown.

[0151] Figure 7 AC show Western blots of sera from Group 3 seronegative non-human primates (P0201, P0202, P0203) as described in Example 1. Animals were not treated with imliase.

[0152] Figure 8 A shows a Western blot of serum from a seropositive nonhuman primate from Group 4 (P0301) with an anti-AAVrh74 baseline titer of 1:1,600 as described in Example 1. Figure 8 B shows a Western blot of serum from a seropositive nonhuman primate from Group 4 (P0302) with an anti-AAVrh74 baseline titer of 1:400 as described in Example 1. Figure 8 C shows a Western blot of serum from a seropositive non-human primate from Group 4 (P0303) with an anti-AAVrh74 baseline titer of 1:800 as described in Example 1. Animals from Group 4 were not treated with imliase.

[0153] Fig. 9 A shows a Western blot of serum from a seropositive nonhuman primate from Group 5 (P0401) with an anti-AAVrh74 baseline titer of 1:1,600 and an anti-AAVrh74 titer of 1:200 after imliase treatment as described in Example 1. Fig. 9 B shows a Western blot of serum from a seropositive nonhuman primate from Group 5 (P0402) with an anti-AAVrh74 baseline titer of 1:800 and an anti-AAVrh74 titer of 1:50 after imlizyme as described in Example 1. Fig. 9 C shows a Western blot of serum from a seropositive nonhuman primate from Group 5 (P0403) with an anti-AAVrh74 baseline titer of 1:800 and an anti-AAVrh74 titer of 1:50 after imlizyme as described in Example 1.

[0154] Fig.10 A shows a Western blot of serum from a highly seropositive nonhuman primate from Group 6 (P0501) with an anti-AAVrh74 baseline titer of 1:6,400 and an anti-AAVrh74 titer of 1:800 after imlizyme as described in Example 1. Fig.10 B shows a Western blot of serum from a highly seropositive nonhuman primate from Group 6 (P0502) with an anti-AAVrh74 baseline titer of 1:3,200 and an anti-AAVrh74 titer of 1:200 after imlizyme as described in Example 1. Fig.10 C shows a Western blot of serum from a highly seropositive nonhuman primate from Group 6 (P0503) with an anti-AAVrh74 baseline titer of 1:3,200 and an anti-AAVrh74 titer of 1:400 after imlizyme as described in Example 1.

[0155] Fig.11 A shows a Western blot of serum from a seronegative nonhuman primate from Group 7 (P0601) with an anti-AAVrh74 baseline titer of 1:204,800 after the first dose of AAVrh74-eGFP and an anti-AAVrh74 titer of 1:6,400 after imliase as described in Example 1. Fig.11 B shows a Western blot of serum from a seronegative nonhuman primate from Group 7 (P0602) with an anti-AAVrh74 baseline titer of 1:400 after the first dose of AAVrh74-eGFP and an anti-AAVrh74 titer of 1:200 after imliase as described in Example 1. Fig.11C shows a Western blot of serum from a seronegative nonhuman primate from Group 7 (P0603) with an anti-AAVrh74 baseline titer of 1:25,600 after the first dose of AAVrh74-eGFP and an anti-AAVrh74 titer of 1:3,200 after imliase as described in Example 1.

[0156] Fig.12 GFP expression in hearts from Group 3, 4, 5, 6, and 7 animals and control Group 2 animals as described in Example 1 is shown.

[0157] Fig.13 Shown is GFP expression in skeletal muscle (gastrocnemius) from Group 3, 4, 5, 6, and 7 animals and control Group 2 animals as described in Example 1.

[0158] Fig.14 GFP expression from diaphragms in Group 3, 4, 5, 6, and 7 animals and control Group 2 animals as described in Example 1 is shown.

[0159] Fig.15 Shown are the average GFP expression of all tissues in Group 3 animals (AAVr74-eGFP vector control, AAVrh74 seronegative); Group 4 animals (AAVrh74-eGFP vector control, AAVrh74 seropositive, titer 1:800-1:1,600); Group 5 animals (Imlirase + AAVrh74-eGFP vector, AAVrh74 seropositive, titer 1:800-1:1,600); Group 6 animals (Imlirase + AAVrh74-eGFP vector, AAVrh74 seropositive, titer 1:3,200-1:25,600); and Group 7 (re-dosing group) as described in Example 1.

[0160] Fig.16 Shown is GFP expression (% positive area) in different muscle types from Group 3, 4, 5, 6, and 7 animals and saline control Group 2 animals as described in Example 1. DETAILED DESCRIPTION

[0161] The present disclosure relates to a method of treating a subject suffering from muscular dystrophy, the method comprising administering to the subject a recombinant AAVrh74 viral vector and an enzyme that cleaves immunoglobulin G (IgG). In some aspects, the muscular dystrophy is DMD. In some aspects, the muscular dystrophy is LGMD.

[0162] I. Terminology

[0163] In order to make the present disclosure more easily understood, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms will have the meanings set forth below. Additional definitions are set forth throughout the application.

[0164] It should be noted that the term "a" or "an" entity refers to one or more of the entities; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a" (or "a kind"), "one or more" and "at least one" can be used interchangeably herein.

[0165] In addition, "and / or" as used herein should be understood as explicitly disclosing each of the two specified features or components with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover 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).

[0166] The term "about" is used herein to mean approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the limits above and below the numerical values ​​set forth. Generally, unless otherwise indicated, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variation of 10% up or down (higher or lower).

[0167] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that can be logically included, as can be clearly seen from 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 indicated property. When at least appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in the numbers in the series or range. "At least" is also not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, 5.18%, without considering the number of significant figures).

[0168] It should be understood that when aspects are described herein with the language “comprising,” other similar aspects described within the terms “consisting of” and / or “consisting essentially of” are also provided.

[0169] Unless otherwise defined, the meaning of all technical terms and scientific terms used herein is the same as that commonly understood by those of ordinary skill in the art of the present disclosure. For example, the following provides a common dictionary of many terms used in the present disclosure for technical personnel: "Concise Dictionary of Biomedicine and Molecular Biology", Juo, Pei-Show, 2nd edition, 2002, CRC Press; "Dictionary of Cell and Molecular Biology", 5th edition, 2013, Academic Press; and "Oxford Dictionary of Biochemistry and Molecular Biology", 2nd edition, 2006, Oxford University Press.

[0170] Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include numbers that define the range. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations on the various aspects of the present disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined immediately below are more fully defined by reference to the entire specification.

[0171] As used herein, the term "enzyme for cutting IgG" refers to a polypeptide (e.g., a protease) that promotes the cutting of immunoglobulin G (IgG). In some aspects, the cutting of the IgG inactivates the IgG. In some aspects, the cutting of the IgG blocks the ability of IgG to bind to an antigen. Any enzyme capable of cutting IgG can be used in the methods disclosed herein. For example, examples of suitable enzymes can be found in International Publication No. WO2020102740 and European Patent Application EP 3768304B1, which are incorporated herein by reference in their entirety. In some aspects, the enzyme for cutting IgG comprises IdeS (immunoglobulin degrading enzyme of Streptococcus pyogenes (S. Pyrogenes); also known as Imliase).

[0172] As used herein, the term "adeno-associated virus" (AAV) is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells, in which certain functions are provided by co-infection helper viruses. AAV serotypes include, but are not limited to, AAV type 1, AAV type 2, AAV type 3 (including type 3A and type 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, AAVrh74, 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 AAV known now or later discovered. See, e.g., FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0173] As used herein, the term "adeno-associated vector nucleic acid" or "AAV vector nucleic acid" refers to a vector comprising one or more polynucleotides of interest (or "transgenes", such as micro-dystrophin) flanked by AAV terminal repeats (ITRs). Such AAV vector nucleic acids can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing the rep and cap gene products.

[0174] As used herein, the terms "AAV vector", "AAV virion", "AAV viral particle", "AAV vector particle", "AAVrh74 vector", "AAVrh74 virion", "AAVrh74 viral particle" or "AAVrh74 vector particle" refer to a viral particle composed of at least one AAV capsid protein or AAVrh74 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), the particle is typically referred to as an "AAV vector particle", "AAVrh74 vector particle" or, in some cases, simply as an "AAV vector" or "AAVrh74 vector". Therefore, the production of AAV vector particles must include packaging the AAV vector nucleic acid into an AAV capsid.

[0175] "Serotypes" of vector or viral capsids are defined by distinct immunological profiles based on capsid protein sequence and capsid structure.

[0176] "AAV cap" means the AAV cap protein, VP1, VP2 and VP3, and their analogs.

[0177] "AAV Rep" means AAV Rep protein and its analogs.

[0178] As used herein, "flanking" with respect to a sequence flanked by other elements indicates the presence of one or more flanking elements located upstream and / or downstream, i.e., 5' and / or 3', relative to the sequence. The term "flanking" is not intended to indicate that a sequence is necessarily continuous. For example, there may be an intervening sequence 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) indicates that one element is located 5' of the sequence and the other element is located 3' of the sequence; however, there may be an intervening sequence in between.

[0179] In some aspects, "AAV" includes derivatives of known AAV. In some aspects, "AAV" includes modified or artificial AAV. In certain aspects, the AAV is AAVrh74. As used herein, "AAVrh74" refers to adeno-associated virus rhesus serotype 74, which is endogenous to rhesus monkeys and was originally isolated from mesenteric lymph nodes and subsequently from the spleen.

[0180] As used herein, the term "derived from" refers to a component separated from a specific molecule or organism or prepared using the specific molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from a specific molecule or organism. For example, a nucleic acid sequence derived from a second nucleic acid sequence may include a nucleotide sequence identical or substantially similar to the nucleotide sequence of the second nucleic acid sequence. In the case of nucleotides or polypeptides, derived species may be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis, or artificial random mutagenesis. Mutagenesis for deriving nucleotides or polypeptides may be intentionally directed or intentionally random, or a mixture of each mutagenesis. Mutagenesis of nucleotides or polypeptides to produce different nucleotides or polypeptides derived from the first nucleotide or polypeptide may be a random event (e.g., caused by polymerase infidelity), and the derived nucleotides or polypeptides may be identified by appropriate screening methods (e.g., as discussed herein). Mutagenesis of polypeptides generally requires manipulation of polynucleotides encoding polypeptides. In some aspects, the nucleotide or amino acid sequence derived from the second nucleotide or amino acid sequence has at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about At least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity, wherein the first nucleotide or amino acid sequence retains the biological activity of the second nucleotide or amino acid sequence.

[0181] The terms "nucleic acid", "nucleic acid molecule", "nucleotide", "nucleotide sequence(s)", and "polynucleotide" are used interchangeably and refer to a phosphate polymeric form of ribonucleosides (adenosine, guanine, uridine, or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecule") or any of their phosphate analogs, such as phosphate polymeric forms of thiophosphates and thioesters, in single-stranded form or double-stranded helices. A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, and specifically DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the conventional convention of giving only the sequence in the 5' to 3' direction along the non-transcribed DNA strand (i.e., the strand with a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has been manipulated by molecular biology. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semisynthetic DNA. A "nucleic acid composition" of the present disclosure comprises one or more nucleic acids as described herein.

[0182] As used herein, the term "expression cassette" refers to any type of gene construct containing nucleic acid, wherein part or all of the nucleic acid coding sequence can be transcribed. Typically, the expression cassette comprises a promoter operably connected to a nucleic acid (e.g., a transgenic being of interest). In some aspects, an "expression cassette" comprises a polynucleotide sequence encoding human micro-dystrophin. In some aspects, an "expression cassette" comprises a polynucleotide sequence that is an intron. In some aspects, an "expression cassette" comprises a polynucleotide sequence that instructs the addition of a polyadenosine (poly (A)) tail.

[0183] As used herein, "coding region" or "coding sequence" is a part of a polynucleotide composed of codons that can be translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are not usually translated into amino acids, the stop codons can be considered as a part of the coding region, but any side sequences (such as promoters, ribosome binding sites, transcription terminators, introns, etc.) are not a part of the coding region. The boundaries of the coding region are usually determined by the start codon at the 5' end of the amino terminal of the encoded polypeptide and the translation stop codon at the 3' end of the carboxyl terminal of the encoded polypeptide. Two or more coding regions can be present in a single polynucleotide construct on a single vector, for example, or in a separate polynucleotide construct on a separate (different) vector. So it can be obtained that a single vector can contain only a single coding region, or include two or more coding regions. As used herein, "transcript" refers to an mRNA sequence formed during DNA transcription, for example, by RNA polymerase.

[0184] The term "downstream" refers to a nucleotide sequence located 3' of a reference nucleotide sequence. In some aspects, a downstream nucleotide sequence refers to a sequence after the transcription start point. For example, the translation start codon of a gene is located downstream of the transcription start site. The term "upstream" refers to a nucleotide sequence located 5' of a reference nucleotide sequence. In some aspects, an upstream nucleotide sequence refers to a sequence located on the 5' side of a coding region or a transcription start site. For example, most promoters are located upstream of the transcription start site.

[0185] A polynucleotide encoding a product (e.g., a micro-dystrophin protein) (e.g., a gene cassette disclosed herein) may include a promoter and / or other expression (e.g., transcription or translation) control elements operably associated with one or more coding regions. In an operably associated association, the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory regions in a manner that places the expression of the gene product under the influence or control of the one or more regulatory regions. For example, a coding region and a promoter are "operably associated" if induction of promoter function results in transcription of an mRNA encoding a gene product encoded by the coding region, and if the nature of the connection between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other expression control elements in addition to promoters, such as enhancers, operators, repressors, and transcription termination signals, may also be operably associated with a coding region to direct the expression of a gene product.

[0186] "Transcription control sequence" refers to a DNA regulatory sequence, such as a promoter, enhancer, terminator, etc., which provides for the expression of a coding sequence in a host cell. A variety of transcription control regions are known to those skilled in the art. The various transcription control regions include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer segments from cytomegalovirus (immediate early promoter, in conjunction with intron-A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences that can control gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters that can be induced by interferons or interleukins).

[0187] Similarly, various translation control elements are known to those of ordinary skill in the art. The translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites or IRES, also known as CITE sequences).

[0188] "Promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence that can control the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' of the promoter sequence. Promoters can all be derived from natural genes, or be composed of different elements derived from different promoters found in nature, or even contain synthetic DNA segments. It will be understood by those skilled in the art that different promoters can direct the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. Promoters that make genes expressed in most cell types at most times are generally referred to as "constitutive promoters". Promoters that make genes expressed in specific cell types are generally referred to as "cell-specific promoters" or "tissue-specific promoters". Promoters that make genes expressed at specific developmental or cell differentiation stages are generally referred to as "developmental specific promoters" or "cell differentiation-specific promoters". Promoters that induce and make gene expression after cells are exposed or treated with reagents, biomolecules, chemicals, ligands, light, etc. that induce promoters are generally referred to as "inducible promoters" or "regulatable promoters". It is further recognized that because in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0189] The promoter sequence is usually defined at its 3' terminus by a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. The transcription initiation site (conveniently defined, for example by mapping with nuclease S1) and the protein binding domains (consensus sequences) responsible for binding RNA polymerase will be found in the promoter sequence.

[0190] In some aspects, the nucleic acid molecule comprises a tissue-specific promoter. In certain aspects, the tissue-specific promoter drives the expression of a therapeutic protein (e.g., dystrophin or micro-dystrophin) in the brain, muscle, kidney, lung, testis, or any combination thereof. In a specific aspect, the promoter is selected from the group consisting of: enhancer 358 bp muscle creatine kinase proximal promoter (Enh358MCK), muscle creatine kinase (CK) promoter (e.g., hCK (SEQ ID NO: 31) or hCK Plus (SEQ ID NO: 32)), truncated muscle creatine kinase promoter (tMCK; SEQ ID NO: 26), myosin heavy chain (MHC), MHCK7 (a hybrid version of MHC and MCK; SEQ ID NO: 25), C5-12 (synthetic promoter), muscle-specific and cardiac-specific enhancer (MHCK), CK8, SPc5-12, human desmin (Des) promoter, human α-myosin heavy chain (α-MHC) promoter, rat myosin light chain 2 (MLC-2) promoter, and human cardiac troponin C (cTnC) promoter. In certain aspects, the promoter comprises a tMCK promoter. In certain aspects, the promoter comprises a MHCK7 promoter.

[0191] Table 1: Promoter sequences

[0192]

[0193]

[0194] In some aspects, the expression of a protein product (e.g., dystrophin or micro-dystrophin) can be enhanced using one or more enhancers. For example, an enhancer can be located in 5' or 3' of an AAV, coding region, or in an intron. In some aspects, the one or more enhancers are tissue-specific enhancers. In some aspects, the one or more enhancers are selected from human skeletal actin gene elements, cardiac actin gene elements, myocyte-specific enhancer binding factor MEF (e.g., MEF2), MyoD enhancer elements, cardiac enhancer factor (CEF) sites, mouse creatine kinase enhancer elements, skeletal fast contraction troponin C gene elements, slow contraction cardiac troponin C gene elements, slow contraction troponin I gene elements, hypoxia-inducible nuclear factors, steroid-inducible elements, glucocorticoid response elements (GREs), and any combination thereof.

[0195] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide. The expression includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of mRNA into a polypeptide. Expression produces a "gene product". As used herein, a gene product can be a nucleic acid, for example, a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications, for example, polyadenylation or splicing, or polypeptides with post-translational modifications, for example, methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage. As used herein, the term "yield" refers to the amount of a polypeptide produced by the expression of a gene.

[0196] As used herein, the terms "dystrophin", "dys" or "dystrophin polypeptide" are intended to refer to full-length dystrophin as well as fragments and / or variants thereof. Human dystrophin is encoded by the DMD gene, which is 2.4 megabases (mb) in size and contains 79 exons. Full-length human dystrophin comprises an N-terminal (NT) domain, four hinge regions (H1-H4), twenty-four spectrin-like repeat regions (R1-R24), a cysteine-rich (CR) domain, and a C-terminal (CT) domain. The dystrophin protein may further comprise various binding domains, including, for example, one or more dystrophin glycan binding domains (Dg BD) located within the H4 and CR domains; one or more neuronal nitric oxide synthase binding domains, for example, located within one or more spectrin-like repeat regions, such as R16 and R17; one or more dystrophin binding protein binding domains (Syn Bd) located within the CT domain; and one or more dystrobrevin binding domains (DB BD) located within the CT domain, for example. Structurally, the spectrin-like repeat sequences form a central rod domain, thereby providing a modular structure for the dystrophin protein. In some aspects, the regions of the dystrophin protein comprising R1-R24 are collectively referred to as "rod domains".

[0197] As used herein, "dystrophin" includes fragments of the full-length dystrophin sequence, including "micro-dystrophin" or "mini-dystrophin" which are used interchangeably. "Micro-dystrophin" is used to refer to any dystrophin polypeptide comprising a deletion relative to the full-length dystrophin sequence. Dystrophin (e.g., micro-dystrophin) may include a deletion at the N-terminus (e.g., a deletion of all or part of the NT domain), a deletion at the C-terminus (e.g., a deletion of all or part of the CT domain), a deletion between the N-terminus and the C-terminus (e.g., a deletion of one or more hinge regions or a portion thereof and / or one or more spectrin-like repeat regions or a portion thereof), or any combination thereof. Dystrophin (e.g., micro-dystrophin) may include a deletion within one or more of the NT domain, hinge region, spectrin-like repeat region, CR domain, and CT domain. In some aspects, the deletion comprises a partial deletion of one or more of the NT domain, hinge region, spectrin-like repeat region, CR domain, and CT domain. In some aspects, the deletion comprises a complete deletion of one or more of the NT domain, hinge region, spectrin-like repeat region, CR domain, and CT domain. Examples of dystrophin and / or micro-dystrophin that can be used in the present disclosure can be found in, for example, International Publication Nos. WO / 2002 / 029056, WO / 2008 / 088895, WO / 2010 / 093784, WO / 2011 / 088081, WO / 2014 / 193716, WO / 2016 / 004319, WO / 2016 / 115543, WO / 2017 / 077451, WO / 2017 / 221145; WO / 2019 / 012336; WO / 2019 / 245973; U.S. Publication No. US2017 / 0368198; and U.S. Patent No. 7,655,467, each of which is incorporated herein by reference in its entirety. In some aspects, the micro-dystrophin retains the function of the full-length dystrophin. In some aspects, the dystrophin comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 40-44. In some aspects, the dystrophin comprises an amino acid sequence encoded by a nucleic acid sequence selected from the nucleic acid sequences shown in SEQ ID NOs: 19 and 33-39. In some aspects, the dystrophin comprises an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO: 19. In some aspects, the AAVrh74 comprises a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO: 19.

[0198] Table 2: Micro-dystrophin sequences.

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207] As used herein, the term "β-sarcoglycan", "b-sarcoglycan", "β-sarcoglycan" or "SGCB" refers to a subcomplex of the dystrophin-glycoprotein complex that forms a connection between the F-actin cytoskeleton and the extracellular matrix. β-sarcoglycan is highly expressed in heart and skeletal muscle, and to a lesser extent in the brain, kidney, placenta, and lung. The typical amino acid sequence of β-sarcoglycan is shown in Table 3 as SEQ ID NO:45 (Uniprot Q16585). In some aspects, the recombinant AAVrh74 viral vector of the present disclosure comprises a gene cassette comprising a nucleic acid molecule encoding β-sarcoglycan. Non-limiting examples of β-sarcoglycan sequences and constructs that can be used in the present disclosure are disclosed in International Publication No. WO2017180976, which is incorporated herein by reference in its entirety. In some aspects, the nucleic acid molecule encoding β-sarcoglycan comprises the nucleic acid sequence shown in SEQ ID NO:21 (Table 3).

[0208] Table 3: Beta-sarcoglycan sequences

[0209]

[0210] As used herein, the term "alpha sarcoglycan", "a-sarcoglycan", "α-sarcoglycan" or "SGCA" refers to a subcomplex of the dystrophin-glycoprotein complex that forms a connection between the F-actin cytoskeleton and the extracellular matrix. Alpha sarcoglycan is highly expressed in skeletal muscle, and to a lesser extent in cardiac muscle, and to a much lesser extent in the lung. A typical amino acid sequence of alpha sarcoglycan is shown in Table 4 as SEQ ID NO:46 (Uniprot Q16586). In some aspects, the recombinant AAVrh74 viral vector of the present disclosure comprises a gene cassette comprising a nucleic acid molecule encoding alpha sarcoglycan. Non-limiting examples of alpha sarcoglycan sequences and constructs that can be used in the present disclosure are disclosed in International Publication No. WO2013078316, which is incorporated herein by reference in its entirety. In some aspects, the nucleic acid molecule encoding alpha sarcoglycan comprises the nucleic acid sequence shown in SEQ ID NO:23 (Table 4).

[0211] Table 4: Alpha sarcoglycan sequences

[0212]

[0213]

[0214] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly connected by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can replace any of these terms or be used interchangeably. The term "polypeptide" is also intended to refer to post-expression modifications of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization of known protection / blocking groups, proteolytic cleavage or modified products by non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides can be generated in any manner, including by chemical synthesis.

[0215] The term "percent identity" as known in the art is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1993). The preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods for determining identity are incorporated into publicly available computer programs.Sequence alignments and percent identity calculations can be performed using sequence analysis software, such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI), the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990), and DNASTAR (DNASTAR Inc., 1228 South Park Street, Madison, WI 53715, USA). In the context of the present disclosure, it is understood that, unless otherwise stated, where sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the program mentioned. As used herein, "default values" shall mean any set of values ​​or parameters that are initially loaded when the software is first initialized. To determine the percent identity between a therapeutic protein (e.g., dystrophin), a sequence of the disclosure, and a reference sequence, only nucleotides in the reference sequence corresponding to nucleotides in the therapeutic protein (e.g., dystrophin) of the disclosure are used to calculate the percent identity.

[0216] As used herein, nucleotides corresponding to nucleotides in a specific sequence of the present disclosure are identified by alignment of the sequences of the present disclosure to maximize the identity with the reference sequence. The number of equivalent amino acids used to identify the reference sequence is based on the number of corresponding amino acids used to identify the sequence of the present disclosure.

[0217] As used herein, the term "connected" refers to a first amino acid sequence or nucleotide sequence that is covalently or non-covalently connected to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly connected or juxtaposed to the second amino acid or nucleotide sequence, or alternatively, an intermediate sequence can covalently connect the first sequence to the second sequence. The term "connected" not only means a fusion of a first amino acid sequence and a second amino acid sequence located at the C-terminus or N-terminus, but also includes inserting the entire first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively). On the one hand, the first amino acid sequence can be connected to the second amino acid sequence by a peptide bond or a joint. The first nucleotide sequence can be connected to the second nucleotide sequence by a phosphodiester bond or a joint. The joint can be any chemical part of a peptide or polypeptide (for a polypeptide chain) or a nucleotide or nucleotide chain (for a nucleotide chain) or (for both polypeptides or polynucleotide chains). The term "connected" is also indicated by a hyphen (-).

[0218] As used herein, the term "Duchenne muscular dystrophy" or "DMD" refers to a type of muscular dystrophy caused by a mutation in the DMD gene, resulting in a reduction in mRNA and the absence of dystrophin, a 427kD sarcoplasmic protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51 (6): 919-28, 1987). DAPC is composed of a variety of proteins at the muscle sarcolemma, forming a structural connection between the extracellular matrix (ECM) and the cytoskeleton through the actin-binding protein dystrophin and the laminin-binding protein α-dystroglycan. These structural connections are used to stabilize the myocyte membrane during contraction and prevent damage caused by contraction. With the loss of dystrophin, membrane fragility leads to sarcoplasmic tearing and calcium influx, thereby inducing calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol. 10 (2): 168-75, 1997). This cycle of uncontrolled muscle degeneration and regeneration eventually depletes the muscle stem cell population (Sacco et al., Cell 2010. 143(7): 1059-71; Wallace et al., Annu Rev Physiol, 2009. 71: 37-57), leading to progressive muscle weakness, endomysial inflammation, and fibrotic scarring. Approximately 1 in 5,000 male newborns suffer from DMD.

[0219] As used herein, the term "limb-girdle muscular dystrophy" or "LGMD" refers to a class of muscular dystrophy that typically manifests in the proximal muscles around the hip and shoulder. LGMD is the fourth most common genetic cause of muscle weakness, with an estimated incidence of approximately 2 per 100,000 individuals. The unifying feature of LGMD is weakness and atrophy of the limb-girdle muscles. However, the age at which symptoms appear, as well as the speed and severity of disease progression, can vary. There are at least nineteen forms of LGMD, and these are classified according to their associated genetic defects: LGMD1A (autosomal dominant; sarcomeric protein gene); LGMD1B (autosomal dominant; lamin A / C genes); LGMD1C (autosomal dominant; caveolin gene); LGMD1D (autosomal dominant; chromosome 7); LGMD1E (autosomal dominant; chromosome 6); LGMD1F (autosomal dominant; chromosome 7); LGMD1G (autosomal dominant; chromosome 4); LGMD2A (autosomal recessive; calpain-3 gene); LGMD2B (autosomal recessive; dysferlin gene); L GMD2C (autosomal recessive; gamma-sarcoglycan gene); LGMD2D (autosomal recessive; alpha-sarcoglycan gene); LGMD2E (autosomal recessive; beta-sarcoglycan gene); LGMD2F (autosomal recessive; delta-sarcoglycan gene); LGMD2G (autosomal recessive; telethonin gene); LGMD2H (autosomal recessive; TRIM32); LGMD2I (autosomal recessive; FKRP gene); LGMD2J (autosomal recessive; titin gene); LGMD2K (autosomal recessive; POMT1 gene); and LGMD2L (autosomal recessive; Fukutin gene).

[0220] As used herein, the term "North Star Ambulatory Assessment (NSAA) score" refers to a clinician-administered scale that scores performance in 17 different functional activities, including 10-meter walk / run (10MWR), standing from a sitting position, standing on 1 leg, climbing box steps, descending box steps, standing from lying to sitting, standing from the floor, lifting head off the floor, standing on heels, and jumping (Mazzone E et al., North Star Ambulatory Assessment, 6-minute walk test and timed items in ambulant boys with Duchenne muscular dystrophy, Neuromuscul Disord. 20(11):712-6, 2010). Subjects will be scored according to the following: 2 = normal, no significant change in activity; 1 = modified method, but achieved the goal independent of physical assistance from others; and 0 = unable to achieve the goal independently.

[0221] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount of a therapeutic compound (such as an AAV vector or enzyme disclosed herein) administered to a mammalian subject as a single dose or as part of a series of doses that is effective to produce the desired therapeutic effect. For enzymes, this effect is typically cleavage of an IgG target. For AAV vectors, the desired therapeutic effect is typically achieved by expression of a heterologous transgene present in the AAV vector and / or persistence of the AAV vector in the subject.

[0222] "Enhance" or "enhancing", or "increase" or "increasing" or "stimulate" or "stimulating" generally refers to the ability of one or more AAVs, enzymes, or pharmaceutical compositions of any of the foregoing to produce or elicit a greater physiological response (i.e., a downstream effect) in a cell or subject relative to the response elicited by no AAV, no enzyme, or administration of a control compound.

[0223] As used herein, the terms "function" and "functional" and the like refer to a biological, enzymatic or therapeutic function.

[0224] The phrase "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising the formulation and / or the subject to be treated therewith.

[0225] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials that can serve as pharmaceutically acceptable carriers are: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered solutions; nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; and antioxidants; according to the judgment of the formulator.

[0226] The term "restoration" with respect to the synthesis or production of a particular protein generally refers to the production of that protein, including truncated forms of the protein, in a patient suffering from muscular dystrophy following treatment with an AAV vector, such as the AAVrh74 vector disclosed herein.

[0227] In some aspects, treatment with an AAV vector, such as an AAVrh74 vector disclosed herein, slows or reduces progressive respiratory muscle dysfunction and / or failure in patients with DMD or LGMD, which would be expected in the absence of treatment. In some aspects, treatment with an AAV vector, such as an AAVrh74 vector disclosed herein, can reduce or eliminate the need for ventilatory assistance, which would be expected in the absence of treatment. In some aspects, respiratory function measurements used to track the course of the disease and evaluation of potential therapeutic interventions include maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and forced vital capacity (FVC). MIP and MEP measure the pressure levels that a person can generate during inspiration and exhalation, respectively, and are sensitive measurements of respiratory muscle strength. MIP is a measure of diaphragmatic muscle weakness.

[0228] In some aspects, MEP may decrease before changes in other lung function tests, including MIP and FVC. In some aspects, MEP can be an early indicator of respiratory dysfunction. In some aspects, FVC can be used to measure the total amount of air expelled during forced exhalation after maximal inspiration. In patients with DMD, FVC increases in parallel with physical growth until the early teenage years. However, as growth slows or is stunted due to disease progression, and muscle weakness progresses, vital capacity enters a phase of decline and decreases at an average rate of approximately 8 to 8.5% per year after the age of 10 to 12 years. In some aspects, MIP percent predicted (MIP adjusted for weight), MEP percent predicted (MEP adjusted for age), and FVC percent predicted (FVC adjusted for age and height) are supportive analyses.

[0229] As used herein, the terms "subject" and "patient" include any animal that exhibits symptoms or is at risk of exhibiting symptoms that can be treated with the AAV disclosed herein, such as the AAVrh74 vector disclosed herein, such as a subject (or patient) suffering from DMD or LGMD or at risk of suffering from the disease or any of the symptoms associated with these conditions (e.g., muscle fiber loss). Suitable subjects (or patients) include laboratory animals (such as mice, rats, rabbits or guinea pigs), farm animals, and livestock or pets (such as cats or dogs). Non-human primates and preferably human patients (or subjects) are included. In some aspects, the subject is male.

[0230] As used herein, the phrases "systemic administration" and "administered systemically" and "peripheral administration" or "administered peripherally" mean administration of a compound, drug, or other material other than directly to the central nervous system so that it enters the patient's system and thereby undergoes metabolism and other similar processes, e.g., subcutaneous administration.

[0231] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0232] "Treatment" of a subject (e.g., a mammal such as a human) or cell is any type of intervention used to attempt to alter the natural course of a subject or cell. Treatment includes, but is not limited to, administration of an AAV vector, such as the AAVrh74 vector disclosed herein, and an enzyme that cleaves IgG, and may be performed prophylactically or after the onset of a pathological event or after contact with a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition associated with dystrophin, such as in certain forms of muscular dystrophy, and may include, for example, a slight change or improvement in one or more measurable markers of the disease or condition being treated. Also included is "preventative" treatment, which may be intended to reduce the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. "Treatment" or "prevention" does not necessarily mean complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0233] In some aspects, treatment with an AAV vector comprising a transgene and an enzyme that cuts IgG according to the disclosure increases the production of a transgene (e.g., microdystrophin, β-sarcoglycan or α-sarcoglycan), delays disease progression, slows or reduces loss of walking, reduces muscle inflammation, reduces muscle damage, improves muscle function, reduces loss of lung function and / or enhances muscle regeneration that can be expected without treatment. In some aspects, treatment maintains, delays or slows disease progression. In some aspects, treatment maintains walking or reduces loss of walking. In some aspects, treatment maintains lung function or reduces loss of lung function. In some aspects, treatment maintains or increases the patient's stable walking distance, as measured by, for example, a 6-minute walk test (6MWT), described in McDonald et al. (Muscle Nerve, 2010; 42: 966-74, the document is incorporated herein by reference). The change in 6-minute walk distance (6MWD) can be expressed as an absolute value, a percentage change, or a change in %-predicted value. The performance of a DMD patient on the 6MWT relative to the typical performance of a healthy peer can be determined by calculating the %-predicted value. For example, for a male, the %-predicted 6MWD can be calculated using the following formula: 196.72 + (39.81 x age) – (1.36 x age) 2 ) + (132.28 x height in meters). For females, %-predicted 6MWD can be calculated using the following formula: 188.61 + (51.50 x age) – (1.86 x age 2 )+(86.10 x height in meters) (Henricson et al., PLoS Curr., 2012, 2nd ed., which is incorporated herein by reference in its entirety).

[0234] In some aspects, treatment maintains or reduces the time of walking / running 10 meters (that is, 10 meters walking / running test). In some aspects, treatment maintains or reduces the time of standing from supine position (that is, standing time test). In some aspects, treatment maintains or reduces the time of climbing four layers of standard stairs (that is, four layers of stairs climbing test). In some aspects, treatment maintains or reduces the muscle inflammation of the patient, as measured by, for example, MRI (for example, MRI of leg muscles). In some aspects, MRI measures T2 and / or fat score to identify muscle degeneration. MRI can identify the changes in muscle structure and composition caused by inflammation, edema, muscle damage and fat infiltration.

[0235] In some aspects, treatment with an enzyme that cleaves IgG of the present disclosure increases the vector copy number (VCN) of AAV following subsequent administration of an AAV described herein. In some aspects, the VCN of AAV is increased relative to the VCN of AAV in a subject to which an enzyme that cleaves IgG is not administered, or relative to the vector copy number in a subject when an enzyme that cleaves IgG is not administered. In some aspects, an increase in AAV VCN is observed in muscle.

[0236] As used herein, the term "neutralizing antibody" refers to any antibody that binds to the AAV capsid protein of the AAV viral vector particles and inhibits the transduction of target cells by the AAV viral vector particles.

[0237] As used herein, the term "non-neutralizing antibody" refers to any antibody that binds to an AAV capsid protein of an AAV viral vector particle and / or an AAV capsid protein that is not a part of an AAV viral vector particle and does not inhibit transduction of target cells by the AAV viral vector particle, but can elicit a proinflammatory response in a subject.

[0238] As used herein, the term "total antibodies" refers to AAV antibodies including neutralizing AAV antibodies and non-neutralizing AAV antibodies.

[0239] As used herein, the term "neutralizing cell assay" refers to an assay for quantifying neutralizing AAV antibodies in a sample of a subject in vitro using cells that bind to and are transduced by AAV virus particles that quantify neutralizing antibodies. In some aspects, the neutralizing cell assay comprises incubating the AAV virus particles that quantify neutralizing antibodies with the sample of the subject to prepare an AAV virus particle sample mixture, and incubating the cells that are bound to and transduced by the AAV virus particles that quantify neutralizing antibodies with the AAV virus particles sample mixture. In some aspects, the assay further comprises washing the cells and detecting the presence of AAV virus particles in the cells, for example, using an enzyme-conjugated or labeled antibody to an AAV virus particle protein, or if the AAV virus particles used are recombinant AAV virus vector particles carrying a transgene, then detecting the expression of the transgene in the cell.

[0240] As used herein, the term "total AAVrh74 antibody ELISA assay" refers to an enzyme-linked immunosorbent assay that uses AAVrh74 viral particles immobilized on a solid phase support to quantify total AAVrh74 antibodies in a sample of a subject in vitro, and detects AAVrh74 antibodies bound to the immobilized AAVrh74 viral particles by an enzyme-conjugated anti-IgG antibody and a substrate that can be quantified. In some aspects, the total AAVrh74 antibody ELISA assay is used as described in Godeeker et al., Ther. Adv. Neurol. Disord., 6:1-7, 2023, which is incorporated herein by reference in its entirety. In some aspects, the total AAVrh74 antibody ELISA assay can be Determination.

[0241] II. Methods of the Disclosure

[0242] Certain aspects of the present disclosure relate to methods for treating a disease or condition in a subject in need thereof, the methods comprising administering to the subject (i) a recombinant AAV viral vector and (ii) an enzyme that cleaves immunoglobulin G (IgG). Without being bound by any particular theory or mechanism, administering an enzyme that cleaves IgG before, simultaneously with, and / or after administering a therapeutic AAV vector comprising a transgene encoding a therapeutic protein disclosed herein, e.g., a recombinant AAVrh74 viral vector, reduces or eliminates an immune response to the AAV vector. Gene therapy using viral vectors such as recombinant AAV viral vectors typically elicits an inhibitory immune response in human subjects. Since many human subjects have been exposed to AAV during their lifetime, the subject may already have inhibitory antibodies prior to the first administration of AAV gene therapy. One way to mitigate the effects of pre-existing inhibitory antibodies is to use an AAV serotype or other viral vector, such as AAVrh74, that most people are unlikely to be exposed to. However, after the first administration of even the rarest serotypes, human subjects in many cases produce inhibitory antibodies to the administered AAV, which limits the duration of the response and almost excludes subsequent administration using the same serotype. The methods disclosed herein reduce the effects of pre-existing inhibitory antibodies and / or the formation of new inhibitory antibodies by destroying IgG polypeptides in a human subject.

[0243] Some aspects of the present disclosure relate to methods of improving the efficacy of an AAV vector in a human subject with muscular dystrophy, the method comprising administering to the subject (i) an enzyme that cleaves IgG and (ii) an AAV vector. Some aspects of the present disclosure relate to methods of improving the efficacy of an AAV vector in a human subject with muscular dystrophy, the method comprising administering to the subject (i) an enzyme that cleaves IgG and (ii) an AAV vector.

[0244] In some aspects, the AAV vector and the enzyme that cleaves IgG are administered simultaneously. In some aspects, the enzyme that cleaves IgG is administered before the AAV vector. In some aspects, the AAV vector is administered before the enzyme that cleaves IgG.

[0245] In some aspects, methods disclosed herein relate to treating a subject suffering from muscular dystrophy. In some aspects, the subject suffers from Duchenne muscular dystrophy (DMD). In some aspects, the subject suffering from DMD clearly diagnoses DMD based on the records of clinical findings and confirmatory gene tests. In some aspects, the subject suffering from DMD is a child of 4 to 8 years old (including 4 years old and 8 years old). In some aspects, the subject does not have a mutation between exon 1-17 of the dystrophin gene or including the exon. In some aspects, the subject does not have a variant of in-frame deletion, in-frame insertion or uncertain significance in the dystrophin gene.

[0246] In some aspects, the subject suffers from limb-girdle muscular dystrophy (LGMD). In some aspects, the subject suffers from LGMD caused by a mutation, in-frame deletion, in-frame insertion, or variant of uncertain significance in an alpha-sarcoglycan gene. In some aspects, the subject suffers from LGMD caused by a mutation, in-frame deletion, in-frame insertion, or variant of uncertain significance in a beta-sarcoglycan gene. In some aspects, the subject suffers from LGMD caused by a mutation, in-frame deletion, in-frame insertion, or variant of uncertain significance in a non-sarcoglycan gene.

[0247] Some aspects of the present disclosure relate to methods for increasing or restoring muscle cell conditions in a human subject having muscular dystrophy and eligible for therapy with an AAV vector, the method comprising administering to the subject an enzyme that cleaves IgG, wherein after administration of the enzyme that cleaves IgG, the subject is administered an AAV vector. Some aspects of the present disclosure relate to methods for increasing or restoring muscle cell conditions in a human subject having muscular dystrophy and eligible for therapy with an AAV vector, the method comprising administering to the subject an AAV vector, wherein the subject was previously administered an enzyme that cleaves IgG. Some aspects of the present disclosure relate to methods for increasing or restoring muscle cell conditions in a human subject having muscular dystrophy and eligible for therapy with an AAV vector, the method comprising administering to the subject an AAV vector, wherein the subject was previously administered an enzyme that cleaves IgG. Some aspects of the present disclosure relate to methods for increasing or restoring muscle cell conditions in a human subject having muscular dystrophy and eligible for therapy with an AAV vector, the method comprising administering to the subject an enzyme that cleaves IgG, wherein after said administration of said enzyme that cleaves IgG, the subject is administered an AAV vector.

[0248] In some aspects, the enzyme that cleaves IgG reduces the titer of IgG antibodies, e.g., neutralizing antibodies, that specifically bind to an antigen expressed on the capsid of the AAV vector. By reducing the titer of one or more neutralizing antibodies, the methods disclosed herein allow for repeated administration of therapeutic AAV vectors with limited or no loss of efficacy in subsequent administrations.

[0249] In some aspects, the disease or condition is characterized by lack of expression of a specific protein, and the AAV viral vector comprises a transgene encoding the specific protein or an analog thereof. In some aspects, the disease or condition is characterized by lack of expression, misexpression, or a combination thereof of dystrophin, and the AAV viral vector comprises a transgene encoding dystrophin or micro-dystrophin disclosed herein. In some aspects, the disease or condition is characterized by lack of expression, misexpression, or a combination thereof of alpha sarcoglycan, and the AAV viral vector comprises a transgene encoding alpha sarcoglycan or an analog thereof disclosed herein. In some aspects, the disease or condition is characterized by lack of expression, misexpression, or a combination thereof of beta sarcoglycan, and the AAV viral vector comprises a transgene encoding beta sarcoglycan or an analog thereof disclosed herein.

[0250] In some aspects, the disease or disorder comprises muscular dystrophy. In some aspects, the muscular dystrophy is DMD. In some aspects, the muscular dystrophy is LGMD.

[0251] II.A. Detection of Anti-AAVrh74 Antibodies

[0252] Methods for detecting neutralizing, non-neutralizing and / or total AAVrh74 antibodies in a subject are provided. The presence of neutralizing and / or non-neutralizing AAVrh74 antibodies in a subject to be treated with AAVrh74 viral vector particles as described herein can reduce the efficacy of transgene transfer of AAVrh74 vector particles to target cells in the subject and / or promote a pro-inflammatory response in the subject. In some aspects, the AAVrh74 antibodies present in the subject bind to the AAVrh74 capsid protein present in the AAVrh74 viral vector particles. In some aspects, the AAVrh74 antibodies in the subject bind to the AAVrh74 capsid protein of the AAVrh74 viral vector particles at a location where the AAVrh74 capsid protein interacts with a cell receptor on a cell to be targeted with the AAVrh74 viral vector particles. In some aspects, AAVrh74 antibodies in a subject that bind to the AAVrh74 capsid protein at a location that interacts with a cellular receptor prevent interaction between the AAVrh74 capsid protein and target cells and block transduction of target cells by AAVrh74 viral vector particles; such transduction-blocking AAVrh74 antibodies are also referred to as neutralizing antibodies.

[0253] In some aspects, the AAVrh74 antibody in the subject binds to the AAVrh74 capsid protein at a location on the AAVrh74 capsid protein that does not interact with a cell receptor on the cell to be targeted, and the AAVrh74 antibody does not block transduction of the target cell by the AAVrh74 viral vector particles; such non-transduction blocking antibodies are also referred to as non-neutralizing antibodies. In some aspects, the non-neutralizing AAVrh74 antibody can promote a pro-inflammatory response in the subject.

[0254] In some aspects, the subject has both neutralizing and non-neutralizing AAVrh74 antibodies. In some aspects, the assays described herein are used to quantify total AAVrh74 antibodies in a subject, including neutralizing and non-neutralizing AAVrh74 antibodies.

[0255] In some aspects, a subject to be treated with the AAVrh74 viral vector particles described herein is tested for the presence of total AAVrh74 antibodies using an assay as described herein, and the subject is further tested for the presence of neutralizing AAVrh74 antibodies using an assay as described herein. In some aspects, the amount of non-neutralizing AAVrh74 antibodies present in a subject is determined by subtracting the amount of neutralizing AAVrh74 antibodies present in the subject from the amount of total AAVrh74 antibodies present in the subject.

[0256] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to treatment with an AAVrh74 viral vector particle described herein.

[0257] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject following treatment with an AAVrh74 viral vector particle described herein.

[0258] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to treatment with an IgG-cleaving enzyme described herein.

[0259] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject following treatment with an IgG-cleaving enzyme described herein.

[0260] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to treatment with AAVrh74 viral vector particles and after a first treatment with an enzyme that cleaves IgG as described herein.

[0261] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to treatment with AAVrh74 viral vector particles and after a second treatment with an enzyme that cleaves IgG as described herein.

[0262] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to treatment with AAVrh74 viral vector particles and after a third treatment with an enzyme that cleaves IgG as described herein.

[0263] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to a second treatment with AAVrh74 viral vector particles after a first treatment with AAVrh74 viral vector particles.

[0264] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to a second treatment with AAVrh74 viral vector particles after a first treatment with AAVrh74 viral vector particles and after a first treatment with an enzyme that cleaves IgG as described herein.

[0265] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject before a second treatment with AAVrh74 viral vector particles after a first treatment with AAVrh74 viral vector particles and after a second treatment with an enzyme that cleaves IgG as described herein.

[0266] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject before a second treatment with AAVrh74 viral vector particles after a first treatment with AAVrh74 viral vector particles and after a third treatment with an enzyme that cleaves IgG as described herein. In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject before a third treatment with AAVrh74 viral vector particles after a second treatment with AAVrh74 viral vector particles.

[0267] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to a third treatment with AAVrh74 viral vector particles after a second treatment with AAVrh74 viral vector particles described herein and after a first treatment with an enzyme that cleaves IgG.

[0268] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject prior to a third treatment with AAVrh74 viral vector particles after a second treatment with AAVrh74 viral vector particles described herein and after a second treatment with an enzyme that cleaves IgG.

[0269] In some aspects, total, neutralizing and / or non-neutralizing AAVrh74 antibodies are determined in a subject before a third treatment with AAVrh74 viral vector particles after a second treatment with AAVrh74 viral vector particles described herein and after a third treatment with an enzyme that cleaves IgG.

[0270] II.B. Detection of Neutralizing Anti-AAVrh74 Antibodies

[0271] In some aspects, neutralizing AAVrh74 antibodies are measured using a neutralizing cell-based assay as described herein. In some aspects, the neutralizing cell assay is an assay described in Enders et al., Clinical and Vaccine Immunology, 20:420-426, 2013, which is incorporated herein by reference in its entirety.

[0272] In some aspects, cells that bind to AAVrh74 viral particles and are transduced by the viral particles are used to quantify neutralizing AAVrh74 antibodies in a sample of a subject in vitro. In some aspects, neutralizing AAVrh74 antibodies in a sample are quantified by incubating AAVrh74 viral particles with a sample of a subject to prepare an AAVrh74 viral particle sample mixture. In some aspects, cells that bind to AAVrh74 viral particles and are transduced by the viral particles are incubated with an AAVrh74 viral particle sample mixture. In some aspects, cells are washed after incubation with an AAVrh74 viral particle sample mixture, and the AAVrh74 viral particles inside the cells are quantified. In some aspects, an enzyme-conjugated antibody to an AAVrh74 viral particle protein is used to quantify the AAVrh74 viral particles in the cell. In some aspects, AAVrh74 viral vector particles carrying a transgene are used in a neutralizing antibody assay, and the AAVrh74 viral vector particles are quantified by quantifying the expression of the transgene in the cell.

[0273] In some aspects, neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and prior to administration of imrase. In some aspects, neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of a first dose of imrase. In some aspects, neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of a second dose of imrase. In some aspects, neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of a third dose of imrase.

[0274] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of AAVrh74 vector particles. In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0275] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0276] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0277] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles. In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0278] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0279] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0280] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles. In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0281] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0282] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0283] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles. In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0284] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0285] In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0286] One of ordinary skill in the art will appreciate that the measurement of neutralizing AAVrh74 antibodies is not limited by the number of administrations of AAVrh74 vector particles or the number of doses of imrase. In some aspects, neutralizing anti-AAVrh74 antibodies are measured at any time when the subject is considered to be administered AAVrh74 vector particles. In some aspects, neutralizing anti-AAVrh74 antibodies are measured at any time when the subject is considered to be administered imrase.

[0287] In some aspects, the assay used to measure neutralizing AAVrh74 antibodies in a subject is a neutralizing cell assay. In some aspects, a subject is determined to have neutralizing anti-AAVrh74 antibodies if the anti-AAVrh74 antibody titer measured in the neutralizing cell assay is greater than 1:400. In some aspects, a subject is determined not to have neutralizing anti-AAVrh74 antibodies if the anti-AAVrh74 antibody titer measured in the neutralizing cell assay is equal to or less than 1:400.

[0288] II.C. Detection of Total Anti-AAVrh74 Antibodies

[0289] In some aspects, total anti-AAVrh74 antibodies comprising neutralizing and non-neutralizing anti-AAVrh74 antibodies are measured. In some aspects, total anti-AAVrh74 antibodies are measured using a total anti-AAVrh74 antibody ELISA assay. In some aspects, a subject having a titer of total anti-AAVrh74 antibodies greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay is seropositive. In some aspects, a subject having a titer of total anti-AAVrh74 antibodies of or less than 1:400 in a total anti-AAVrh74 antibody ELISA assay is seronegative.

[0290] In some aspects, the total anti-AAVrh74 antibody ELISA assay is the assay described in Godeeker et al., Advances in Therapeutics of Neurological Disorders 16:1-7, 2023, which is incorporated herein by reference in its entirety.

[0291] In some aspects, total anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and prior to administration of the imrase. In some aspects, total anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of the first dose of the imrase. In some aspects, total anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of the second dose of the imrase. In some aspects, total anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of the third dose of the imrase.

[0292] In some aspects, total anti-AAVrh74 antibodies are measured after administration of AAVrh74 vector particles. In some aspects, total anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0293] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0294] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0295] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles. In some aspects, total anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0296] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0297] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0298] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles. In some aspects, total anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0299] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0300] In some aspects, total anti-AAVrh74 antibodies are measured after administration of a third dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0301] In some aspects, total anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles. In some aspects, total anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of the first dose of imlizyme.

[0302] In some aspects, total anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a second dose of imlizyme.

[0303] In some aspects, total anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0304] One of ordinary skill in the art will appreciate that the measurement of total AAVrh74 antibodies is not limited by the number of administrations of AAVrh74 vector particles or the number of doses of imlizyme. In some aspects, total anti-AAVrh74 antibodies are measured at any time a subject is considered to be administered AAVrh74 vector particles. In some aspects, total anti-AAVrh74 antibodies are measured at any time a subject is considered to be administered imlizyme.

[0305] In some aspects, the assay used to measure total AAVrh74 antibodies in a subject is a total AAVrh74 antibody ELISA assay. In some aspects, a subject is determined to have total anti-AAVrh74 antibodies if the anti-AAVrh74 antibody titer measured in the total AAVrh74 antibody ELISA assay is greater than 1:400. In some aspects, a subject is determined to not have total anti-AAVrh74 antibodies if the anti-AAVrh74 antibody titer measured in the total AAVrh74 antibody ELISA assay is equal to or less than 1:400.

[0306] II.D. Detection of Non-Neutralizing Anti-AAVrh74 Antibodies

[0307] In some aspects, a subject to be treated with the AAVrh74 viral vector particles described herein is tested for the presence of non-neutralizing AAVrh74 antibodies. In some aspects, total AAVrh74 antibodies are quantified in a sample of a subject as described herein, and neutralizing AAVrh74 antibodies are quantified in a sample as described herein. In some aspects, the amount of non-neutralizing AAVrh74 antibodies present in a sample of a subject is determined by subtracting the amount of neutralizing AAVrh74 antibodies present in the sample from the amount of total AAVrh74 antibodies present in the sample.

[0308] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and prior to administration of the imrase. In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of a first dose of the imrase. In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of a second dose of the imrase. In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured prior to administration of the AAVrh74 vector particles and after administration of a third dose of the imrase.

[0309] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of AAVrh74 vector particles. In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0310] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a second dose of imrplase.

[0311] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a first dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0312] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles. In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0313] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a second dose of imlizyme.

[0314] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a second dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0315] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of anti-AAVrh74 vector particles. In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of anti-AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0316] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of anti-AAVrh74 vector particles and after administration of a second dose of imrplase.

[0317] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of a third dose of anti-AAVrh74 vector particles and after administration of a third dose of imrplase.

[0318] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles. In some aspects, neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a first dose of imlizyme.

[0319] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a second dose of imlizyme.

[0320] In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured after administration of an additional dose of AAVrh74 vector particles and after administration of a third dose of imrplase.

[0321] One of ordinary skill in the art will appreciate that the measurement of non-neutralizing AAVrh74 antibodies is not limited by the number of administrations of AAVrh74 vector particles or the number of doses of imlizyme. In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured at any time when a subject is considered to be administered AAVrh74 vector particles. In some aspects, non-neutralizing anti-AAVrh74 antibodies are measured at any time when a subject is considered to be administered imlizyme.

[0322] III. Enzymes that cleave IgG

[0323] Certain aspects of the present disclosure relate to a method for treating a disease or condition in a subject in need thereof, such as muscular dystrophy, the method comprising administering to the subject (i) an AAV vector and (ii) an enzyme that cuts IgG. In some aspects, the enzyme targets and cuts human IgG. In some aspects, the enzyme targets and cuts human IgG selected from IgG1, IgG2, IgG3, IgG4, and any combination thereof. In some aspects, the enzyme targets and cuts human IgG1. In some aspects, the enzyme targets and cuts human IgG2. In some aspects, the enzyme targets and cuts human IgG3. In some aspects, the enzyme targets and cuts human IgG4. In some aspects, the enzyme targets and cuts human IgG1, human IgG2, human IgG3, and human IgG4. In some aspects, the IgG binds to an AAV vector. In some aspects, the IgG binds to an AAVrh74 vector. In some aspects, the subject has one or more IgG antibodies that specifically bind to a protein expressed by AAVrh74. In some aspects, the subject has one or more IgG antibodies that specifically bind to an AAVrh74 capsid protein. In some aspects, the one or more IgG antibodies are AAVrh74 neutralizing antibodies.

[0324] In some aspects, the enzyme for cutting IgG comprises a protease. In some aspects, the enzyme for cutting IgG comprises a cysteine ​​protease. In some aspects, the enzyme for cutting IgG comprises a sulfhydryl protease. In some aspects, the enzyme for cutting IgG inactivates the IgG when cutting. In some aspects, the enzyme cuts the IgG at the hinge region of the IgG. In some aspects, after cutting, the IgG can no longer bind to the antigen. In some aspects, after cutting, the IgG can no longer be recognized by human immune cells.

[0325] Any enzyme known in the art that can promote the cleavage of IgG can be used in the methods disclosed herein. In some aspects, the enzyme is a naturally occurring enzyme. In some aspects, the enzyme is derived from a naturally occurring enzyme. In some aspects, the enzyme is a synthetase. In some aspects, the enzyme is recombinant. In some aspects, the enzyme is separated from or derived from bacteria. In some aspects, the enzyme is separated from or derived from bacteria of the genus Streptococcus. In some aspects, the enzyme is separated from or derived from Streptococcus pyogenes. In some aspects, the enzyme is separated from or derived from Streptococcus equi. In some aspects, the enzyme is separated from or derived from bacteria of the genus Mycoplasma. In some aspects, the enzyme is separated from or derived from Mycoplasma canis.

[0326] In some aspects, the enzyme for cleaving IgG comprises an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 1-18 with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity (Table 5). In some aspects, the enzyme for cleaving IgG comprises an amino acid sequence with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity. In some aspects, the enzyme for cleaving IgG comprises an amino acid sequence shown in SEQ ID NO: 2. In some aspects, the enzyme that cleaves IgG comprises the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the enzyme that cleaves IgG comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs 3-18.

[0327] Table 5: Sample enzymes that cleave IgG (amino acid sequences)

[0328]

[0329]

[0330]

[0331] In some aspects, the enzyme for cutting IgG comprises Imliase. In some aspects, the enzyme for cutting IgG consists of Imliase. Imliase (also known as "IdeS" and "Mac-1"; UniProt F8V4V0) is an endopeptidase derived from Streptococcus pyogenes, which targets and cuts all human sub-branches of IgG without cutting IgA (gD, IgE or IgM). Imliase is a cysteine ​​protease with an active site residue consisting of a single Cys residue. Imliase cuts the heavy chain of IgG located at the hinge region to produce two Fc and one F (ab 1 )2 fragments. Cleavage of IgG by imliase inhibits complement activation and Fc receptor-mediated processes, including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). Within hours of administration of imliase, the IgG pool is cleaved into F(ab')2 and Fc fragments, creating a window for the administration of gene therapy.

[0332] It was previously shown that administration of imrilase to highly HLA-sensitive patients prior to renal transplantation eliminated or reduced donor-specific antibodies. Imrilase is also known to interact with vitronectin and platelet receptors through its RGD motif.

[0333] IV. AAV Viral Vector Particles

[0334] Certain aspects of the present disclosure relate to a method for treating a disease or condition in a subject in need thereof, such as muscular dystrophy, the method comprising administering to the subject (i) an AAV vector and (ii) an enzyme that cleaves IgG. The AAV vector may comprise a known vector, or may comprise a variant, fragment, or fusion thereof. In some aspects, the AAV vector is selected from the group consisting of AAV type 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, and any combination thereof. In some aspects, the AAV vector is derived from an AAV vector selected from the group consisting of AAV type 1 (AAV1), AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, and any combination thereof. In certain aspects, the AAV vector comprises regions of at least two different AAV vectors known in the art. In some aspects, the AAV vector comprises an inverted terminal repeat sequence from a first AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV) and a second inverted terminal repeat sequence from a second AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh.74, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV).

[0335] In some aspects, the AAV vector is an AAV1 vector. In some aspects, the AAV vector is derived from an AAV1 vector. In some aspects, the AAV vector comprises a portion of an AAV1 vector. In some aspects, the AAV vector is an AAV2 vector. In some aspects, the AAV vector is derived from an AAV2 vector. In some aspects, the AAV vector comprises a portion of an AAV2 vector. In some aspects, the AAV vector is an AAV3 vector. In some aspects, the AAV vector is derived from an AAV3 vector. In some aspects, the AAV vector comprises a portion of an AAV3 vector. In some aspects, the AAV vector is an AAV8 vector. In some aspects, the AAV vector is derived from an AAV8 vector. In some aspects, the AAV vector comprises a portion of an AAV8 vector. In some aspects, the AAV vector is an AAV9 vector. In some aspects, the AAV vector is derived from an AAV9 vector. In some aspects, the AAV vector comprises a portion of an AAV9 vector. In some aspects, the AAV vector is an AAVrh.74 vector. In some aspects, the AAV vector is derived from an AAVrh.74 vector. In some aspects, the AAV vector comprises a portion of an AAVrh.74 vector. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is present in Srivastava et al., Journal of Virology 45:555-564 (1983), as corrected by Ruffing et al., Journal of General Virology (J Gen Virol) 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 related to AAV-8); the AAV-9 genome is provided in Gao et al., Journal of Virology, 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004).The cloning of the AAVrh.74 serotype is described in Rodino-Klapac et al., Journal of Translational Medicine 5:45 (2007).

[0336] In certain aspects, the AAV vector nucleic acid comprises the nucleic acid sequence of the capsid gene of AAVrh.74. In some aspects, the AAV vector nucleic acid comprises a capsid gene, wherein the capsid gene comprises a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:47. In some aspects, the AAV vector nucleic acid comprises a capsid gene, wherein the capsid gene comprises the nucleic acid sequence shown in SEQ ID NO:47. In some aspects, the AAV vector nucleic acid comprises a capsid gene, wherein the capsid gene comprises a nucleic acid sequence encoding a polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 48. In some aspects, the AAV vector nucleic acid comprises a capsid gene, wherein the capsid gene comprises a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 48.

[0337] In certain aspects, the AAV vector nucleic acid comprises a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:49. In some aspects, the AAV vector nucleic acid comprises a nucleic acid sequence as shown in SEQ ID NO:49. In some aspects, the AAV vector nucleic acid comprises a portion of the nucleic acid sequence shown in SEQ ID NO:49. SEQ ID NO:49 represents the nucleic acid sequence of the AAVrh.74 genome. Nucleotides 210-2147 of SEQ ID NO:49 are the Rep 78 gene open reading frame. In some aspects, the AAV vector nucleic acid comprises the Rep 78 gene open reading frame of AAVrh.74. Nucleotides 882-2081 of SEQ ID NO:49 are the Rep52 open reading frame. In some aspects, the AAV vector nucleic acid comprises the Rep52 open reading frame of AAVrh.74. Nucleotides 2079-2081 of SEQ ID NO:49 are the Rep52 terminator. In some aspects, the AAV vector nucleic acid comprises the Rep52 terminator of AAVrh.74. Nucleotides 2145-2147 of SEQ ID NO:49 are the Rep78 terminator. In some aspects, the AAV vector nucleic acid comprises the Rep78 terminator of AAVrh.74. Nucleotides 1797-1800 of SEQ ID NO:49 are the splice donor site. In some aspects, the AAV vector nucleic acid comprises the splice donor site of AAVrh.74. Nucleotides 2094-2097 of SEQ ID NO:49 are the splice acceptor site. In some aspects, the AAV vector nucleic acid comprises a splice acceptor site corresponding to nucleotides 2094-2097 (SEQ ID NO:49) of AAVrh.74. Nucleotides 2121-2124 of SEQ ID NO:49 are splice acceptor sites. In some aspects, the AAV vector nucleic acid comprises a splice acceptor site corresponding to nucleotides 2121-2124 (SEQ ID NO:49) of AAVrh.74. Nucleotides 174-181 of SEQ ID NO:49 are predicted p5 promoter +1. In some aspects, the AAV vector nucleic acid comprises the predicted p5 promoter +1 of AAVrh.74. Nucleotides 145-151 of SEQ ID NO:49 are p5 TATA box. In some aspects, the AAV vector nucleic acid comprises the Rep52 open reading frame of AAVrh.74. Nucleotides 758-761 of SEQ ID NO:49 are predicted p19 promoter +1.In some aspects, the AAV vector nucleic acid comprises the Rep52 open reading frame of AAVrh.74. Nucleotides 732-738 of SEQ ID NO:49 are the p19 TATA box. In some aspects, the AAV vector nucleic acid comprises the Rep52 open reading frame of AAVrh.74. Nucleotides 1711-1716 of SEQ ID NO:49 are the p40 TATA box. In some aspects, the AAV vector nucleic acid comprises the p40 TATA box of AAVrh.74. Nucleotides 2098-4314 of SEQ ID NO:49 are the VP1 cap gene open reading frame. In some aspects, the AAV vector nucleic acid comprises the VP1 cap gene open reading frame of AAVrh.74. Nucleotides 2509-2511 of SEQ ID NO:49 are the VP2 initiator. In some aspects, the AAV vector nucleic acid comprises the VP2 initiator of AAVrh.74. Nucleotides 2707-2709 of SEQ ID NO:49 are the VP3 initiator. In some aspects, the AAV vector nucleic acid comprises the VP3 initiator of AAVrh.74. Nucleotides 4328-4333 of SEQ ID NO:49 are the polyA signal. In some aspects, the AAV vector nucleic acid comprises the polyA signal of AAVrh.74.

[0338] Table 6: AAVrh.74 sequences

[0339]

[0340]

[0341]

[0342]

[0343] In some aspects, the AAV vector nucleic acid comprises a promoter. Any promoter known in the art can be used in the AAV vector nucleic acid of the present disclosure. In some aspects, the AAV vector nucleic acid comprises a constitutively active promoter. Any constitutively active promoter can be used in the AAV vector nucleic acid, including but not limited to cytomegalovirus immediate gene (CMV) promoter, EF1a promoter, SV40 promoter, PGK1 promoter, Ubc promoter, human beta actin promoter, CAG promoter, TRE promoter, UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, GAL1 promoter, GAL10 promoter, TEF promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, Ubi promoter, H1 promoter, U6 or any combination thereof. In some aspects, the AAV vector comprises a CMV promoter. In some aspects, the AAV vector nucleic acid comprises an SV40 promoter.

[0344] In some aspects, the AAV vector nucleic acid comprises a tissue-specific promoter. In some aspects, a tissue-specific promoter drives the expression of therapeutic molecules encoded by AAV (e.g., micro-dystrophin), α-sarcoglycan and / or β-sarcoglycan in the brain, muscle, kidney, lung, testis, or any combination thereof. In some aspects, a tissue-specific promoter drives the expression of cells of the quadriceps of a subject, the myocardium of a subject, the diaphragm of a subject, or any combination thereof. In specific aspects, a tissue-specific promoter drives the expression of therapeutic molecules encoded by AAV (e.g., micro-dystrophin), α-sarcoglycan and / or β-sarcoglycan in cells of the quadriceps of a subject. In specific aspects, a tissue-specific promoter drives the expression of therapeutic molecules encoded by AAV (e.g., micro-dystrophin), α-sarcoglycan and / or β-sarcoglycan in cells of the myocardium of a subject. In specific aspects, a tissue-specific promoter drives the expression of therapeutic molecules encoded by AAV (e.g., micro-dystrophin), α-sarcoglycan and / or β-sarcoglycan in cells of the diaphragm of a subject.

[0345] In specific aspects, the promoter is selected from the group consisting of: enhancer 358 bp muscle creatine kinase proximal promoter (Enh358MCK), muscle creatine kinase (CK) promoter (e.g., hCK (SEQ ID NO: 31) or hCK Plus (SEQ ID NO: 32)), truncated muscle creatine kinase promoter (tMCK; SEQ ID NO: 26), myosin heavy chain (MHC), MHCK7 (a hybrid version of MHC and MCK; SEQ ID NO: 25), C5-12 (synthetic promoter), muscle-specific and cardiac-specific enhancer (MHCK), CK8, SPc5-12, human desmin (Des) promoter, human α-myosin heavy chain (α-MHC) promoter, rat myosin light chain 2 (MLC-2) promoter, and human cardiac troponin C (cTnC) promoter. In certain aspects, the promoter comprises an MCK promoter. In certain aspects, the promoter comprises an MHCK promoter. In some aspects, the tissue-specific promoter is Enh358MCK. In some aspects, the tissue-specific promoter is the CK promoter. In some aspects, the tissue-specific promoter is the muscle-specific and cardiac-specific enhancer 7 (MHCK7) promoter. In some aspects, the tissue-specific promoter is the CK8 promoter. In some aspects, the tissue-specific promoter is the SPc5-12 promoter.

[0346] In some aspects, the promoter comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 25. In some aspects, the promoter comprises the nucleic acid sequence shown in SEQ ID NO: 25.

[0347] In some aspects, the promoter comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 26. In some aspects, the promoter comprises the nucleic acid sequence shown in SEQ ID NO: 26.

[0348] In some aspects, the AAV vector nucleic acid comprises one or more enhancers. In some aspects, one or more enhancers are present in AAV alone or together with a promoter disclosed herein. In some aspects, at least one enhancer in the one or more enhancers is a tissue-specific enhancer. In some aspects, the one or more enhancers are selected from human skeletal actin gene elements, cardiac actin gene elements, myocyte-specific enhancer binding factor MEF (e.g., MEF2), MyoD enhancer elements, cardiac enhancer factor (CEF) sites, mouse creatine kinase enhancer elements, skeletal fast contraction troponin C gene elements, slow contraction cardiac troponin C gene elements, slow contraction troponin I gene elements, hypoxia-inducible nuclear factor, steroid-inducible elements, glucocorticoid response elements (GREs) and any combination thereof. In some aspects, the AAV vector nucleic acid comprises MEF2. In some aspects, the AAV vector nucleic acid comprises MyoD enhancer elements. In some aspects, the AAV vector comprises a CEF site.

[0349] In some aspects, the AAV vector nucleic acid further comprises an intron sequence, i.e., an intron. In some aspects, the intron sequence is located at the 5' of the nucleic acid sequence encoding the dystrophin. In some aspects, the intron sequence is located at the 3' of the promoter. In some aspects, the intron sequence comprises a synthetic intron sequence. In some aspects, the intron comprises a nucleic acid sequence selected from SEQ ID NO:27-29 having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity. In some aspects, the intron comprises the nucleic acid sequence shown in SEQ ID NO:27. In some aspects, the intron comprises the nucleic acid sequence shown in SEQ ID NO:28. In some aspects, the intron comprises the nucleic acid sequence shown in SEQ ID NO:29.

[0350] Table 7: Sample intron sequences

[0351]

[0352] In some aspects, the AAV vector nucleic acid comprises a post-transcriptional regulatory element. In some aspects, the post-transcriptional regulatory element is located at the 3' position of the nucleic acid sequence encoding the dystrophin protein. In some aspects, the post-transcriptional regulatory element comprises a mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a microRNA binding site, a DNA nuclear targeting sequence (DTS) or any combination thereof. In some aspects, the microRNA binding site comprises a binding site for miR142-3p. In other aspects, the miRNA binding site is selected from Rennie et al., RNA Biol. 13(6):554-560 (2016), and can be in

[0353] In some aspects, the DTS comprises an SV40 enhancer sequence. In some aspects, the DTS comprises a c-Myc enhancer sequence.

[0354] In some aspects, the AAV vector nucleic acid comprises a 3'UTR poly(A) tail sequence. In some aspects, the 3'UTR poly(A) tail sequence is selected from the group consisting of bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. In some aspects, the 3'UTR poly(A) tail sequence comprises bGH poly(A). In some aspects, the 3'UTR poly(A) tail sequence comprises the same sequence as SEQ ID NO:30

[0355] In some aspects, the 3'UTR poly (A) tail sequence comprises the nucleic acid sequence shown in SEQ ID NO:30.

[0356] In some aspects, the AAV vector nucleic acid of the present disclosure further comprises a gene cassette encoding a therapeutic molecule. In some aspects, the therapeutic molecule is selected from a polypeptide, an RNA molecule or a DNA molecule. In some aspects, the therapeutic molecule encoded by the gene cassette is a therapeutic RNA molecule. In some aspects, the therapeutic RNA molecule is selected from miRNA, siRNA, mRNA or any combination thereof. In some aspects, the therapeutic molecule is an antisense oligomer.

[0357] In some aspects, the therapeutic molecule comprises a polypeptide. In some aspects, the polypeptide is a polypeptide that is not expressed or misexpressed in a human subject. In certain aspects, the gene cassette encodes a therapeutic polypeptide selected from micro-dystrophin, beta sarcoglycan, alpha sarcoglycan, and any combination thereof.

[0358] IV.A. Micro-dystrophin transgene

[0359] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a micro-dystrophin polypeptide. In some aspects, the gene cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19. In some aspects, the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO: 19.

[0360] In some aspects, the gene cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a nucleic acid sequence selected from the group consisting of the nucleic acid sequences shown in SEQ ID NOs: 33-39. In some aspects, the gene cassette comprises a nucleic acid sequence selected from the group consisting of the nucleic acid sequences shown in SEQ ID NOs: 33-39.

[0361] In some aspects, the gene cassette comprises a nucleic acid sequence encoding a micro-dystrophin polypeptide, wherein the micro-dystrophin polypeptide comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 40-44. In some aspects, the gene cassette comprises a nucleic acid sequence encoding a micro-dystrophin polypeptide, wherein the micro-dystrophin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 40. In some aspects, the gene cassette comprises a nucleic acid sequence encoding a micro-dystrophin polypeptide, wherein the micro-dystrophin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 41. In some aspects, the gene cassette comprises a nucleic acid sequence encoding a micro-dystrophin polypeptide, wherein the micro-dystrophin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 42. In some aspects, the gene cassette comprises a nucleic acid sequence encoding a micro-dystrophin polypeptide, wherein the micro-dystrophin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 43. In some aspects, the gene cassette comprises a nucleic acid sequence encoding a micro-dystrophin polypeptide, wherein the micro-dystrophin polypeptide comprises the amino acid sequence shown in SEQ ID NO: 44.

[0362] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette and a promoter encoding a micro-dystrophin polypeptide, e.g., any promoter disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding a micro-dystrophin disclosed herein, and (ii) an MHCK7 promoter. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence as shown in SEQ ID NO: 19, and (ii) an MHCK7 promoter. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence as shown in SEQ ID NO: 19, and (ii) a promoter comprising a nucleic acid sequence as shown in SEQ ID NO: 25.

[0363] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a micro-dystrophin polypeptide and an intron sequence, e.g., any intron sequence disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding a micro-dystrophin disclosed herein, and (ii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO: 27. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence shown in SEQ ID NO: 19, and (ii) an intron sequence comprising a nucleic acid sequence shown in SEQ ID NO: 27. In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO:19; (ii) a promoter comprising the nucleic acid sequence shown in SEQ ID NO:25; and (iii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO:27.

[0364] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a micro-dystrophin polypeptide and a 3'UTR poly (A) tail sequence, for example, any 3'UTR poly (A) tail sequence disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding a micro-dystrophin disclosed herein, and (ii) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO: 30. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO: 19, and (ii) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO: 30. In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO:19; (ii) a promoter comprising the nucleic acid sequence shown in SEQ ID NO:25; (iii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO:27; and (iv) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO:30.

[0365] In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises a gene cassette encoding a micro-dystrophin polypeptide, wherein the AAV vector (e.g., an AAVrh74 vector) comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 20. In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises a gene cassette encoding a micro-dystrophin polypeptide, wherein the AAV vector (e.g., an AAVrh74 vector) comprises the nucleic acid sequence shown in SEQ ID NO: 20.

[0366] Table 8: Example AAVrh74-micro-dystrophin nucleic acid sequences

[0367]

[0368]

[0369] IV.A.1. Beta-sarcoglycan transgene

[0370] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a beta sarcoglycan polypeptide. In some aspects, the gene cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21. In some aspects, the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO: 21.

[0371] In some aspects, the gene cassette comprises a nucleic acid sequence encoding a β-sarcoglycan polypeptide, wherein the β-sarcoglycan polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 45. In some aspects, the gene cassette comprises a nucleic acid sequence encoding a β-sarcoglycan polypeptide, wherein the β-sarcoglycan polypeptide comprises the amino acid sequence shown in SEQ ID NO: 45.

[0372] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette and a promoter encoding a β-sarcoglycan polypeptide, e.g., any promoter disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding β-sarcoglycan disclosed herein, and (ii) an MHCK7 promoter. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence as shown in SEQ ID NO: 21, and (ii) an MHCK7 promoter. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence as shown in SEQ ID NO: 21, and (ii) a promoter comprising a nucleic acid sequence as shown in SEQ ID NO: 25.

[0373] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a β-sarcoglycan polypeptide and an intron sequence, e.g., any intron sequence disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding β-sarcoglycan disclosed herein, and (ii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO: 28. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO: 21, and (ii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO: 28. In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO:21; (ii) a promoter comprising the nucleic acid sequence shown in SEQ ID NO:25; and (iii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO:28.

[0374] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a β-sarcoglycan polypeptide and a 3'UTR poly (A) tail sequence, for example, any 3'UTR poly (A) tail sequence disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding β-sarcoglycan disclosed herein, and (ii) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO: 30. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO: 21, and (ii) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO: 30. In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO:21; (ii) a promoter comprising the nucleic acid sequence shown in SEQ ID NO:25; (iii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO:28; and (iv) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO:30.

[0375] In certain aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a β-sarcoglycan polypeptide, wherein the AAV vector (e.g., AAVrh74 vector) comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 22. In certain aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding a β-sarcoglycan polypeptide, wherein the AAV vector (e.g., AAVrh74 vector) comprises the nucleic acid sequence shown in SEQ ID NO: 22.

[0376] Table 9: Example AAVrh74-β-sarcoglycan nucleic acid sequences

[0377]

[0378]

[0379] IV.A.2. Alpha-sarcoglycan transgene

[0380] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding an alpha sarcoglycan polypeptide. In some aspects, the gene cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 23. In some aspects, the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO: 23.

[0381] In some aspects, the gene cassette comprises a nucleic acid sequence encoding an alpha sarcoglycan polypeptide, wherein the alpha sarcoglycan polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 46. In some aspects, the gene cassette comprises a nucleic acid sequence encoding an alpha sarcoglycan polypeptide, wherein the alpha sarcoglycan polypeptide comprises the amino acid sequence shown in SEQ ID NO: 46.

[0382] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding an alpha sarcoglycan polypeptide and a promoter, e.g., any promoter disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding an alpha sarcoglycan disclosed herein, and (ii) a tMCK promoter. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence as shown in SEQ ID NO: 23, and (ii) a tMCK promoter. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence as shown in SEQ ID NO: 23, and (ii) a promoter comprising a nucleic acid sequence as shown in SEQ ID NO: 26.

[0383] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding an alpha sarcoglycan polypeptide and an intron sequence, e.g., any intron sequence disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding an alpha sarcoglycan disclosed herein, and (ii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO: 29. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO: 23, and (ii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO: 29. In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO:23; (ii) a promoter comprising the nucleic acid sequence shown in SEQ ID NO:26; and (iii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO:29.

[0384] In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding an alpha sarcoglycan polypeptide and a 3'UTR poly (A) tail sequence, e.g., any 3'UTR poly (A) tail sequence disclosed herein. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising a nucleic acid sequence encoding an alpha sarcoglycan disclosed herein, and (ii) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO: 30. In some aspects, the AAV vector (e.g., AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO: 23, and (ii) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO: 30. In some aspects, the AAV vector (e.g., an AAVrh74 vector) comprises (i) a gene cassette comprising the nucleic acid sequence shown in SEQ ID NO:23; (ii) a promoter comprising the nucleic acid sequence shown in SEQ ID NO:26; (iii) an intron sequence comprising the nucleic acid sequence shown in SEQ ID NO:29; and (iv) a 3'UTR poly (A) tail sequence comprising the nucleic acid sequence shown in SEQ ID NO:30.

[0385] In certain aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding an alpha sarcoglycan polypeptide, wherein the AAV vector (e.g., AAVrh74 vector) comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 24. In certain aspects, the AAV vector (e.g., AAVrh74 vector) comprises a gene cassette encoding an alpha sarcoglycan polypeptide, wherein the AAV vector (e.g., AAVrh74 vector) comprises the nucleic acid sequence shown in SEQ ID NO: 24.

[0386] Table 10: Example AAVrh74-α-sarcoglycan nucleic acid sequences

[0387]

[0388]

[0389] V. Methods for Producing AAV Viral Vector Particles

[0390] The present disclosure provides a composition comprising recombinant adeno-associated virus (rAAV) viral vector particles, wherein the rAAV vector particles are produced in adherent mammalian cells, and wherein the adherent cells are cultured in N-1 containers under suspension conditions. In some aspects, the rAAV vector particles are vector particles of serotype AAVrh.74 (e.g., rAAVrh74.MHCK7. micro-dystrophin).

[0391] The present disclosure also provides a method for producing rAAV viral vector particles, such as rAAVrh74.MHCK7.micro-dystrophin, in adherent mammalian cells by a suspension seeding process. In some aspects, the suspension and seeding process comprises: (a) culturing cells in a first growth medium containing serum in an N-2 container; (b) removing cells from the first culture medium; (c) inoculating cells from step (b) into a second culture medium in an N-1 container that does not contain serum or has a lower serum concentration than the first culture medium; (d) culturing cells in an N-1 container under suspension conditions; and (e) inoculating a third culture medium in a bioreactor with cells from step (d).

[0392] In some aspects, the suspension seeding process further comprises: (f) transfecting the adherent cells with a transgenic plasmid comprising a recombinant MHCK7. micro-dystrophin construct between AAV ITRs, a plasmid comprising an AAV rep gene and an AAV cap gene, and an adenovirus helper plasmid.

[0393] In some aspects, the plasmid comprising the AAV rep gene and the AAV cap gene comprises the AAV2 rep gene and the AAVrh74 cap gene. In some aspects, the plasmid comprising the AAV rep gene comprises an AAV rep gene having the same AAV serotype as the AAV ITR in the plasmid comprising the recombinant MHCK7. micro-dystrophin construct. For example, the recombinant MHCK7. micro-dystrophin construct comprises the AAV2 ITR, and the plasmid comprising the AAV rep gene comprises the AAV2 rep gene. In some aspects, the plasmid comprising the AAV2 rep gene further comprises the AAVrh74 cap gene. In some aspects, the adenovirus helper plasmid comprises the adenovirus 5E2A, E4ORF6 and VA RNA genes.

[0394] In some aspects, the suspension seeding process further comprises: (g) lysing the adherent cells. In some aspects, the adherent cells are lysed by freeze-thaw, solid shear, hypertonic and / or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis or a combination thereof.

[0395] In some aspects, the suspension seeding process further comprises (h) purifying the rAAVrh74 viral vector particles 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.

[0396] In some aspects, the suspension seeding process further comprises culturing cells in the N-3 container with a first growth medium. In some aspects, the suspension seeding process further comprises culturing cells in the N-4 container with a first growth medium.

[0397] In some aspects, the bioreactor is an adherent bioreactor. In some aspects, rAAVrh74 viral vector particles are purified from a culture produced in an adherent bioreactor.

[0398] In some aspects, the third culture medium in the bioreactor comprises at least one factor that promotes cell adhesion. In some aspects, at least one factor that promotes cell adhesion is selected from the group consisting of: serum, FBS, fibronectin, collagen, laminin, calcium ions, proteoglycans or non-proteoglycan polysaccharides of the extracellular matrix and combinations thereof. In some aspects, the third culture medium in the bioreactor comprises DMEM and 10% FBS.

[0399] In some aspects, the adherent cells are cultured in suspension conditions for about 48-72 hours.

[0400] In some aspects, the N-1 container is a suspension shake flask.

[0401] In some aspects, the adherent cells are selected from the group consisting of: 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 suspended adapted. In some aspects, culturing cells under suspension conditions does not change the cell's attachment dependence. In some aspects, culturing does not change the cell to produce a new cell line.

[0402] In some aspects, a suspension seeding process for producing rAAVrh74.MHCK7.micro-dystrophin in adherent mammalian cells comprises: (a) culturing cells in a first growth medium comprising 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 that does not contain serum or has a lower serum concentration than the first medium; (d) culturing the cells in the N-1 container under suspension conditions; (e) inoculating a third medium in a bioreactor with the cells from step (d); (f) transfecting the cells with a transgenic plasmid comprising a recombinant MHCK7.micro-dystrophin construct located between AAV ITRs, a plasmid comprising an AAV rep gene and an AAV cap gene, and an adenovirus helper plasmid; (g) lysing the cells; and (h) purifying rAAV by at least one column chromatography step. In some aspects, the transgenic plasmid comprises a recombinant MHCK7. micro-dystrophin construct positioned between AAV2 ITRs, and the plasmid comprising the AAV rep gene and the AAV cap gene comprises the AAV2 rep gene and the AAVrh74 cap gene.

[0403] In some aspects, the DNA plasmid of the present disclosure comprises a recombinant AAV (rAAV) genome of the present disclosure. In some aspects, the DNA plasmid is transferred to a cell that allows infection with a helper virus (e.g., adenovirus, adenovirus or herpes virus deleted by E1) of AAV to assemble the rAAV genome into infectious rAAV viral vector particles. The technology for producing rAAV viral vector particles is standard in the art, wherein the rAAV genome to be packaged, the rep gene and the cap gene, and the helper virus function are provided to the cell. The production of rAAV vector particles requires the following components to be present in a single cell (represented herein as packaging cells): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not therein), and the helper virus function. The AAV rep and cap genes can be from any AAV serotype from which recombinant viruses can be derived and can be from an AAV serotype different from the rAAV genome ITRs (generating pseudotyped rAAV viral vector particles), including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAV vector particles is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0404] In some aspects, the method for producing packaging cells is to produce a cell line that stably expresses all necessary components for the production of rAAV viral vector particles. For example, the rAAV genomic nucleic acid comprising lacking AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genomic nucleic acid, and the plasmid (or multiple plasmids) of selectable markers such as neomycin resistance genes are integrated into the genome of the cell. In some aspects, the AAV rep gene is the AAV2 rep gene, and the AAV cap gene is the AAVrh74 cap gene. In some aspects, the AAV rep gene has the same AAV serotype as the AAV ITR in the rAAV plasmid comprising the rAAV genome lacking AAV rep and AAV cap genes. In some aspects, the AAV rep gene has the same AAV serotype as the AAV ITR in the recombinant MHCK7. micro-dystrophin plasmid, and the AAV cap gene is the AAVrh74 cap gene.

[0405] The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., Proc. Natl. Acad. S6. USA 79:2077-2081 (1982)), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., Gene, 23:65-73 (1983)), or by direct blunt-end ligation (Senapathy and Carter, J. Biol. Chem. 259:4661-4666 (1984)). In some aspects, the packaging cell line is infected with a helper virus such as adenovirus. This approach has the advantage that the cells are selectable and suitable for large-scale production of rAAV viral vector particles. Other examples of suitable methods use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0406] The general principles of rAAV viral vector particle production are reviewed in, e.g., Carter, Current Opinions in Biotechnology, 1533-539 (1992); and Muzyczka, N, Curr. Topics Microbial. Immunol., 158:97-129 (1992). Various methods are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al., J. Virol., 63:3822-3828 (1989); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658.776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. Vaccine 13:1244-1250 (1995); Paul et al. Human Gene Therapy 4:609-615 (1993); Clark et al. Gene Therapy Therapy) 3: 1124-1132 (1996); U.S. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the document relating to the production of rAAV vector particles.

[0407] In some aspects, at least one of a plasmid comprising a transgene located between AAV ITRs, a plasmid comprising an AAV rep gene and an AAV cap gene, and / or an adenovirus helper plasmid used to produce AAVrh74 viral vector particles as described herein is stably integrated into the genome of a cell that produces AAVrh74 viral vector particles.

[0408] In some aspects, the transgenic plasmid comprises an expression cassette comprising enhanced green fluorescent protein (eGFP) located between the AAV2 ITRs and is used to prepare AAVrh74 viral vector particles.

[0409] VI. Combination therapy

[0410] In some aspects, a method of treatment is provided, the method comprising administering an initial dose of imrase to a subject in need thereof, followed by administration of rAAVrh74 vector particles. In some aspects, the rAAVrh74 vector particles are administered no later than 54 hours after the initial dose of imrase. In some aspects, the method further comprises measuring the titer of antibodies against rAAVrh74 in the subject before administering imrase. In some aspects, the method further comprises measuring the titer of antibodies against rAAVrh74 in the subject after administering imrase. A useful rAAVrh74 vector particle is the androestrogens Moxpavovec.

[0411] In some aspects, a method of treatment is provided, the method comprising administering an initial dose of about 0.25 mg / kg of imrase intravenously to a subject in need thereof. In some aspects, the method further comprises measuring the titer of antibodies against rAAVrh74 in the subject before administering imrase. In some aspects, the method further comprises measuring the titer of antibodies against rAAVrh74 in the subject after administering imrase. In some aspects, the method further comprises administering rAAVrh74 vector particles if the titer of anti-rAAVrh74 antibodies after administering imrase is or is less than 1:400. In some aspects, the rAAVrh74 vector particles are administered no later than 54 hours after administering imrase. In some aspects, the rAAVrh74 vector particles are the androestrogens Moxpavovek.

[0412] In some aspects, the method further comprises administering a second dose of imrase if the titer of anti-rAAVrh74 antibodies is above 1:400 after the first administration of imrase. In some aspects, the method further comprises measuring the titer of anti-rAAVrh74 antibodies after the second imrase administration, and if the titer of anti-rAAVrh74 antibodies is at or below 1:400, the method further comprises administering rAAVrh74. In some aspects, the rAAVrh74 vector particles are administered no later than 54 hours after the second imrase administration. In some aspects, the rAAVrh74 vector particles are the androestrogens Moxpavovir.

[0413] In some aspects, if the titer of anti-rAAVrh74 antibodies is above 1:400 after administration of the second dose of imrplase, the subject will not be administered any rAAVrh74 vector particles.

[0414] In some aspects, a method of treatment is provided, the method comprising administering an initial dose of 0.25 mg / kg of imrase intravenously. In some aspects, the method further comprises measuring the titer of antibodies against rAAVrh74 in the subject after administration of imrase. In some aspects, the method further comprises administering 1.33×10 intravenously if the titer of antibodies against rAAVrh74 in the subject after administration of imrase is at or below 1:400. 14 vg / kg rAAVr74. In some aspects, rAAVrhH74 is administered within 48 hours after administration of the first dose of imrase. In some aspects, rAAVrh74 vector particles are administered no later than 54 hours from the first dose of imrase. In some aspects, rAAVrh74 is administered within 48 hours from the time point at which the titer of anti-rAAVrh74 antibodies measured is or is less than 1:400. In some aspects, rAAVrh74 is administered within 48 hours from the time point at which the titer of anti-rAAVrh74 antibodies measured after the first dose of imrase is or is less than 1:400. In some aspects, rAAVrh74 is administered within 48 hours from the time point at which the titer of anti-rAAVrh74 antibodies measured after the second dose of imrase is or is less than 1:400. In some aspects, the rAAVrh74 vector particle is administered no later than 54 hours after the first dose or the second dose of imlizyme. In some aspects, the rAAVrh74 vector particle is the diastolic estrogen moxepavik.

[0415] In some aspects, the method comprises administering an initial dose of 0.25 mg / kg of imrase intravenously. In some aspects, the method further comprises measuring the titer of antibodies against rAAVrh74 in the subject after administering the initial dose of imrase. In some aspects, the method further comprises administering a second dose of 0.25 mg / kg of imrase intravenously within 60 hours of measuring the titer of antibodies against rAAVrh74 in the subject to be higher than 1:400. In some aspects, the method further comprises measuring the titer of antibodies against rAAVrh74 in the subject after administering the second dose of imrase. In some aspects, the method further comprises administering 1.33×10 intravenously if the titer of antibodies against rAAVrh74 in the subject measured within 48 hours after administering the second dose of imrase is or is lower than 1:400. 14vg / kg rAAVrh74. In some aspects, the rAAVrh74 vector particles are administered no later than 54 hours after the second dose of imlizyme. In some aspects, if the titer of antibodies against rAAVrh74 in the subject measured within 48 hours after administration of the second dose of imlizyme is higher than 1:400, the subject will not be administered rAAVrh74.

[0416] In some aspects, the method further comprises administering a steroid. In some aspects, the method further comprises administering an oral steroid. In some aspects, the oral steroid is administered daily for at least 12 weeks prior to administering AAVrh74. In some aspects, the additional steroid is administered for at least one day prior to administering AAVrh74. In some aspects, the additional steroid is administered for at least one day prior to administering AAVrh74 and continues to be administered for about 60 days after administering AAVrh74. In some aspects, the oral steroid is prednisone or prednisolone.

[0417] VII. Kit

[0418] Certain aspects of the present disclosure relate to kits for treating muscular dystrophy in human subjects in need thereof. In some aspects, the kit comprises (i) an enzyme disclosed herein that cleaves IgG, (ii) an AAV vector disclosed herein, e.g., an AAVrh74 vector; and (iii) instructions for administering the enzyme and the AAV vector according to any method disclosed herein. In some aspects, the kit comprises (i) an AAV vector disclosed herein, e.g., an AAVrh74 vector; and (ii) instructions for administering the AAV vector to a subject who has previously been administered an enzyme that cleaves IgG according to any method disclosed herein.

[0419] All of the various aspects, aspects and options described herein may be combined in any and all variations.

[0420] Examples

[0421] Example 1 : Safety and efficacy evaluation of AAVrh74.CMV.eGFP (rAAVrh74.CMV.eGFP) after single or repeated intravenous administration to female cynomolgus monkeys, followed by a single intravenous injection of imlimidizumab and then observed for at least 59 days

[0422] The purpose of this study was to evaluate the safety and efficacy of AAVrh74.CMV.eGFP ("Test Article 2", hereinafter referred to as AAVrh74-eGFP) when administered to female cynomolgus monkeys as a single (Groups 2 to 6) or repeated (Group 7) intravenous injections, followed by a single intravenous injection of imrilase ("Test Article 1"). Following administration of AAVrh74.CMV-eGFP, followed by imrilase, the animals underwent an observation period of at least 59 days.

[0423] Study Design

[0424] Female cynomolgus monkeys were assigned to nine groups and dosed as shown in Table 19. Animals in Phase I were administered a single intravenous injection of imrase at a volume of 1 mL / kg to examine the initial tolerability of imrase in monkeys. Animals in Phase II were administered a single dose of prednisolone at a volume of 0.33 mL / kg / day on day 1 (Groups 2 to 7) or once daily for 14 days (Groups 8 and 9 only) by oral gavage. To achieve the primary objective of the study, animals in Phase III were administered daily doses of prednisolone at a volume of 0.33 mL / kg / day by oral gavage for 62 days (Groups 3 to 6) or 96 days (Groups 2 and 7), and single or repeated doses of control products, imrase, and / or AAVrh74.eGFP were administered by intravenous injection at a volume of 1 mg / mL (control product or imrase) or 5.47 mL / kg (control product or AAVrh74.eGFP). The control product for imrase and AAVrh74.eGFP was sterile saline. The control product for prednisolone was reverse osmosis water. Due to the complexity of the study design, animals are referred to by group number, and in the Summary, Results, Discussion, and Conclusions sections, AAVrh74.CMV.eGFP is referred to as AAVrh74.eGFP. Furthermore, since dosing phase II consisted of a single dose of prednisolone to all animals and lasted for one day, dosing phase II was barely discussed in the Summary, Results, Discussion, and Conclusions sections.

[0425] Administration of imlizyme and AAVrh74-eGFP vector

[0426] In this example, non-human primates (NHPs) were dosed with AAVrh74-eGFP vector and imrase to determine whether imrase administered prior to treatment would reduce anti-AAVrh74 total antibody titers to allow safe administration of AAV gene therapy.

[0427] One caveat to the study is that the efficacy of Imritase in NHPs was about 70%, compared to humans where Imritase efficacy was about 99%.

[0428] NHPs were grouped according to pre-existing antibody titers and treatment. Group 2 and 3 animals were seronegative with total AAVrh74 antibody titers less than 1:400.

[0429] Animals in Groups 4 and 5 had total AAVrh74 antibody titers ranging from 1:800 to 1:1,600 and were considered AAVrh74 seropositive animals.

[0430] Group 6 animals had total AAVrh74 antibody titers ranging from 1:3,200 to 1:25,600 and were considered highly seropositive for AAVrh74. Group 7 animals (re-dosing group) had total AAVrh74 antibody titers less than 1:400 before any treatment and were considered seronegative for AAVrh74. After the first dose of AAVrh74 vector, one Group 7 animal had a total AAVrh74 antibody titer less than 1:400, while the other two Group 7 animals had total AAVrh74 antibody titers of 1:204,800 and 1:25,600, respectively.

[0431] Treatments were as follows: Group 2 animals received saline only, and Groups 3 and 4 animals received the AAVrh74-eGFP vector but no imliase (vector control).

[0432] Animals in groups 5 and 6 received imlizyme (Hansa Biopharma AB, Sweden). ) and AAVrh74-eGFP vector.

[0433] Animals in Group 7 received the AAVrh74-eGFP vector followed by imlizyme and then a second dose of the AAVrh74-eGFP vector.

[0434] In groups 5 and 6, imlizyme was administered at 10 mg / kg / dose on day 1, and AAVrh74-EGFP was administered at 1.33 x 10 14 Vector genomes / kg / dose were administered.

[0435] In group 7, AAVrh74-eGFP was expressed at 1.33 x 10 14 Vector genomes / kg / dose were administered, and imlizyme was administered at 10 mg / kg / dose on day 36.

[0436] The dosing schedule is shown in Table 19.

[0437] Table 19

[0438] Dosing Phase III: Test Article 1 (Imlizumab), Test Article 2 (AAVrh74.CMV.eGFP), and Control Article

[0439]

[0440] AAV- = anti-AAVrh74 negative animals, AAV+ = anti-AAVrh74 positive animals, TA1 = test article 1 (Immyrase), TA2 = test article 2 (AAVrh74.CMV.eGFP); vg = vector genome

[0441] aAnimals in Groups 2 through 7 were administered prednisolone at a dose level of 1 mg / kg / day, a dose volume of 0.33 mL / kg, and a dose concentration of 3 mg / mL throughout the duration of the dosing period and until the day before final sacrifice. On days when the control article or test article was administered, prednisolone was administered approximately 1 hour prior to dosing.

[0442] b Anti-AAVrh74 antibody status is based on titer values ​​obtained during the pre-dose period. Anti-AAVrh74 negative animals in Groups 2 and 3 are defined as animals with anti-AAVrh74 antibody titers ≤ 1:400. Anti-AAVrh74 positive animals in Groups 4 and 5 are defined as animals with anti-AAVrh74 antibody titers greater than 1:400 (i.e., 1:800-1:1600). Anti-AAVrh74 positive animals in Group 6 are defined as animals with anti-AAVrh74 antibody titers of 1:3200-1:25600. Anti-AAVrh74 negative animals in Group 7 are defined as animals with anti-AAVrh74 antibody titers ≤ 1:400 within 7 days prior to administration of Test Article 1 on Day 36 and anti-AAVrh74 antibody titer levels ≥ 1:51200.

[0443] c Animals in Groups 3, 4, and 7 received a control product (sterile saline) only on Day 1. Animals in Group 2 received a control product (sterile saline) only on Days 1, 3, 36, and 38.

[0444] dOn Day 1 (Groups 2 to 7) and Day 36 (Groups 2 and 7), animals were dosed with the control article or Test Article 1 at a volume of 1 mL / kg.

[0445] eOn Day 3 (Groups 2 to 7) and Day 38 (Groups 2 and 7), animals were dosed with Control Article or Test Article 2 at a volume of 5.47 mL / kg.

[0446] f Animals in Groups 3, 4, 5, and 6 underwent an observation period of 60 days after administration of the control article or Test Article 2 on Day 3 and were sacrificed on Day 63. Animals in Groups 2 and 7 underwent an observation period of 59 days after administration of the control article or Test Article 2 on Day 38 and were sacrificed on Day 97.

[0447] In summary, animals at three different seropositivity levels were administered control product (saline) or 10 mg / kg / dose imlidase by bolus intravenous injection once on day 1 (Groups 3 to 6) or once on days 1 and 36 (Groups 2 and 7), followed by control product or 1.33 x 10 14 vg / kg / dose AAVrh74.eGFP. As standard immunosuppressive treatment, all animals were also administered a single dose of prednisolone once daily via oral gavage for the duration of the study. Clinical observations associated with AAVrh74.eGFP included rapid breathing, corresponding to macroscopic observations of fluid in the abdomen and thorax noted in one animal dosed with AAVrh74.eGFP (anti-AAVrh74 titer ≤ 1:400) (Group 3).

[0448] Safety and efficacy were assessed based on mortality, clinical observations, body weight, weight change, qualitative food consumption, nerve conduction velocity (NCV) measurements, and clinical and anatomical pathology. Blood samples were collected for clinical pathology, anti-AAVrh74 antibodies, imliase exposure (toxicokinetics) and efficacy (pharmacodynamics), cytokine, peripheral blood mononuclear cells, and complement analysis. Muscle biopsies and selected tissues were collected for vector genome analysis and direct fluorescence evaluation.

[0449] All animals survived until their scheduled sacrifice. No AAVrh74.eGFP-related changes in body weight or food consumption, alterations in sensory NCV, or peripheral blood mononuclear cell analysis were observed in animals administered AAVrh74.eGFP, regardless of prior administration of imlimidase. AAVrh74.eGFP-related clinical observations of rapid breathing were noted in one Group 3 animal, corresponding to macroscopic observations of fluid in the thoracic and peritoneal cavities.

[0450] Total anti-AAVrh74 antibody responses in animals treated with AAVrh74-eGFP and imlibrilase

[0451] The total antibody response to AAVrh74-eGFP is shown in Figure 4. As expected, Group 2 animals that had no pre-existing antibodies and received only saline did not develop anti-AAVrh74 antibodies. Group 3 animals that had a total AAVrh74 antibody titer below 1:400 before any treatment showed an increase in total anti-AAVrh74 antibody titer after receiving the AAVrh74-eGFP vector ( Figure 4 , Group 3).

[0452] Animals in Group 4, whose total AAVrh74 antibody titers before treatment ranged from 1:800 to 1:1,600, also showed an increase in anti-AAVrh74 antibody titers after receiving the AAVrh74-eGFP vector ( Figure 4 , Group 4).

[0453] Animals in Group 5, whose total AAVrh74 antibody titers were 1:800 to 1:1,600 before treatment, showed a decrease in total AAVrh74 antibody titers to less than 1:400 after administration of imlimidase on day 1. After administration of AAVrh74-eGFP on day 3, total AAVrh74 antibody titers increased ( Figure 4 , Group 5).

[0454] Animals in Group 6 with total AAVrh74 antibody titers ranging from 1:3,200 to 1:25,600 before treatment showed a decrease in AArh74 antibody titers to approximately 1:400 after administration of imlimidase on day 1. After administration of AAVrh74-eGFP on day 3, total AAVrh74 antibody titers increased ( Figure 4 , Group 6).

[0455] Group 7 animals with total AAVrh74 antibody titers of / less than 1:400 before treatment developed anti-AAVrh74 antibody titers after receiving the first dose of AAVrh74-eGFP vector on day 3. Animals were treated with imliase on day 36, which reduced total anti-AAVrh74 antibody titers. On day 38, animals received a second dose of AAVrh74-eGFP, which resulted in a moderate increase in anti-AAVrh74 antibody titers until approximately day 50, after which time point total anti-AAVrh74 antibody titers increased further.

[0456] In summary, these results indicate that when administered to NHPs on day 1, imrase maintained total AAVrh74 antibody titers at or below 1:400 until at least day 3. Furthermore, the data indicate that a second dose of imrase administered prior to the second dose of AAVrh74-eGFP resulted in a less significant increase in anti-AAVrh74 antibody titers.

[0457] Total anti-AAVrh74 antibody titers after imlizyme

[0458] Animals in groups 2, 4, 5, and 6 were treated with imlimidase, and total anti-AAVrh74 antibody titers were measured starting at 5 min to 5760 min (96 h) after imlimidase treatment ( Figure 5 In group 2 control animals, no AAVrh74 antibodies were detected. In group 4 animals, where AAVrh74 antibody titers ranged from 1:800 to 1:1,600 before treatment, imlimidase treatment resulted in titers below 1:400 ( Figure 5 In animals in group 5, where pre-treatment AAVrh74 antibody titers ranged from 1:800 to 1:1,600, and in animals in group 6, where pre-treatment AAVrh74 antibody titers ranged from 1:3,200 to 1:25,600, imlidase treatment resulted in AAVrh74 antibody titers below 1:400 (5 and 30 minutes later, respectively). Figure 5 ).

[0459] In group 7 animals with anti-AAVrh74 antibody titers of 1:204,800; 1:25,600; and 1:400, respectively, imlimidase treatment resulted in a decrease in anti-AAVrh74 antibody titers in all animals after 5 minutes, with the lowest titers measured between 30 and 360 minutes (6 hours) ( Fig. 6A Importantly, animals in Group 7 did not experience a significant increase in anti-AAVrh74 antibody titers for at least 72 hours after reintroduction of nti-AAVrh74-eGFP ( Figure 6B ).

[0460] In summary, animals administered a single dose of AAVrh74-eGFP had increased anti-AAVrh74 capsid antibody titers noted in both anti-AAVrh74-negative and -positive animals, with slightly higher titers observed in seropositive animals (Group 4). Imretinase treatment reduced titers to cutoff (titer cutoff defined as ≤1:400) in animals with low (titer levels ≤1:400) and moderate (titer levels 1:800–1:1600) seropositivity. However, for animals with high seropositivity levels (titer ≥1:51200, Group 7) that were re-administered with AAVrh74-eGFP, imretinase was unable to reduce high seropositivity titers to cutoff, except for 1 of 3 animals. Dosing of low-seropositive and moderate-seropositive animals was able to reduce titers to cutoff following imretinase treatment. The ability of imretinase treatment to reduce titers to ≤1:400 depended on the total load of anti-AAVrh74 antibodies prior to AAV administration.

[0461] Cleavage efficiency of Imlase

[0462] Western blot was used to measure the cleavage efficiency of imliase in serum samples from all animals. Serum samples of seronegative group 3 animals (P0201, P0202, P0203) and seropositive group 4 animals (ID Nos.: P0301, P0302, P0303) not treated with imliase are shown in Figure 7 and Figure 8 middle.

[0463] Seropositive Group 5 animals (P0401, P0402, P0403) with baseline AAVrh74 antibody titers of 1:400, 1:800, and 1:1,600, when treated with imliase, began to display scIgG, F(ab)2, and Fc fragments 5 minutes after imliase treatment and resulted in AAVrh74 antibody titers of 1:200, 1:50, and 1:50, respectively, after imliase treatment. Fig. 9 Furthermore, scIgG, F(ab)2, and Fc fragments predominated over whole IgG for at least 24 hours in all animals.

[0464] Similarly, seropositive Group 6 animals (P0501, P0502, P0503) with baseline AAVrh74 antibody titers of 1:6,400, 1:3,200, and 1:3,200, when treated with imliase, began to display scIgG, F(ab)2, and Fc fragments 5 minutes after imliase treatment and resulted in AAVrh74 antibody titers of 1:800, 1:200, and 1:400, respectively, after imliase treatment ( Fig.10 ScIgG, F(ab)2, and Fc fragments predominated over whole IgG for at least 24 hours in all animals.

[0465] The AAVrh74 antibody titers of the seronegative group 7 animals that received the first dose of the AAVrh74-eGFP vector on day 36 were 1:204,800, 1:400, and 1:25,600 (P0601, P0602, and P0603), respectively. Fig.11 ). After imliase treatment on day 36, animals in group 7 began to express scIgG, F(ab)2, and Fc fragments 5 minutes after imliase treatment, and the AAVrh74 antibody titers after imliase treatment were 1:6,400, 1:200, and 1:3,200, respectively ( Fig.11 ScIgG, F(ab)2, and Fc fragments predominated over whole IgG for at least 24 hours in all animals.

[0466] eGFP transgene expression

[0467] Immunolabeling for green fluorescent protein (GFP) was used to detect eGFP transgene expression in cells and was annotated in the heart (cardiomyocytes), skeletal muscle (myocytes and cells within inflammatory infiltrates), liver (hepatocytes, Kupffer cells, and / or cells within small clusters of infiltrates), spleen (monocytes in germinal centers and / or randomly scattered mononuclear cells), and kidney (tubular epithelium, macula densa cells, and / or interstitial cells of renal papillae, occasionally in collecting duct epithelium, and / or rarely in apical epithelium of glomeruli) of animals from all AAVrh74-eGFP-treated groups, and in the gallbladder (reactive mesothelium) of one Group 7 animal.

[0468] eGFP transgene expression was assessed by fluorescence microscopy. Seronegative Group 3 animals administered AAVrh74-eGFP vector showed Fig.12 ), skeletal muscle ( Fig.13 ) and the diaphragm ( Fig.14 ) with high GFP expression.

[0469] Seropositive Group 4 animals administered AAVrh74-eGFP vector showed Fig.12 ) and the diaphragm ( Fig.14 ) and moderate GFP expression in skeletal muscle ( Fig.13 ). A trend toward increased GFP expression was observed in animals with low anti-AAVrh74 antibody titers.

[0470] Seropositive group 5 animals treated with imlichase and administered with AAVrh74-eGFP vector showed Fig.12 ), skeletal muscle ( Fig.13 ) and the diaphragm ( Fig.14 ), where animals with an anti-AAVrh74 antibody titer of 1:50 had the highest GFP expression after imrase treatment, and animals with a titer of 1:200 had slightly lower GFP expression after imrase treatment.

[0471] In contrast, highly seropositive group 6 animals administered with the AAVrh74-eGFP vector after imliase treatment showed significant changes in the heart ( Fig.12 ), skeletal muscle ( Fig.13 ) and the diaphragm ( Fig.14 ). As discussed above, Group 6 animals had post-Immunase anti-AAVrh74 antibody titers of 1:800, 1:200, and 1:400, respectively.

[0472] One group 7 animal with an anti-AAVrh74 antibody titer of 1:400 after the first dose of AAVrh74-eGFP and 1:200 after imlidase treatment showed significant changes in the heart ( Fig.12 ), skeletal muscle ( Fig.13 ) and the diaphragm ( Fig.14 ). In contrast, two animals of group 7 with high anti-AAVrh74 antibody titers after the first dose of AAVrh74-eGFP and anti-AAVrh74 antibody titers of 1:6,400 and 1:3,200, respectively, after imlimidase treatment showed high GFP expression in the heart ( Fig.12 ) and low GFP expression in skeletal muscle ( Fig.13 ) and the diaphragm ( Fig.14 ) in the medium GFP expression.

[0473] To determine the transduction level in all tissues, the average GFP expression in all tissues was normalized to Group 3 (anti-AAVrh74 antibody negative and AAVrh74eGFP administered). Groups 4, 5, 6, and 7 showed reduced GFP expression compared to Groups 3 and 5 (anti-AAVrh74 antibody titers of 1:800-1:1,600 and imliase + AAVrh74-eGFP treatment), showing the highest GFP in all tissues compared to Groups 4, 6, and 7 ( Fig.15 ).

[0474] GFP expression was also measured in different muscle tissues. For the seronegative AAVrh74-eGFP treated Group 3 animals, GFP expression was highest in all muscle tissues tested ( Fig.16 Seropositive Group 4 animals treated with AAVrh74-eGFP showed moderate GFP expression in the heart, biceps interfemoral nerve, diaphragm, and vastus medialis muscle ( Fig.16 Thus, animals with moderate levels of pre-existing anti-AAVrh74 antibodies (1:800-1:1,600 anti-AAVrh74 antibody titers) prior to treatment with AAVrh74-eGFP had low levels of eGFP expression, consistent with the understanding that pre-existing antibodies prevent AAVrh74-eGFP expression. Low-level seropositive Group 5 animals treated with imlimidase prior to AAVrh74-eGFP showed moderate GFP expression in the heart, diaphragm, lateral and medial gastrocnemius, and vastus lateralis and medialis muscles ( Fig.16Thus, administration of imrase to animals with moderate levels of pre-existing antibodies (1:800-1:1600 anti-AAVrh74 antibody titers) (Group 5) resulted in increased eGFP expression compared to animals with moderate levels of pre-existing antibodies that did not receive imrase (Group 4). In contrast, animals with high levels of pre-existing antibodies (1:3,200-1:25,600 anti-AAVrh74 antibody titers) treated with imrase prior to AAVrh74-eGFP (Group 6) showed low levels of GFP expression only in the heart, diaphragm, triceps, lateral and medial gastrocnemius, and no GFP expression in other tissues ( Fig.16 ).

[0475] In addition, those animals with pre-existing low antibody titers treated twice with imrase and AAVrh74-eGFP produced moderate levels of GFP expression (Group 7). These data demonstrate the effectiveness of imrase in increasing the transduction rate of AAVrh74-eGFP in seropositive animals according to antibody titers.

[0476] Vector genome analysis

[0477] Data from vector genome analysis of tissues showed that animals without pre-existing anti-AAVrh74 antibodies (titer levels ≤ 1:400, Group 3) achieved the highest levels of vector genome in animals. For animals with pre-existing moderate antibodies (1:800-1:1600 anti-nti-AAVrh74 antibody titer range, Group 5), administration of imrase prior to administration of AAVrh74-aeGFP resulted in increased GFP expression compared to animals with the same pre-existing antibody range that did not receive imrase (Group 4), confirming the effectiveness of imrase in reducing pre-existing antibodies and enhancing AAV transduction and subsequent GFP expression in seropositive animals.

[0478] However, despite the administration of imrplase, animals with pre-existing high anti-AAVrh74 antibody titer levels (1:3200-1:25600 titers) produced the second lowest vector genome transduction among the groups. One animal (Group 7) showed evidence of seroconversion prior to the administration of imrplase, resulting in the lowest transduction level of Group 7 animals. Following 2 doses of AAVrh74-eGFP, Group 7 exhibited the lowest transduction levels in all tissues analyzed and was most similar to Group 6 in terms of transduction.

[0479] Cytokine analysis

[0480] No changes in any of the cytokines examined were noted at 24 or 48 hours post-dose for animals administered imrilase compared to pre-dose period values ​​on day 1, controls, or animals not administered imrilase.

[0481] Interleukin-1 receptor antagonist (IL-1RA) and monocyte chemoattractant protein-1 (MCP-1)

[0482] AAVrh74-eGFP-single dose

[0483] Robust increases in IL-1RA levels (70.75-fold) and MCP-1 levels (4.38-fold) were noted on day 43 of dosing phase III (40 days post-AAVrh74-eGFP) in AAVrh74-negative animals (Group 3) administered AAVrh74-eGFP compared to pre-dose phase values. Increases several fold higher were noted in two animals within Group 3. IL-1RA levels decreased at subsequent time points compared to day 43 dosing phase III values, but remained greater than 18-fold above pre-dose values. MCP 1 levels continued to remain similar to day 43 dosing phase III values ​​for the remainder of the study.

[0484] In AAVrh74-positive animals (Groups 4, 5, and 6) administered control article or imlizyme and AAVrh74-eGFP, less robust increases in IL-1RA (greater than 4.5-fold) and MCP-1 levels (greater than 0.96-fold) were noted at 72 hours post-dose on Day 1 of Dosing Phase III (24 hours post-AAVrh74-eGFP dose). In anti-AAVrh74 antibody-positive animals administered control article and AAVrh74-eGFP (Group 4) or imlizyme and AAVrh74-eGFP (Group 6), IL 1RA and MCP-1 levels tended to return to pre-dose levels and remained at levels similar to or slightly higher than pre-dose values ​​at Day 43 of Dosing Phase III and subsequent time points. However, in anti-AAVrh74 antibody-positive animals administered imlizyme and AAVrh74-eGFP (Group 5), IL-1RA and MCP-1 levels remained increased in 2 of 3 animals and 1 of 3 animals, respectively, at day 43 and subsequent time points during dosing phase III compared to baseline values.

[0485] In summary, AAVrh74-eGFP-related increases in IL-1RA and MCP-1 were noted in both AAVrh74-negative and -positive animals (Groups 3 to 6), and delayed but robust and prolonged increases were noted in AAVrh74-negative animals administered AAVrh74-eGFP (Group 3). Changes in IL-1RA and MCP-1 levels in AAVrh74-positive animals administered imrhase and AAVrh74-eGFP (Groups 5 and 6) were smaller in magnitude and not consistent among animals within the same group compared to anti-AAVrh74 antibody-positive animals administered imrhase and AAVrh74-eGFP (Group 4); therefore, these were considered unrelated to imrhase.

[0486] AAVrh74-eGFP - Repeated doses

[0487] An increase in IL-1RA levels was noted in one anti-AAVrh74 antibody negative animal at 72 hours post-dose on Day 1 of dosing phase III (24 hours after the first AAVrh74-eGFP dose). A more robust increase in IL-1RA (17.87-fold) was noted in all anti-AAVrh74 antibody negative animals (Group 7) administered AAVrh74-eGFP on Day 15 of dosing phase III (12 days after the first AAVrh74-eGFP dose) compared to pre-dose values. On Day 29 of dosing phase III (26 days after AAVrh74-eGFP administration), IL-1RA levels trended back to pre-dose values, but remained above pre-dose IL-1RA values ​​(2.58-fold). A further increase in IL-1RA levels was noted in only one animal administered a second dose of AAVrh74-GFP on Day 57 of Dosing Phase III (Day 21 after the imrase dose and Day 19 after the second AAVrh74-eGFP dose, respectively), whereas IL-1RA values ​​in the other animals administered a second dose of AAVrh74-eGFP (Group 7) remained close to the levels noted on Day 29 of Dosing Phase III (before administration of imrase) for the remainder of the study. In summary, an AAVrh74-eGFP-related increase in IL-1RA was noted in anti-AAVrh74 antibody-negative animals administered a first dose of AAVrh74.eGFP. Among animals within this same group, changes in IL-1RA following the imrase dose and the second AAVrh74-eGFP dose were smaller in magnitude and less consistent; thus, the changes were not considered to be related to AAVrh74-eGFP or imrase.

[0488] Compared with the pre-dose value, in an animal using AAVrh74.eGFP, the reduction of MCP-1 levels was noted at the 29th day of the dosing phase (26 days after the first AAVrh74-eGFP dose). At all subsequent time points, MCP-1 levels continued to remain reduced in this animal. Compared with baseline values, an animal using the second dose of AAVrh74-eGFP observed an increase in MCP-1 levels at the 43rd day of dosing phase III (7 days after the imliase dose and 5 days after the second AAVrh74-eGFP dose, respectively) and at all subsequent time points during dosing phase III. In general, the change of MCP-1 levels in animals (Group 7) after the first and second doses of AAVrh74-eGFP was inconsistent in animals and was considered to be unrelated to AAVrh74-eGFP.

[0489] Other cytokines

[0490] At all time points evaluated, even before dosing, for all animals, the levels of IL-8 were detectable or above the upper limit of quantitation. Changes in IL-8 were comparable to pre-dose period values ​​and were noted in controls; therefore, these changes were attributed to normal biological changes and were considered independent of the test article.

[0491] Increases in MIP-1β were observed in animals administered AAVrh74.eGFP (Group 7) on day 15 of dosing phase III (12 days after the first AAVrh74-eGFP dose). These increases were small in magnitude and were not observed at subsequent time points and were therefore considered unrelated to the test article.

[0492] Levels of GM-CSF, IFN-γ, L-1β, IL-2, IL-5, IL-6, IL-10, IL12 / IL23p40, IL-13, IL 15, IL 17A, MIP-1α, and TGF-α were occasionally elevated above the limit of quantitation in animals administered control preparations or AAVrh74-eGFP. The magnitude of changes in these cytokines was small, observed in the control group, and not consistent among animals in the same group; therefore, the changes were considered normal biological variation and were not related to imliozinase or AAVrh74-eGFP.

[0493] In summary, in animals administered a single dose of AAVrh74-eGFP, AAVrh74-eGFP-associated increases in IL-1RA and MCP-1 were noted in both anti-AAVrh74 antibody-negative animals (Group 3) and positive animals (Groups 4, 5, and 6), with delayed but prolonged and more robust increases noted in negative animals. No consistent imliase-associated changes were noted in IL-1RA or MCP-1 levels in anti-AAVrh74 antibody-positive animals (Groups 5 and 6) administered imliase and AAVrh74-eGFP compared to animals administered saline and AAVrh74-eGFP (Group 4). In animals administered repeated doses of AAVrh74-eGFP (Group 7), an AAVrh74-eGFP-associated increase in IL-1RA, but not MCP-1, was noted in anti-AAVrh74 antibody-negative animals following the first dose of AAVrh74.eGFP. No consistent AAVrh74-eGFP-associated changes were noted in IL-1RA or MCP-1 levels in these animals following administration of imlimidase and a second dose of AAVrh74-eGFP.

[0494] No imlichase- or AAVrh74.eGFP-related changes in GM-CSF, IFN-γ, IL-1β, IL-2, IL-5, IL-6, IL-8, IL-10, IL12 / IL23p40, IL-13, IL-15, IL-17A, MIP-1α,β, or TGF-α levels were noted in animals administered single or repeated doses of AAVrh74-eGFP.

[0495] Thus, only AAVrh74-eGFP-related changes in the levels of IL-1RA and MCP-1 were noted in anti-AAVrh74 antibody negative and positive animals. The increases were more robust and prolonged in anti-AAVrh74 antibody negative animals compared to anti-AAVrh74 antibody positive animals. No imliase-related changes were noted in the levels of any of the cytokines examined. No relevant effects of AAVrh74-eGFP or imliase on the levels of other cytokines and complement activation were noted.

[0496] Clinical Pathology

[0497] Clinical pathology findings associated with AAVrh74-eGFP in animals from Groups 2 to 7 were mild to moderate in magnitude and consisted primarily of increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, often associated with increased creatine kinase (CK) activity. Clinical pathology was consistent with skeletal muscle damage, which correlated with microscopic findings of degeneration / necrosis in cardiac and skeletal muscle. Cardiomyocyte degeneration / necrosis in the heart may also contribute to the above-mentioned increases in CK and / or AST activity. Enzyme activities (ALT, AST, and CK) in all AAVrh74-eGFP-treated groups generally had the highest magnitude on days 22 and / or 36, regardless of antibody titer or co-administration of imliase, and generally showed a trend of decreasing values ​​over time (days 36 to 63) in Groups 4, 5, and 6 compared to animals in Groups 3 and 7.

[0498] Additional AAVrh74-eGFP-related effects were noted in one animal in Group 3 and Group 7, with decreased albumin and total protein concentrations; decreased albumin concentration, cardiomyocyte degeneration, necrosis, and inflammation that may have led to pleural effusions were considered contributing mechanisms to fluid accumulation in two or more of these individual cavities in these animals. The AAVrh74-eGFP-related effects limited to Group 7 animals consisted of decreased red blood cell mass, decreased mean cell volume (decreased red blood cell [RBC] size), decreased mean corpuscular hemoglobin concentration, and decreased mean corpuscular hemoglobin (decreased RBC hemoglobin content); increased RBC distribution width and moderately increased absolute reticulocyte counts, suggesting red blood cell regeneration; and increased platelet counts, thought to be secondary to decreased red blood cell mass and regenerative responses. Decreased food consumption may have contributed to these findings and was associated with decreased cholesterol concentrations in this animal. In addition, evidence of hepatobiliary perturbations (increased gamma-glutamyltransferase and alkaline phosphatase activities and increased total bilirubin concentrations) was noted in Group 7 animals in the absence of microscopic evidence of hepatobiliary changes. Additional AAVrh74-eGFP-related effects limited to Group 3 animals consisted of increased urea nitrogen and inorganic phosphorus concentrations, indicative of renal perturbations, which were not affected by microscopic correlates in the kidney.

[0499] In anatomical pathology, vector-related microscopic findings were noted in the AAVrh74-eGFP treated groups at terminal sacrifice in the peripheral nervous system (sciatic, left and right plantar nerves, and left and right radial nerves), heart, skeletal muscle (biceps femoris, left gastrocnemius [medial and lateral], right gastrocnemius [medial and lateral], tibialis anterior, and skeletal muscle of the tongue and esophagus), and liver. Vector-related microscopic findings in the central nervous system (brainstem and spinal cord) and lumbar dorsal root ganglia were noted in animals from Groups 3, 5, and 7. These microscopic findings in the spinal cord and dorsal root ganglia are well-characterized changes observed with the administration of AAV vectors (Hinderer et al., 2018; Hutt et al., 2022). Additionally, these findings were not associated with clinical observations or observations during nerve conduction velocity testing.

[0500] Microscopic findings associated with AAVrh74-eGFP noted in all AAVrh74-eGFP-treated groups, whether or not before imlimitase, included neurofibrillary degeneration (mild to moderate) in peripheral nerves (sciatic, left and right plantar, and left and right radial); oval cell hyperplasia (mild to moderate) in the liver; degeneration / necrosis (mild to moderate) and mononuclear inflammation (mild to marked) in the heart; and degeneration and necrosis (mild to marked), mononuclear inflammation (mild to marked), regeneration (mild to mild), and / or fibrosis (mild to moderate) in skeletal muscle (biceps femoris, left gastrocnemius [medial and lateral], right gastrocnemius [medial and lateral], tibialis anterior, and / or skeletal muscle of the tongue and esophagus).

[0501] Microscopic findings in skeletal muscle are consistent with the recognized role of GFP reporter gene expression in skeletal muscle and oval cell hyperplasia in liver, and correlate with clinical pathological findings of increased aspartate aminotransferase (AST), creatine kinase (CK), and alanine aminotransferase (ALT). Degeneration and necrosis in the heart may also contribute to increased AST and / or CK activity. Macroscopic findings associated with the AAVrh74 vector were noted at the intravenous injection site (thickened tissue around the right saphenous vein) and right medial gastrocnemius muscle (discolored tan) in one Group 5 animal, and correlated with microscopic findings of skeletal muscle degeneration, necrosis, and mononuclear inflammation in skeletal muscle deep to the right saphenous vein and right medial gastrocnemius muscle.

[0502] In summary, these results suggest that animals with higher AAVrh74 titers had less severe AAVrh74-eGFP-related effects noted in clinical pathology test results, consistent with the well-known effect of pre-existing anti-AAV antibodies to reduce AAV transduction. No AAVrh74-eGFP-related effects were noted in coagulation test results during dosing phase III, either with or without imlibrilase. AAVrh74-eGFP-related hematologic effects during dosing phase III were limited to the aforementioned Group 7 animals.

[0503] Overview

[0504] Regardless of seropositivity levels, animals receiving a single dose of AAVrh74eGFP noted an increase in anti-capsid antibody titers. Treatment with Imretinase reduced anti-AAVrh74 antibody titer levels to a defined cutoff (≤1:400) in animals with low and moderate pre-existing titer levels. After the second dose sequence, Imretinase was unable to reduce high anti-AAVrh74 titer levels to the cutoff in 2 of 3 animals in Group 7. The ability of Imretinase treatment to reduce titers to the cutoff value depends on the total load of anti-AAVrh74 antibodies before AAV administration. Similarly, vector genome analysis of tissues and quantification of GFP percentage also showed that, depending on pre-existing antibody titer levels, Imretinase was effective in reducing pre-existing antibodies, enhancing AAV transduction and subsequent eGFP expression, and enhancing the transduction rate of AAVrh74-eGFP in seropositive animals.

[0505] Overall, imlizyme was found to be a safe and effective way to reduce pre-existing anti-AAVrh74 titer levels in seropositive animals administered AAVrh74-eGFP, however, the reduction was dependent on pre-existing anti-AAVrh74 titer levels prior to imlizyme administration. Repeated administration of imlizyme on day 36 followed by a second administration of AAVrh74-eGFP on day 38 reduced anti-AAVrh74 antibody titer levels in 1 of 3 animals and is therefore less effective in a repeat dosing scenario.

[0506] In conclusion, this NHP study proved the concept that pretreatment imrilase reduced total AAVrh74 antibody titers to safely administer AAVrh74 gene therapy. No adverse clinical events, no deaths related to imrilase or AAV were observed, and no immunotoxicology or histopathology was observed, including in reproductive organs, in this NHP study.

[0507] Reduction of AAVrh74 antibody titers by imlichase pretreatment allowed efficient transduction and expression of the GFP transgene in muscle, heart, and diaphragm.

[0508] Example 2 : An open-label, systemic gene delivery study to evaluate the safety, tolerability, and expression of the diastolic estradiol moxepavir versus imliprove in subjects with Duchenne muscular dystrophy with pre-existing antibodies to rAAVrh74

[0509] Goals and End Points

[0510] Main Objectives

[0511] 1) Evaluation of micro-dystrophin expression at 12 weeks following administration of the diandroestrogen moxepavik in subjects pretreated with imlimax. Endpoints: Change from baseline to week 12 in the amount of micro-dystrophin expression in biopsied muscle tissue as measured by Western blot; immunofluorescence (IF) fiber intensity; and IF dystrophin positive fiber percentage (PDPF) (Part 1).

[0512] 2) To evaluate micro-dystrophin transduction at 12 weeks after administration of the diandroestrogen moxepavik in subjects pretreated with imlimitrase.

[0513] Endpoint: Vector genome copies in muscle tissue biopsies using polymerase chain reaction.

[0514] Secondary Objectives

[0515] 1) Establishment of the pharmacokinetic (PK) profile of imrilase. Endpoint: PK of imrilase in serum up to 7 days after imrilase administration.

[0516] 2) Establishment of the pharmacodynamic (PD) profile of imrase (cleavage and recovery of IgG). Endpoint: Imrase PD (total IgG) in serum up to 12 weeks after imrase administration.

[0517] 3) Evaluation of rAAVrh74 antibody titers after imrplase administration. Endpoint: rAAVrh74 antibody titers up to 120 hours after imrplase administration.

[0518] 4) Evaluation of rAAVrh74 genome concentrations in systemic circulation. Endpoint: Vector genome copies using polymerase chain reaction in serum up to 7 days after administration of the androestrogen Moxepavik.

[0519] 5) Evaluate the safety of imlizyme. Endpoints: the incidence of treatment-emergent adverse events; deterioration of vital signs or physical examination findings; the incidence of adverse events of special concern; the incidence of serious adverse events; and clinically significant abnormalities in safety laboratory assessments.

[0520] 6) To evaluate the safety of imlizyme followed by combination with the diandroestrogen moxepavir. Endpoints: incidence of treatment-emergent adverse events; worsening of vital signs or physical examination findings; incidence of adverse events of special interest; incidence of serious adverse events; clinically significant abnormalities in safety laboratory assessments; electrocardiogram (ECG); and echocardiogram (ECHO).

[0521] Exploratory goals

[0522] 1) To evaluate the effect of the diandroestrogen moxepavir on creatine kinase (CK) levels. Endpoint: Change in CK from baseline over 104 weeks.

[0523] 2) To evaluate the effect of the diandroestrogen moxepavik on assessments of physical function as assessed by the North Star Ambulatory Assessment (NSAA) score. Endpoint: Change in NSAA total score from baseline to Week 104.

[0524] 3) To evaluate the effect of the diandroestrogen moxepavik on timed functional tests as assessed by measuring: 100 meter walk / run (100MWR); time to ascend 4 steps; time to get up from floor; and 10 meter walk / run (10MWR). Endpoints: Change in time to 100MWR from baseline to 104 weeks; change in time to ascend 4 steps from baseline to 104 weeks; change in time to get up from floor from baseline to 104 weeks; change in time to 10MWR from baseline to 104 weeks.

[0525] 4) Evaluation of the immunogenicity of the diandroestrogen moxpavoviride as assessed by detecting cellular immune responses to rAAVrh74 and the micro-dystrophin transgene; and antibodies to rAAVrh74 and the micro-dystrophin. Endpoints: Cellular immune responses to rAAVrh74 and the micro-dystrophin transgene as measured by ELISpot over 104 weeks, see Slota et al., Expert Rev Vaccines 10(3):299-206, 2011; and antibody titers to rAAVrh74 and the micro-dystrophin transgene over 104 weeks.

[0526] 5) Establish the immunogenicity profile of imrase (anti-drug antibodies [ADA]). Endpoint: Imrase ADA serum levels within 104 weeks after imrase administration.

[0527] method

[0528] This is an open-label study in ambulatory male subjects aged ≥4 to ≤8 years with DMD and pre-existing antibodies to rAAVrh74. Approximately 6 subjects will be treated with imlimidase followed by treatment with the diastolic estradiol, moxepavir. To ensure 6 subjects receive both therapies, additional subjects (up to a maximum of 6) may be recruited to account for subjects who received imlimidase but did not meet the criteria for dosing with the diastolic estradiol, moxepavir.

[0529] Subjects will receive an initial intravenous dose of imrplase (0.25 mg / kg) followed by the diandroestrogen moxepavir (1.33 × 10 14 vg / kg) intravenous infusion, or if the subject's post-Imlase rAAVrh74 result remains reactive, a second intravenous dose of Imlase (0.25 mg / kg) within 60 hours of their initial Imlase dose. For subjects who receive the second intravenous dose of Imlase, if the post-Imlase rAAVrh74 result is not reactive, the subject will receive the intravenous androgen moxepavik (1.33×10 14 vg / kg). Subjects whose rAAVrh74 results return to responsiveness after the second dose of imlimitrase will not receive the diandrosensitizer Moxepavik.

[0530] The first three subjects dosed with both imlizyme and the diandroestrogen moxepavir are sentinel subjects and will enter the imlizyme infusion period, separated by at least one week. Safety, PK / PD, and 12-week expression data for the three sentinel subjects will be reviewed by the SRC before additional subjects enter the infusion period.

[0531] The study will consist of 4 periods:

[0532] - A screening period of up to approximately 3 weeks (pre-infusion) during which disease characteristics, baseline therapy, imrplase antibody concentrations, and rAAVrh74 antibody titers will be assessed, and pre-infusion evaluations will be completed.

[0533] ● An approximately 1-week baseline period (pre-infusion) during which baseline assessments will be completed. This period begins on the day of enrollment and ends the day before the first imlimidase infusion.

[0534] - Up to a 5-day imrilase infusion period, during which up to 2 intravenous doses of imrilase will be administered.

[0535] o Subjects will begin a 4-week course of prophylactic antibiotics beginning on the day of the first imlimidase infusion.

[0536] ○ Subjects will receive an antihistamine prior to each imlizyme infusion.

[0537] Follow-up period:

[0538] o Subjects whose rAAVrh74 antibody results remain reactive despite two doses of imlimidazole infusion will not receive the diandrozole and will be followed for 52 weeks, during which safety parameters will be monitored. For these subjects, the last study procedure will be performed at Week 52.

[0539] ○ Subjects whose rAAVrh74 antibody results return to non-reactive after imipramine infusion will receive a single dose of the diandroestrogen moxepavir (1.33 × 10 14 vg / kg) intravenous infusion, during which safety and expression parameters will be evaluated.

[0540] -Subjects will receive at least 1 mg / kg of glucocorticoids (prednisone equivalents) daily starting at least 1 day before the dexamethasone infusion and continuing until at least 60 days after the dexamethasone infusion, unless earlier taper is necessary to manage adverse events.

[0541] - Part 1 of the follow-up period will begin with the diandroestrogen Moxepavik infusion (Day 1) until Week 12.

[0542] - Part 2 of the follow-up period will begin after Week 12 and continue until Week 104. For subjects who complete the study, the last study visit will be at Week 104.

[0543] Efficacy, safety, and immunogenicity assessments will be performed throughout the study and are listed in the Timeline of Events, Tables 11-14.

[0544] Number of subjects:

[0545] Up to 12 subjects may be enrolled; however, enrollment will cease once approximately 6 ambulatory subjects aged ≥4 to ≤8 years are dosed with both imlizyme and diastolic steroids. At least three subjects enrolled in the study will have diastolic steroid antibody titers of ≥1:1600.

[0546] Inclusion criteria:

[0547] Meet the following standards all Criteria Subjects will be eligible to participate in the study:

[0548] 1. Male at birth, ambulatory, and aged ≥4 to ≤8 years at screening.

[0549] 2. Have a clear diagnosis of DMD based on clinical findings and documentation of confirmatory genetic testing using clinical diagnostic genetic testing prior to screening. The genetic report must describe a frameshift deletion, frameshift duplication, premature stop ("nonsense"), canonical splice site mutation, or other pathogenic variant in the DMD gene that is completely contained between exons 18 to 79 (inclusive) that is expected to result in the absence of dystrophin.

[0550] a. Mutations between or including exons 1-17 are not eligible.

[0551] b. In-frame deletions, in-frame duplications, and variants of uncertain significance (VUS) are ineligible.

[0552] 3. Ability to cooperate with movement assessment tests.

[0553] 4. All regionally recommended immunizations for the encapsulated organisms are current.

[0554] 5. A stable dose of oral corticosteroids for at least 12 weeks prior to screening, and the dose is expected to remain constant (except for potential adjustments to accommodate changes in body weight) until Week 52 of the study.

[0555] 6. Have a total rAAVrh74 antibody result that is reactive as determined by the total anti-AAVrh74 antibody ELISA at screening.

[0556] 7. Sexually active subjects must agree to use condoms throughout the study, and female sexual partners must also use a medically acceptable form of birth control (e.g., oral contraception).

[0557] 8. Whether those under the age of consent (<18 years old) have one or more parents or legal guardians who can understand and comply with the study visit schedule and all other study protocol requirements.

[0558] 9. Whether the subject is willing to provide informed consent (if applicable) and has one or more parents or legal guardians who are willing to provide written informed consent / assent for the subject to participate in the study.

[0559] Exclusion criteria

[0560] Meet the following criteria any Subjects will be excluded from this study if:

[0561] 1. Previous treatment with imlizyme.

[0562] 2. High-dose IVIG therapy (2 g / kg BW) within 28 days prior to imlimidase treatment.

[0563] 3. Left ventricular ejection fraction <40% on screening ECHO or clinical signs and / or symptoms of cardiomyopathy.

[0564] 4. Major surgery within 3 months before Day 1 or planned surgery at any time during the study period.

[0565] 5. The presence of any other clinically significant disease, including cardiac, pulmonary, hepatic, renal, hematological, immunologic, or behavioral disease, or infection or malignancy or concomitant disease, or requirement for chronic medication that, in the opinion of the investigator, creates an unnecessary risk of receiving study drug or medical condition, or extenuating circumstances that, in the opinion of the investigator, may impair the subject's ability to comply with tests or procedures required by the study protocol or impair the subject's health, safety, or clinical interpretability.

[0566] 6. Have serological evidence of current, chronic or active human immunodeficiency virus, hepatitis C or hepatitis B infection.

[0567] 7. The subject has a history of major thrombotic events, active peripheral vascular disease, or confirmed hypercoagulable state.

[0568] 8. Subjects with active pulmonary tuberculosis.

[0569] 9. The patient has a history of thrombotic thrombocytopenic purpura (TTP) or a known family history of TTP.

[0570] 10. Have a medical condition or extenuating circumstances that, in the opinion of the investigator, may impair the subject's ability to comply with the tests or procedures required by the study protocol or compromise the subject's health, safety, or clinical interpretability.

[0571] 11. Symptomatic infection within 4 weeks prior to Day 1 (e.g., upper respiratory tract infection, pneumonia, pyelonephritis, meningitis, tuberculosis).

[0572] 12. Demonstrates cognitive delays or impairments that, in the opinion of the investigator, may confound motor development.

[0573] 13. Treatment with any of the following therapies according to the specified time frame:

[0574] Any time: gene therapy, cell-based therapy (e.g., stem cell transplant), CRISPR / Cas9, or any other form of gene editing.

[0575] Day 1 during 12 weeks and any time during the study: Use human growth factor.

[0576] Within 6 months of Day 1: any study drug; any treatment designed to increase dystrophin expression (eg, Translarna TM ).

[0577] 14. Received a live virus vaccine within 4 weeks of the Day 1 visit or an inactivated vaccine within 2 weeks, or is expected to receive a vaccination within the first 3 months after Day 1.

[0578] 15. Have abnormal laboratory values ​​considered clinically significant by the investigator, including but not limited to:

[0579] ○ Gamma-glutamyl transferase > 2 times the upper limit of normal (ULN).

[0580] ○Total bilirubin > ULN. Note: Confirmation of elevated total bilirubin due to Gilbert's syndrome is not exclusionary.

[0581] ○ White blood cell count >18,500 per μl.

[0582] ○ Platelets < lower limit of normal.

[0583] 16. Known hypersensitivity to the diandroestrogen moxepavik or its excipients or to imlizyme or its excipients.

[0584] 17. The family does not wish the subject's research participation to be disclosed to general practitioners / primary care physicians and other healthcare providers.

[0585] Study Drug, Dosage and Mode of Administration:

[0586] Imritivir (0.25 mg / kg) intravenously; and the diandroestrogen moxepavik (1.33 × 10 14 vg / kg).

[0587] Study duration:

[0588] The expected duration of study participation for each subject is approximately 108 weeks; the pre-infusion period is approximately 4 weeks, and the treatment and follow-up period is 104 weeks. For subjects not receiving the diandroestrogen moxepavik, the expected duration of participation is approximately 56 weeks; the pre-infusion period is approximately 4 weeks, and the infusion and follow-up period is 52 weeks.

[0589] Statistical methods:

[0590] Sample Size Determination: Analysis of the primary endpoint of this study will be descriptive. A total of up to 12 subjects may be enrolled with approximately 6 ambulatory subjects ≥ 4 to ≤ 8 years old dosed with both imlizyme and the diandroestrogen moxepavik.

[0591] Statistical Analysis of Primary Endpoints: Statistical analysis of micro-dystrophin expression and transduction will be performed following determination of micro-dystrophin expression at baseline and Week 12, and determination of micro-dystrophin transduction completed at Week 12. Observed values ​​and changes from baseline values ​​for the primary endpoint will be summarized descriptively.

[0592] Statistical Analysis of Secondary Endpoints:

[0593] PK and / PD Data Analysis

[0594] The PK parameter Cmax will be estimated based on the actual values. The remaining PK parameters will be estimated by non-compartmental or compartmental analysis based on the measured results of the serum concentration-time data of imlizyme, and if possible, the following parameters (but not limited to) will be estimated: AUC, Tmax, t1 / 2, CL and Vz.

[0595] PD analysis will be descriptive based on serum concentration-time data of intact IgG, absolute concentrations of IgG (intact + singly cleaved IgG [scIgG]), and measurements of the proportion of remaining IgG.

[0596] Security Analysis

[0597] Adverse events, clinical laboratory evaluations, vital signs, ECG, ECHO, physical examination findings, and weight and height will be summarized descriptively.

[0598] rAAVrh74 Antibody and Vector Genome Analysis

[0599] Antibody titers to rAAVrh74 and vector genome copies in serum will be summarized descriptively.

[0600] Study Design

[0601] Figure 1 The study design for subjects receiving imliase alone is shown in .

[0602] The overall study design is shown in Figure 2 middle.

[0603] The design of the pre-infusion period and the imlidase infusion period is shown in Figure 3During the pre-infusion period and the imlizyme infusion period, certain events were scheduled as shown in Table 11. Scheduled events for subjects who did not receive the diandroestrogen moxepavik during the follow-up period are shown in Table 12. Scheduled events for subjects who did not receive the diandroestrogen moxepavik during the follow-up period are shown in Table 13 (Part 1) and Table 14 (Part 2).

[0604] Screening / Baseline Assessment

[0605] Demographics and medical history

[0606] Demographic information (eg, age, sex at birth, race, ethnicity, weight, height, body mass index) and medical history will be obtained for all subjects.

[0607] Genetic diagnosis

[0608] Subjects must have a definitive DMD diagnosis based on clinical findings and documentation of previous confirmatory genetic testing using clinical diagnostic genetic testing prior to screening. The genetic report must describe a frameshift deletion, frameshift duplication, premature stop, or other pathogenic variant in the DMD gene that is completely contained between exons 18 to 79 (inclusive) that is expected to result in the absence of dystrophin.

[0609] Mutations between or including exons 1-17 are not eligible.

[0610] In-frame deletions, in-frame duplications, and variants of uncertain significance ("VUS") were not eligible.

[0611] Mutations contained entirely within exon 45 (inclusive) were not eligible.

[0612] Efficacy evaluation

[0613] Micro-dystrophin expression

[0614] As shown in Tables 11 and 13, muscle biopsies will be collected from all subjects at baseline and Week 12 to evaluate micro-dystrophin expression. Muscle biopsies should be collected using open biopsy or VACORA core biopsy with sponsor approval. The biopsy will require muscle tissue to be collected from the medial gastrocnemius muscle. If this muscle is not feasible, use of an alternative muscle will require prior approval from the sponsor.

[0615] Biopsy samples will be used to quantify transgene expression by Sarepta's Western blot adjusted for muscle content, IF fiber strength, and PDPF. Please refer to the Surgical and Laboratory Biopsy Manual for more details on the handling and processing of biopsy tissue.

[0616] Functional assessment

[0617] Every effort must be made to have the same clinical assessor perform the physical functional assessment (NSAA and timed functional test) for each assessed subject throughout the study. Functional assessments should be scheduled in the morning and should be the first assessment performed during a given visit.

[0618] Functional assessments will be administered at the time points indicated in Tables 11, 13, and 14.

[0619] North Star Ambulatory Assessment Scale

[0620] The NSAA is a clinician-administered scale that scores performance in a variety of functional activities (Mazzone E et al., Neuromuscul Disord. 20(11):712-6 (2010), which is incorporated herein by reference in its entirety). The scale was designed for use in boys with DMD who are able to stand, and has been used in DMD boys in the age range of this study (≥3 and <8 years, and ≥8 years) (Connolly et al. Neuromuscul Disord. 2013; 23(7):529-39; Mrecuri E et al., PLoS One. 016; 11(8):e0160195; Muntoni F et al., PLoS One. 2019 Sep 3; 14(9):e0221097, which are incorporated herein by reference in their entirety).

[0621] During this assessment, subjects performed 17 different functional activities including 10MWR, rising from sit to stand, standing on 1 leg, climbing a box step, descending a box step, rising from lying to sit, rising from the floor, lifting head off the floor, standing on heels, and jumping.

[0622] Subjects will be scored as follows: 2 = normal, no significant change in activity; 1 = modified method, but achieves goal independent of physical assistance from others; and 0 = unable to achieve goal independently.

[0623] Time to get up from the floor

[0624] Testing the time to get up from the floor is part of the NSAA (item 11) and quantifies the time required for the subject to stand in an upright position with the side arms, starting from a supine position with the side arms (Henricson 2013, which is incorporated herein by reference in its entirety). The time required for the subject to complete the task will be recorded during the NSAA administration.

[0625] 10-meter walk / run

[0626] The timed 10MWR is part of the NSAA (item 17) and quantifies the time required for a subject to run or walk 10 meters (on a straight sidewalk) from a stationary position (Mc Donald CM et al., Muscle Nerve 2013 Sep; 48(3):357-68, which is incorporated herein by reference in its entirety). The subject is encouraged to run past the 10-meter mark. The time required for the subject to complete the distance will be recorded during the NSAA administration.

[0627] Time to climb 4 steps

[0628] The timed 4 steps test quantifies the time required for a subject to ascend 4 standard steps (each step is 6 inches high) (Bushby K et al., Clin Investig (Lond). 2011; 1(9): 1217-35, which is incorporated herein by reference in its entirety). The time required for a subject to climb 4 standard size steps will be recorded.

[0629] 100m walk / run

[0630] The 100MWR quantifies the time it takes a subject to run or walk 100 meters (on a straight sidewalk) from a stationary position (Alfano LN et al., Neuromuscular Diseases 2017; 27(5):452-7, which is incorporated herein by reference in its entirety). The subject is encouraged to run past the 100-meter mark. The time it takes the subject to complete the distance will be recorded.

[0631] Vector genome and quantification

[0632] Vector quantification in serum will be performed using polymerase chain reaction in all subjects at study visits as shown in Tables 11, 13, and 14. On Day 2, samples should be collected approximately 22 to 26 hours after the end of the diastolic estrogen moxepavik infusion.

[0633] Vector genome copies will be measured in muscle biopsy samples at baseline and week 12 using polymerase chain reaction.

[0634] Creatine kinase

[0635] Creatine kinase levels after infusion of the diandroestrogen moxpavovec will be used as an exploratory efficacy measure.

[0636] Safety Assessment

[0637] Physical examination

[0638] A complete or brief (symptom-oriented) physical examination will be performed by the Investigator or qualified study staff at the time points specified in Tables 11-14.

[0639] A comprehensive physical examination will include examination of the general appearance, head, ears, eyes, nose, throat (HEENT), heart, chest (respiratory), abdomen (gastrointestinal), skin, lymph nodes, extremities, and the musculoskeletal and nervous systems.

[0640] A brief physical examination will include general appearance; HEENT; cardiac; chest; abdominal; and skin evaluation.

[0641] Electrocardiogram and echocardiogram

[0642] A 12-lead ECG will be obtained in triplicate at the time points specified in Tables 11, 13, and 14 using an ECG machine that automatically calculates heart rate and measures PR, QRS, and QT intervals. All ECGs should be performed before any invasive procedure (e.g., blood sampling, study drug infusion, or biopsy). All ECGs are performed only after the subject has rested and been silent for approximately 5 minutes. On Day 1, an ECG will be performed in triplicate before and after the end of the infusion of the androstenone moxpavovec. The investigator (or designee) will review the ECG results and determine whether the findings are clinically significant. The Day 1 pre-infusion review should be completed and recorded before the infusion of the androstenone moxpavovec.

[0643] Standard 2-dimensional ECHO will be obtained at the time points specified in Tables 11 and 14. The echocardiogram should be performed prior to any invasive procedure (e.g., blood sampling, study drug infusion, or biopsy). The ECHO will be reviewed and interpreted by local medically qualified personnel. Left ventricular ejection fraction will be recorded.

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656] Table 15: List of abbreviations and definitions of terms

[0657]

[0658]

[0659]

[0660] Example 3: Study Design in Example 2

[0661] Overall study design

[0662] This is an open-label study in male subjects aged ≥4 to ≤8 years with DMD and pre-existing antibodies to rAAVrh74. Approximately 6 subjects will be treated with imliprose followed by the diandroestrogen moxepavir.

[0663] To ensure that at least 6 subjects receive both therapies, additional subjects (up to a maximum of 6) may be recruited to account for subjects who receive imlizyme but do not meet the criteria for dosing with the diastolic steroid, moxepavir.

[0664] The first three subjects dosed with both imlizyme and the diandroestrogen moxepavir are sentinel subjects and will enter infusion periods separated by at least one week. Safety, PK / PD, and expression data for the three sentinel subjects will be reviewed by the SRC before additional subjects enter the infusion period.

[0665] The total duration of participation for subjects who received both imrizine and the diandroestrogen moxpavovir is expected to be approximately 108 weeks, including an approximately 4-week pre-infusion period and up to approximately 5 days of imrizine infusion period, and 104 weeks of diandroestrogen moxpavovir treatment and follow-up period. The total duration of participation for subjects who did not receive the diandroestrogen moxpavovir is expected to be approximately 56 weeks, including an approximately 4-week pre-infusion period and up to approximately 5 days of imrizine infusion period, and 52 weeks of follow-up period.

[0666] A schematic diagram of the study design is provided in Figure 1-3 A timeline of events is provided in Tables 11-14.

[0667] Pre-infusion period

[0668] Prior to any study procedures, subjects will provide informed consent / assent, and (for subjects <18 years of age) one or more parents or one or more legal guardians will provide written informed consent for the subject's participation in the study.

[0669] Screening period

[0670] During a screening period of up to approximately three weeks, various assessments will be performed to confirm eligibility, including collection of the subject's medical history, demographics, DMD genotyping records, vital signs, physical examination, and blood draw for enzyme-linked immunosorbent assay (ELISA) and select clinical laboratory evaluations.

[0671] During the Screening Period, additional assessments included ECG, ECHO, RNA samples, ELISpot samples, and review of prior and concomitant medications. Please refer to the timeline of events in Tables 11-14 for a complete overview of the assessments performed during the Screening Period.

[0672] If permitted by the informed consent / assent form, all laboratory samples obtained will be stored for future research on DMD regardless of screening results and stored for up to 15 years after the study is concluded or in accordance with local regulations.

[0673] Baseline period

[0674] The baseline period will begin at confirmation of eligibility and end the day before the first imlimidase infusion. During this period, vital signs, select clinical laboratory assessments, and a brief physical examination will be performed.

[0675] Physical functional assessments to be performed include the NSAA (including time to get up from the floor and a timed functional test of 10 MWR, although the timing component is not part of the NSAA), time to ascend 4 steps, and 100 MWR. Functional assessment data collected at the Baseline Visit will be used for efficacy endpoint analyses as described in Section 5.2 and detailed in the Statistical Analysis Plan (SAP).

[0676] After completing all other pre-infusion assessments and confirming eligibility, muscle biopsies will be performed on all subjects. The baseline biopsy will be a baseline biopsy of the medial gastrocnemius, preferably on the right leg. If the medial gastrocnemius is not feasible, the use of an alternative muscle of the lower limb requires prior approval from the sponsor. If possible, the biopsy at Week 12 will be in the same muscle group as the muscle group used on the contralateral side at baseline. For subjects who may have had a pre-infusion biopsy before entering the study, this can be used as a "baseline" biopsy. An overview of the assessments performed during the baseline period is shown in Table 11.

[0677] Imliase infusion time

[0678] The Imrease infusion period consists of up to two doses of Imrease administered at intervals of up to 60 hours. All enrolled subjects will receive one dose of Imrease. Approximately six hours later, blood samples will be drawn to test antibodies against rAAVrh74. If the rAAVrh74 antibody titer is ≤1:400 and no more than 48 hours have passed since the completion of the Imrease infusion, the subject will enter the 260-week treatment and follow-up period of the androestrogens Moxpavovirk at approximately 48 hours after the Imrease infusion. If the antibody titer result is achieved or is >1:400 after 48 hours, the subject will receive a second Imrease infusion approximately 60 hours after the first infusion. The antibody titer will be drawn approximately 6 hours after the second dose of Imrease; if the antibody titer result is achieved and is ≤1:400 within 48 hours, the subject will enter the treatment and follow-up period of the androestrogens Moxpavovirk at approximately 48 hours after the Imrease infusion. If the antibody titer result is achieved or is >1:400 after 48 hours, the subject will not receive the diandroestrogen moxepavir and will enter the 52-week follow-up period 48 hours after the second imlimidizumab infusion.

[0679] On the day of the first imlimidase infusion, subjects will begin a 4-week course of prophylactic antibiotics to mitigate the possibility of increased risk of infection caused by temporary IgG depletion. Because respiratory tract infections are the most common infections in patients with hypogammaglobulinemia, the antibiotics selected should have coverage for upper respiratory tract pathogens, such as amoxicillin 10 mg / kg twice daily (up to a maximum dose of 500 mg twice daily). Alternative agents with appropriate coverage (e.g., cephalosporins, macrolides) may be used at the discretion of the investigator and may be based on regional or institutional resistance patterns and guidelines.

[0680] To reduce the risk of infusion reactions, an antihistamine (e.g., oral diphenhydramine 1.25 mg / kg up to a maximum of 50 mg) will be administered up to approximately one hour prior to each imlizyme infusion. The choice and dose of antihistamine is at the discretion of the investigator and may be based on local or institutional guidelines.

[0681] Each imlidase infusion is administered as an intravenous infusion of 0.25 mg / kg over 15 minutes using an infusion pump. Please refer to the pharmacy manual for additional information. Body weight recorded the day before (or on the day of) infusion should be used for dosage calculations.

[0682] During the imrilase infusion period, subjects will undergo selected laboratory studies to monitor safety and evaluate the PK / PD of imrilase and the effect of imrilase on rAAVrh74 titers. A summary of the assessments performed during the imrilase infusion period is shown in Table 11.

[0683] Treatment with the diandroestrogen moxepavir and 260-week follow-up period: Parts 1 and 2

[0684] The follow-up period will consist of 2 parts: Part 1 and Part 2. Part 1 of the follow-up period will begin with the infusion of the androestrogen Moxepavox (Day 1) and continue until Week 12. Part 2 of the follow-up period will begin after Week 12 and continue until Week 104.

[0685] Follow-up period: Part 1

[0686] At least one day before the infusion of the androestrogens Moxpavovek, in addition to their chronic DMD-related steroid doses, the subjects will also start additional corticosteroid treatment (prednisone or prednisolone) at 1 mg / kg (Table 11-14). The maximum total daily dose (including chronic and additional doses) is 60 mg per day. The additional steroid will continue until the 60th day; however, with prior approval by the medical supervisor, earlier taper may be allowed to manage AEs. If the gamma-glutamyl transferase (GGT) level is confirmed to be ≥150U / L or there are other clinically significant liver function abnormalities after infusion, the glucocorticoids added after the infusion for immunosuppression should be increased to 2 mg / kg per day. For subjects with 2 mg / kg of additional corticosteroids per day, the maximum total daily dose is 120 mg per day. As assessed by monitoring liver function with GGT, the taper dose of glucocorticoids is implemented based on the individual subject's response to the infusion.

[0687] The diandroestrogen Moxepavir infusion will be administered via a peripheral limb vein over 1-2 hours on Day 1. Body weight obtained the day before or on the day of dosing should be used for dose calculations.

[0688] On Day 1, vital signs and blood samples for selected laboratory studies will be collected. A brief physical examination and ECG will be performed. A summary of the assessments performed on Day 1 is shown in Tables 11-14.

[0689] During Part 1, safety will be assessed by monitoring vital signs, physical examination, ECG, TEAEs, SAEs, and selected laboratory assessments. Other assessments during this period include blood samples for deoxyribonucleic acid (DNA) analysis (obtained from those who consent to this assessment), RNA analysis, PK / PD modeling, functional assessments, and muscle biopsies. A summary of the assessments performed during the follow-up period, Part 1, is shown in Tables 11-14.

[0690] Follow-up period: Part 2

[0691] Part 2 of the follow-up period will begin after Week 12 and continue until the follow-up visit at Week 104. From Week 16 to Week 48, monthly telephone calls will be made between the 12-week in-clinic visits.

[0692] Safety will be assessed by monitoring vital signs, physical examination, ECG, ECHO, TEAEs, SAEs, and selected laboratory assessments.

[0693] Immunogenicity will be assessed by measuring antibody titers against rAAVrh74 using ELISA. In addition, an interferon gamma ELISpot assay will be performed to detect cellular immune responses to the rAAVrh74 capsid and the micro-dystrophin transgene.

[0694] Functional physical assessments will be performed throughout the study and will include the NSAA (which will also be used to measure floor rise time and 10MWR time, although the timing component is not part of the NSAA), 4-step ascent, and 100MWR time.

[0695] Follow-up period for subjects not receiving the diandroestrogen moxepavik

[0696] Subjects who receive an antibody titer of ≥1:400 or an antibody titer >1:400 greater than 48 hours after completing the second dose of imliase will not receive diastolic steroids and will enter the 52-week follow-up period 48 hours after the imliase infusion. Administering the diastolic steroid infusion greater than approximately 48 hours after completing the imliase infusion increases the likelihood that diastolic steroids will be administered outside of the expected IgG nadir induced by imliase.

[0697] Safety monitoring of vital signs, physical examinations, TEAEs, SAEs, and selected laboratory assessments, as outlined in Tables 11-14, will continue through 52 weeks to ensure adequate reconstitution of IgG levels and subsequent anti-imlidase antibodies.

[0698] Study Termination / Early Termination

[0699] For subjects who receive both imlizyme and the diandroestrogen moxepavir and complete the study, the last study visit will be at Week 104. For subjects who terminate the study early, an early termination visit will be required as shown in the event schedule (Table 14).

[0700] After completing 104 weeks of follow-up, subjects will be enrolled in an extension study to evaluate the long-term safety and efficacy of the androestrogens Moxpavovir. Subjects will be followed for at least five years following their Moxpavovir infusion.

[0701] For subjects who did not receive the diandroestrogen moxpavovek and completed the study, the last study procedure will be performed at Week 52. Subjects who did not receive the diandroestrogen moxpavovek will not be enrolled in the extension study.

[0702] Study completed

[0703] The study will be considered completed when all subjects complete the Week 104 or Week 52 visit, as appropriate, or are otherwise discontinued from the study.

[0704] Scientific rationale for study design

[0705] The diandrozole serotype Moxepavir was designed to treat the underlying biological etiology of DMD, however, to date, the presence of pre-existing antibodies to rAAVrh74 has limited its potential for benefit in this population because the antibodies interfere with AAV-mediated gene delivery and attenuate or eliminate its therapeutic effect. Imretinase represents a well-tolerated therapy with an appropriate mechanism of action to deplete pre-existing antibodies to rAAVrh74 and provide a safe therapeutic window for treating seropositive patients with gene therapy.

[0706] The subject dosing schedule allows for evaluation of acute safety on a subject-by-subject basis, and this small open-label study enables collection of acute and subacute clinical safety data with commercially representative process materials. Subjects will be allowed to continue standard of care steroid therapy as well as non-pharmacological interventions, such as physical therapy, during the study. The 12-week duration of Part 1 provides sufficient time to obtain primary endpoint expression data. The total study duration of 104 weeks allows for long-term safety follow-up following infusion of the dexamethasone oxaliplatin.

[0707] Study dose selection rationale

[0708] Androgenes Moxpavovec

[0709] Based on the malnutrition animal model (DMD MDX A broad dose range was evaluated in a mouse model (4.43 × 10 13 vg / kg to 4.01×10 14 vg / kg), a dose-dependent increase in tissue vehicle exposure was observed. MDXIn mice, a robust and nonlinear relationship with a saturated response was demonstrated between tissue vehicle exposure and treatment response (diastolic estradiol protein expression and functional improvement). 14 vg / kg (the clinically recommended dose) compared with the highest dose studied (4.01×10 14 vg / kg) were significantly overlapped in the ER curves for both protein expression and functional improvement, with 1.33×10 14 vg / kg-related therapeutic responses approached the plateau achieved at the highest dose studied. 14 vg / kg, which is the recommended clinical dose, compared with 4.01×10 14 vg / kg-related increases in vehicle exposure resulted in a modest increase (less than 17%) in the expression of the diandroestrogen moxepavir protein, which did not translate into functional improvements in animal models of malnutrition.

[0710] In 3 clinical studies (Diesel Moxpavovek-101, Diesel Moxpavovek-102, and Diesel Moxpavovek-103) and 3 clinical doses (including 6.29 × 10 13 vg / kg and 8.94×10 13 vg / kg and the clinically recommended dose of 1.33×10 14 vg / kg), the generic PK, PD, and population-based exposure response (efficacy and safety) of the diandroestrogen moxepavirque have been characterized in patients with DMD.

[0711] 1.33×10 14 The clinically recommended dose of 1.33 × 10 vg / kg achieved robust tissue transduction and dystrophin expression, which translated into clinically meaningful benefits in motor function outcomes as measured by NSAA total score 1 year after dosing. Importantly, similar to evidence demonstrated in the basic nonclinical studies, 1.33 × 10 14 The dose of 1.33×10 vg / kg also showed a plateau of biological efficacy in patients with DMD (dystrophin expression measured as IF PDPF). These findings support the idea that 1.33×10 14vg / kg of dystrophin is approaching maximization of membrane localization and functional dystrophin expression, and higher doses (above 1.33 x 10 14 vg / kg) is not expected to produce a significant increase in biological efficacy.

[0712] In wild type and DMD MDX Nonclinical GLP toxicology studies in mice using commercially representative materials can be found in the Investigator's Manual for the diandroestrogen Moxpavovek. Using clinical data, an evaluation of the relationship between drug administration (total capsid load and serum vector genome exposure) and clinical safety biomarkers with diandroestrogen Moxpavovek has been performed. In general, no significant relationship was found between drug administration with diandroestrogen Moxpavovek and changes in levels of biomarkers of liver injury (GGT, GLDH), biomarkers of cardiac injury (troponin), and importantly, immune system responses including complement components and platelet counts. This is in line with the current 1.33×10 14 Further support is provided by the overall safety evidence demonstrated for the diandroestrogen moxepavir at doses of vg / kg.

[0713] Imritiase

[0714] This study used the commercially indicated dose of 0.25 mg / kg, up to 2 doses.

[0715] During the clinical development of Imlizumab, a dosage range of 0.12, 0.25 and 0.50 mg / kg was studied in healthy adults and patients with end-stage renal disease. Clinical results show that 0.12 mg / kg is not effective enough in cutting IgG, and compared with 0.25 mg / kg, 0.50 mg / kg does not provide additional efficacy. In addition, one or two doses of 0.25 mg / kg are supported by toxicological studies in non-clinical species, wherein in rabbits and dogs, there is a NOAEL (no adverse effect level) of 2 mg / kg. In addition, in clinical studies, 0.25 mg / kg (up to 2 doses) shows a favorable safety profile.

[0716] The dosage extrapolation to pediatric patients is supported by population PK / PD modeling and simulation, which is displayed after 0.25mg / kg, predicting that the pediatric patients of different ages and weight groups between 0-18 years old have a curve consistent with the PK / PD curve in adults. In addition, the IgG cleavage determination of the imliase effect in the serum of pediatric serum pool (covering 3-11 years old, 12-17 years old or 3-17 years old) and the serum from adult healthy donors shows that there is no difference in effect. The proposed dosage of 0.25mg / kg is clinically evaluated in the study 20-HMedIdeS-21 in the target pediatric population of highly sensitive children with a positive cross-matching test and the age of intending to carry out renal transplantation>1 to<18 years old.

[0717] The same dose of 0.25mg / kg can be extended to patients with DMD, because mutations in the DMD gene and disease progression are not expected to bring renal or liver damage, which will change the metabolism, excretion or clearance of the imliase protein and affect its overall PK curve. Therefore, it is reasonable that the above-mentioned pediatric dose extrapolation can be extended and support the initial evaluation of imliase in patients with DMD. In addition, the pharmacokinetics of imliase are similar to other protein therapeutics, so it is expected that the systemic clearance rate and distribution parameter volume of imliase are proportional to body weight, and the dose of 0.25mg / kg will also be sufficient to reach the effective concentration in the target pediatric population. If a single dose of imliase fails to reduce the titer of total anti-rAAVrh74 to less than 1:400, a second dose can be given within 60 hours of the first infusion. In this study, 0.25mg / kg will be used to evaluate the PK and PD of imliase in patients with DMD to confirm the dose extrapolation.

[0718] Stopping rule or completing the study described in Example 2

[0719] Study enrollment will be interrupted when any of the following sentinel events occur: Emergence of a new serious safety (ADR) alert or signal. Serious patient outcome: Death, life-threatening eventa, hospitalization, or major medical event.

[0720] A subject will be considered to have completed his or her participation in the study after completing the last study visit of his or her follow-up period (Week 104 or Week 52, depending on whether the subject is receiving the androestrogen moxepavir) or has withdrawn from the study.

[0721] Example 4: One or more treatments of patients in the study described in Example 2

[0722] One or more study treatments administered

[0723] Androgenes Moxpavovec

[0724] The diandroestrogen Moxpavovek is supplied as a sterile single-use frozen liquid for intravenous infusion. The frozen drug product must be thawed prior to clinical administration.

[0725] The administration of the diandroestrogen Moxpavovirk will be performed through a peripheral limb vein. The subjects will be closely monitored for at least 6 hours after the infusion is completed (see Table 13). A local anesthetic cream (e.g., lidocaine 2.5%, prilocaine 2.5%, LMX4 cream) may be applied to the skin before inserting an intravenous catheter for each site and subject preference infusion.

[0726] Imritiase

[0727] Imritinase IMP is supplied as a freeze-dried (lyophilized) powder for concentrate for infusion, 11 mg per vial. After reconstitution with sterile water for injection, each mL of concentrate contains 10 mg of imritinase. The concentrate is then diluted in sodium chloride 9 mg / mL (0.9%) infusion solution and administered as an infusion. Please refer to the study-specific pharmaceutical brochure for more detailed information.

[0728] Table 16: One or more study treatments administered

[0729]

[0730] Non-study treatments

[0731] The exact selection and dispensing of non-study medications is at the discretion of the investigator and will be supplied from the study center pharmacy.

[0732] Corticosteroids

[0733] All subjects will receive a stable daily dose of oral corticosteroids for at least 12 weeks prior to the screening visit as required by the inclusion criteria. Each subject will start additional oral steroids (prednisone or prednisolone) for immunosuppression one day before the infusion of androestrogens Moxpavovir and for approximately 60 days after the infusion of androestrogens Moxpavovir.

[0734] antibiotic

[0735] On the day of the first imlimidase infusion, subjects will begin a 4-week course of prophylactic antibiotics to mitigate the possibility of increased risk of infection due to IgG depletion. The antibiotic chosen should have coverage for upper respiratory tract pathogens, such as amoxicillin 10 mg / kg twice daily (up to a maximum dose of 500 mg twice daily). Alternative agents (e.g., cephalosporins, macrolides) may be used at the discretion of the investigator and may be based on local or institutional resistance patterns or guidelines.

[0736] Antihistamines

[0737] To reduce the risk of infusion reactions, an antihistamine (e.g., oral diphenhydramine 1.25 mg / kg up to a maximum of 50 mg) will be administered up to approximately one hour prior to each imlizyme infusion. The choice and dose of antihistamine is at the discretion of the investigator and may be based on local or institutional guidelines.

[0738] Study Drug Handling / Storage

[0739] store

[0740] The diandroestrogen Moxpavovek must be transported and stored at ≤ -60°C.

[0741] Prior to reconstitution, IMBRYASE should be stored refrigerated at +2°C to +8°C protected from light.

[0742] Safety Monitoring

[0743] Safety Monitoring of Liver Chemistry Tests

[0744] Monitoring of liver chemistry tests is indicated as described in Tables 11-14. Confirmation of abnormal liver chemistry test result(s) is indicated if:

[0745] GGT >3× ULN at any time during the study

[0746] AST or ALT measurement >2× baseline value if baseline value is >ULN; or >3× ULN if baseline value is within the normal range

[0747] Subjects with confirmed liver chemistry test results (as described above) should have liver chemistry tests (GGT, ALT, AST, alkaline phosphatase, and total and direct bilirubin) retested at least 2 times per week, with additional testing beyond that determined by the investigator. If the abnormality is stable and the subject is asymptomatic, the frequency of retesting may be reduced to once a week or less.

[0748] Additional investigation

[0749] It is recommended that subjects with confirmed abnormal liver chemistry test results (described above) undergo the following evaluations:

[0750] ● Obtain a more detailed history of symptoms and prior or concurrent illnesses;

[0751] ● Obtain a history of concomitant medication use (including over-the-counter medications and herbal and dietary supplement preparations), alcohol use, recreational drug use, and special diets;

[0752] ●Exclude acute viral hepatitis A, B, C, D, and E; autoimmune hepatitis; nonalcoholic steatohepatitis; hypoxic / ischemic liver disease; and biliary tract disease;

[0753] ●Consider other viral illnesses associated with hepatitis (eg, Epstein-Barr virus [EBV], cytomegalovirus [CMV], parvovirus B19, human herpesvirus 6 [HH6], varicella-zoster virus);

[0754] ● Obtain a history of exposure to environmental chemical agents.

[0755] Additional liver evaluations including gastroenterology / hepatology consultation, liver computed tomography, or magnetic resonance imaging scans may be performed at the discretion of the investigator in consultation with the sponsoring medical monitor.

[0756] Safety Monitoring for Immune-Mediated Myositis

[0757] The occurrence of immune-mediated myositis in subjects will be monitored by monitoring according to the AEs specified in Tables 11-14. In the limited cases reported so far, the onset time is 24-42 days (Bonnemann et al. 2022 Muscular Dystrophy Association Clinical & Scientific Conference; March 13-16, 2022; Nashville, TN. Poster 44.). Subjects will be instructed to report any new muscle weakness in a timely manner, and researchers need to closely evaluate all potential causes, including concomitant diseases.

[0758] Additional investigation

[0759] Subjects who experience rapidly progressive muscle weakness need to be discussed with the sponsor medical monitor to determine if additional monitoring or laboratory testing is necessary. Additional evaluations including immunology consultation, ELISpot and ELISA testing for transgenic micro-dystrophin, muscle biopsy, muscle MRI, and / or electromyographic nerve conduction studies may be performed at the discretion of the investigator in consultation with the sponsor medical monitor.

[0760] Adverse events of particular interest related to immune-mediated myositis include any myositis, including immune-mediated myositis or autoimmune myositis; any severe (CTCAE grade ≥3) weakness or muscle weakness; dysphagia or dysphonia secondary to muscle weakness; rhabdomyolysis, myoglobinuria; chromaturia; and acute onset or exacerbation of myalgia.

[0761] Ultra-sensitive safety monitoring

[0762] Subjects will be monitored for the occurrence of allergic reactions by monitoring the AEs specified in Tables 11 to 14. Subjects will be instructed to promptly report any signs or symptoms of fever or systemic symptoms that may occur during the study, and the investigator is required to closely evaluate all potential causes, including concomitant diseases.

[0763] Additional investigation

[0764] Subjects who experience significant or persistent systemic symptoms, including any CTCAE grade ≥3 hypersensitivity reactions, need to be discussed with the sponsor medical monitor to determine if additional monitoring or laboratory testing is necessary. Additional evaluations, including immunology consultation, allergy testing (e.g., absolute eosinophils, eosinophil cationic protein, serum / plasma tryptase), may be performed at the discretion of the investigator in consultation with the sponsor medical monitor.

[0765] Safety Monitoring of Thrombotic Microangiopathy (TMA)

[0766] The occurrence of complement-mediated reactions in subjects should be monitored primarily by monitoring AEs and complement levels. Subjects will be instructed to promptly report any signs or symptoms of fever or systemic symptoms that may occur during the study, and investigators need to closely evaluate all potential causes, including concomitant diseases. In addition to monitoring AEs, routine laboratory monitoring of complement-mediated reactions, including complement levels, should be performed according to the protocol (see Tables 11-14).

[0767] Additional investigation

[0768] Subjects diagnosed with TMA will need to discuss with the sponsor medical monitor to determine additional monitoring and laboratory testing. Additional evaluations including nephrology consultation and testing for complement activation and other etiologies (C3a, C5a, sC5b-9, Bb, prothrombin time, activated partial thromboplastin time, FDP, D-dimer, ADAMTS13, CFH Ab, STEC, stool culture swab, antiphospholipid antibodies, ECG, echocardiogram, ANA, anticentromere antibodies, anti-scl-70, anti-ds-DNA, plasma homocysteine, plasma and urine methylmalonic acid, G6PD activity, T antigen, influenza testing, CMV and / or EBV) may be performed at the discretion of the investigator in consultation with the sponsor medical monitor.

[0769] Safety Monitoring of Platelet Count Results

[0770] Platelet counts will be monitored as specified in Tables 11-14.

[0771] Confirmed platelet count <75,000 / mm 3The subjects should be evaluated as follows:

[0772] ● Complete blood count with reticulocytes

[0773] ●Peripheral blood smear

[0774] Coagulation tests (prothrombin time / INR, activated partial thromboplastin time)

[0775] hsCRP

[0776] Additional investigation

[0777] Additional platelet evaluation for confirmed, unexplained, significant platelet count decreases including hematology consultation, fibrinogen, fibrinogen split products / D-dimer, von Willebrand factor, total immunoglobulins, complement levels, viral serology, autoantibody screening, antiplatelet antibodies, antiplatelet factor 4 assay, and platelet function tests may be performed at the discretion of the investigator in consultation with the sponsoring medical monitor.

[0778] Safety Monitoring of Rhabdomyolysis

[0779] Rhabdomyolysis must be monitored by urine dipstick and AEs as specified in Tables 11-14.

[0780] Subjects with confirmed heme+dipstick urinalysis will require evaluation of urine microscopy and the following AEs:

[0781] Rhabdomyolysis

[0782] Myoglobinuria

[0783] Color urine

[0784] Acute onset or worsening of myalgia

[0785] Additional investigation

[0786] In the presence of rhabdomyolysis, myoglobinuria, or chromaturia, subjects will require evaluations of myoglobinuria, CK, renal function (e.g., serum cystatin C), and serum chemistries 2 or 3 times per week until values ​​reach usual / pre-event levels or stabilize. If myalgia develops acutely or worsens, these evaluations should be performed at the discretion of the investigator in the absence of rhabdomyolysis, myoglobinuria, or chromaturia.

[0787] In addition, the investigator should obtain more detailed symptoms, previous activity and hydration status, concomitant medication use, and history of recent or concurrent infection. Additional evaluations including rheumatology / immunology consultation, anti-muscle antibodies or ELISpot may be performed at the discretion of the investigator in consultation with the sponsoring medical monitor.

[0788] If acute onset or worsening of weakness is characteristic of the rhabdomyolytic event, subjects should be assessed for rapidly progressive weakness as described in Section 4.4.2.

[0789] Risk of anesthesia-induced rhabdomyolysis

[0790] Participation in this study involves a biopsy (Section 9, 2, 1), which may require anesthesia. Investigators should be aware that subjects with DMD have specific risks and requirements when receiving anesthesia (American Academy of Pediatrics 2005, Birnkrant 2009, Bushby 2010). For example, there are absolute contraindications to the use of depolarizing muscle relaxants (e.g., succinylcholine, also known as suxamethonium) due to the risk of rhabdomyolysis, and avoidance of inhaled anesthetics (e.g., sevoflurane, desflurane, enflurane, halothane, isoflurane) is strongly recommended due to the risk of malignant hyperthermia-like reactions and rhabdomyolysis. There is no evidence that exposure to the desmospavovirin affects this risk.

[0791] Safety monitoring of troponin increases

[0792] Cardiac troponin levels need to be monitored as specified in the timeline of events. Confirmation of the initial cardiac troponin level result is indicated if:

[0793] Cardiac troponin level >3× ULN (or 3× baseline for subjects with elevated baseline values)

[0794] ●Subjects with confirmed abnormal cardiac troponin levels will need to have their cardiac troponin levels retested until the level returns to baseline or has been stable for at least 6 weeks. The frequency of retesting is at the discretion of the investigator.

[0795] Additional investigation

[0796] Additional evaluations including ECG, cardiology consultation, ECHO, cardiac MRI, CKMB, and myoglobin studies may be performed at the discretion of the Investigator in consultation with the Sponsoring Medical Monitor (see also Section 5.3.4).

[0797] Safety monitoring of infection

[0798] Signs and symptoms of potential infection will be monitored throughout the study using vital signs, physical examination, selected laboratories, and patient reports. The following signs and symptoms should prompt consideration of infectious workup:

[0799] Fever; chills and sweats; headache; cough with and without mucus; stuffy or runny nose; sore throat or new mouth sores; hoarseness; swollen lymph nodes in the neck; shortness of breath, chest tightness, or wheezing; burning or pain when urinating; increased urination; unusual vaginal discharge or irritation; redness, soreness, or swelling anywhere, including surgical wounds and mouth; vomiting; diarrhea; abdominal or rectal pain; fatigue, muscle aches, and feeling generally unwell.

[0800] Additional investigation

[0801] Laboratory studies such as CBC, virome, blood, urine, or stool cultures, and imaging studies such as chest X-ray may be performed at the investigator's discretion, and subjects should be managed according to standard of care and applicable guidelines (see also Section 5.3.4).

[0802] Example 5 - Mixed with the above research evaluation

[0803] A time chart outlining the study assessments and the timing of the assessments is provided in Tables 11-14.

[0804] Adverse Events of Special Interest

[0805] In this study, AESI was defined as follows:

[0806] Hepatotoxicity

[0807] GGT>8×ULN

[0808] GGT > 5 × ULN for ≥ 2 weeks

[0809] GGT>3×ULN and total bilirubin>2×ULN or INR>1.5

[0810] GGT > 3 × ULN and new onset of fatigue, nausea, vomiting, right upper quadrant pain or tenderness, fever, rash, or eosinophilia (ie, onset consistent with changes in liver enzymes) (> 5%) considered by the investigator to be possibly related to liver inflammation.

[0811] Myositis

[0812] Any myositis including immune-mediated or autoimmune myositis; any severe (CTCAE grade ≥3) weakness or muscle weakness; dysphagia or dysphonia secondary to muscle weakness.

[0813] Thrombotic microangiopathy (TMA)

[0814] TMA, acute kidney injury, and / or atypical hemolytic uremic syndrome.

[0815] Hypersensitivity

[0816] Anaphylactic reaction, anaphylactoid reaction or angioedema; any severe (CTCAE grade ≥3) allergic reaction; any severe (CTCAE grade ≥3) inflammatory event (e.g., myocarditis, pneumonitis, vasculitis, etc.); any severe (grade ≥3) infusion-related reaction occurring within 24 hours of infusion of the androgen moxepavik; any infusion-related reaction occurring within 48 hours of infusion of imlimitase.

[0817] Thrombocytopenia

[0818] Platelet count < 75,000 / mm 3

[0819] Rhabdomyolysis

[0820] Rhabdomyolysis; myoglobinuria; chromaturia; acute onset or exacerbation of myalgia.

[0821] Troponin evaluation

[0822] Troponin I > 3× ULN (or 3× baseline for subjects with elevated baseline values).

[0823] Infect

[0824] Any severe (grade ≥3) infection occurring within 30 days of imlizyme infusion; Any infection requiring hospitalization occurring within 30 days of imlizyme infusion

[0825] Dosage interruption

[0826] Any event leading to interruption (suspension and / or discontinuation) of imlimitrase administration

[0827] Pharmacokinetics, pharmacodynamics, and laboratory evaluations

[0828] Pharmacokinetic and pharmacodynamic evaluation

[0829] Blood samples for pharmacokinetic and pharmacodynamic analysis will be drawn. Note that the IgG analysis methods typically available in clinical chemistry laboratories (turbidimetry / nephelometric methods) will not distinguish between the different IgG fragments generated after imlizyme treatment. The actual collection date and time of each sample will be recorded in the eCRF. PK samples will be analyzed by a validated electroluminescence-based immunoassay using MesoScale Diagnostics (MSD) technology. PD samples will be analyzed by a validated electroluminescence-based immunoassay using MesoScale Diagnostics (MSD) technology and SDS-PAGE.

[0830] Laboratory evaluation

[0831] Routine clinical laboratory tests will be performed at the time points specified in Tables 11 to 14. Protocol-required safety laboratory assessments are listed in Table 18.

[0832] NOTE: At week 12, samples will be collected prior to biopsy.

[0833] Table 17

[0834]

[0835] Laboratory abnormalities that are considered clinically significant by the Investigator or designee should be recorded as AEs. Clinically significant abnormalities are generally abnormalities confirmed by repeat testing that are sufficiently changed from screening / baseline that, at the discretion of the Investigator, a change in management is warranted.

[0836] Safety laboratory evaluation required by the protocol

[0837] The tests detailed in Table 18 will be performed by a central laboratory.

[0838] Table 18: Safety laboratory assessments required by the protocol

[0839]

[0840] Additional testing may be performed at any time during the study if determined by the investigator to be necessary or required by local regulations.

[0841] It should be understood that the Detailed Description section, rather than the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present invention contemplated by the inventors, and therefore, are not intended to limit the present invention and the appended claims in any way.

[0842] The present invention has been described above with the aid of functional building blocks, which illustrate embodiments of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries may be defined as long as the specified functions and their relationships are properly performed.

[0843] The foregoing description of the specific embodiments will fully reveal the general nature of the invention so that others can easily modify and / or adapt various applications, such as specific embodiments, by applying knowledge in the art without departing from the general concept of the invention, without having to perform unnecessary experiments. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the wording or terminology herein is for descriptive and non-restrictive purposes, so that the terms or wording of this specification will be interpreted by the technician in view of teaching and guidance.

[0844] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0845] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for treating a subject suffering from muscular dystrophy, the method comprising administering to the subject a recombinant AAVrh74 viral vector and an enzyme that cleaves immunoglobulin G (IgG).

2. The method of claim 1, wherein the recombinant AAVrh74 viral vector and the IgG-cleaving enzyme are administered to the subject simultaneously.

3. The method of claim 1, wherein the recombinant AAVrh74 viral vector and the enzyme that cleaves IgG are administered to the subject sequentially.

4. The method of claim 1, wherein the subject is determined to have anti-AAVrh74 antibodies.

5. The method of claim 4, wherein the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

6. The method of claim 4, wherein the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

7. The method of claim 4, wherein the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

8. The method of claim 7, wherein the subject's anti-AAVrh74 antibody titer is greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

9. The method of claim 5, wherein the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

10. The method according to any one of claims 1 and 3 to 9, wherein the recombinant AAVrh74 viral vector is administered after the enzyme that cleaves IgG.

11. The method of claim 10, wherein the subject is administered the recombinant AAVrh74 viral vector no more than 54 hours after the administration of the enzyme that cleaves IgG.

12. The method of claim 10, further comprising administering to the subject a second dose of the IgG-cleaving enzyme prior to administering the recombinant AAVrh74 viral vector if the subject is determined to have anti-AAVrh74 antibodies after administration of the first dose of the IgG-cleaving enzyme.

13. The method of claim 12, wherein the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

14. The method of claim 12, wherein the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

15. The method of claim 12, wherein the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

16. The method of claim 15, wherein the subject's anti-AAVrh74 antibody titer is greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

17. The method of claim 13, wherein the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

18. The method of claims 12 to 17, wherein the recombinant AAVrh74 viral vector is administered after the second dose of the IgG-cleaving enzyme.

19. The method of claim 18, wherein the subject is administered the recombinant AAVrh74 viral vector no more than 54 hours after the administration of the second dose of the IgG-cleaving enzyme.

20. The method of any one of claims 12 to 19, wherein the subject is administered the second dose of the enzyme no more than 60 hours after the first dose of the enzyme that cleaves IgG.

21. The method of claim 15, wherein the subject has an anti-AAVrh74 antibody titer of about 1:1600 to about 1:3200 in a total anti-AAVrh74 antibody ELISA assay.

22. The method of claim 21, further comprising administering to the subject a third dose of the IgG-cleaving enzyme prior to administering the recombinant AAVrh74 viral vector.

23. The method of claim 22, wherein the recombinant AAVrh74 viral vector is administered after the third dose of the IgG-cleaving enzyme.

24. The method of claim 23, wherein the subject is administered the recombinant AAVrh74 viral vector no more than 54 hours after the administration of the third dose of the IgG-cleaving enzyme.

25. The method of any one of claims 22 to 24, wherein the subject is administered the third dose of the enzyme no greater than 60 hours after the second dose of the IgG-cleaving enzyme.

26. A method of treating a subject having muscular dystrophy, the method comprising administering to the subject a recombinant AAVrh74 viral vector, wherein the subject has previously been administered an enzyme that cleaves IgG.

27. The method of claim 26, wherein the subject has been administered an enzyme that cleaves IgG no more than 54 hours prior to administration of the recombinant AAVrh74 viral vector.

28. A method of preparing a subject for gene therapy for muscular dystrophy, the method comprising administering to the subject an enzyme that cleaves IgG prior to administering a recombinant AAVrh74 viral vector.

29. A method of removing anti-AAVrh74 antibodies in a subject, the method comprising administering an enzyme that cleaves IgG, and measuring the titer of anti-rAAVrh74 antibodies in the subject after administering the enzyme that cleaves IgG.

30. The method of claim 29, further comprising measuring the titer of anti-rAAVrh74 antibodies in the subject prior to administering the IgG-cleaving enzyme.

31. The method of claim 29 or 30, further comprising administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is at or below 1:

400.

32. The method of claim 29 or 30, wherein the recombinant AAVrh74 viral vector is not administered to the subject if the anti-rAAVrh74 antibodies in the subject are above 1:400 in a total anti-AAVrh74 antibody ELISA assay.

33. The method of claim 32, further comprising administering to the subject a second dose of the IgG-cleaving enzyme.

34. The method of claim 33, further comprising measuring the titer of anti-rAAVrh74 antibodies in the subject after the second dose of the IgG-cleaving enzyme.

35. The method of claim 34, wherein the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

36. The method of claim 34, wherein the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

37. The method of claim 34, wherein the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

38. The method of claim 37, wherein the subject has an anti-AAVrh74 antibody titer of 1:400 or greater in a total anti-AAVrh74 antibody ELISA assay.

39. The method of claim 35, wherein the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

40. The method of claim 34 or 37, further comprising administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is at or below 1:

400.

41. The method of claim 40, wherein the recombinant AAVrh74 viral vector is administered no more than 54 hours after the second dose of the IgG-cleaving enzyme.

42. The method of claim 34 or 37, wherein the recombinant AAVrh74 viral vector is not administered to the subject if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is higher than 1:

400.

43. The method of any one of claims 1 to 42, wherein the muscular dystrophy is selected from Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy (LGMD).

44. The method of claim 43, wherein the muscular dystrophy is DMD.

45. The method of any one of claims 1 to 44, wherein the IgG is human IgG.

46. ​​The method of any one of claims 1 to 45, wherein the IgG-cleaving enzyme specifically targets and cleaves human IgG selected from IgGl, IgG2, IgG3, IgG4, and any combination thereof.

47. The method of any one of claims 1 to 46, wherein the IgG-cleaving enzyme specifically targets and cleaves human IgGl, human IgG2, human IgG3, and human IgG4.

48. The method of any one of claims 1 to 47, wherein the IgG is associated with the recombinant AAVrh74 viral vector.

49. The method according to any one of claims 1 to 48, wherein the enzyme that cleaves immunoglobulin IgG comprises a protease.

50. The method of claim 49, wherein the protease comprises a cysteine ​​protease or a thiol protease.

51. The method of any one of claims 1 to 50, wherein the enzyme that cleaves IgG inactivates the IgG.

52. The method of any one of claims 1 to 51, wherein the IgG-cleaving enzyme cleaves human IgG at the hinge region.

53. A method according to any one of claims 49 to 52, wherein the protease is isolated or derived from a protease expressed by Streptococcus pyogenes, Streptococcus equi or Mycoplasma canis.

54. The method of any one of claims 1 to 53, wherein the enzyme that cleaves IgG comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 1 or 2.

55. The method of any one of claims 1 to 54, wherein the enzyme that cleaves IgG comprises the amino acid sequence shown in any one of SEQ ID NOs: 3-18.

56. The method according to any one of claims 1 to 55, wherein the enzyme that cleaves IgG comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

57. The method of any one of claims 1 to 56, wherein the enzyme is Imlifilase.

58. The method of any one of claims 1 to 57, wherein the subject has one or more IgG antibodies that specifically bind to a protein present in the recombinant AAVrh74 viral vector.

59. The method of any one of claims 1 to 58, wherein the subject has one or more IgG antibodies that specifically bind to a capsid protein of a recombinant AAVrh74 viral vector.

60. The method of claim 58 or 59, wherein the one or more IgG antibodies are AAVrh74 neutralizing antibodies.

61. The method of claim 58 or 59, wherein the one or more IgG antibodies are AAVrh74 non-neutralizing antibodies.

62. The method of claim 58 or 59, wherein the one or more IgG antibodies are total AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

63. The method of claim 62, wherein the subject's anti-AAVrh74 antibody titer is greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

64. The method of claim 60, wherein the subject has anti-AAVrh74 antibodies in a neutralizing cellular assay.

65. The method of any one of claims 1 to 64, wherein the subject has previously been administered a recombinant AAVrh74 viral vector.

66. The method of any one of claims 1 to 65, wherein the recombinant AAVrh74 viral vector comprises a gene cassette encoding a therapeutic molecule.

67. The method of claim 66, wherein the therapeutic molecule comprises a polypeptide, an RNA molecule, or a DNA molecule.

68. The method of claim 66 or 67, wherein the gene cassette encodes a therapeutic polypeptide selected from micro-dystrophin, beta sarcoglycan, alpha sarcoglycan, and any combination thereof.

69. The method of any one of claims 66 to 68, wherein the gene cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:

19.

70. The method of claim 69, wherein the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO:

19.

71. The method of any one of claims 66 to 68, wherein the gene cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:

21.

72. The method of claim 71, wherein the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO:

21.

73. The method of any one of claims 66 to 68, wherein the gene cassette comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:

23.

74. The method of claim 73, wherein the gene cassette comprises the nucleic acid sequence shown in SEQ ID NO:

23.

75. The method of any one of claims 68 to 74, wherein the recombinant AAVrh74 viral vector further comprises a promoter.

76. The method of claim 75, wherein the promoter is a tissue-specific promoter.

77. The method of claim 75 or 76, wherein the promoter is selected from the group consisting of MHCK7 promoter and tMCK promoter.

78. The method of any one of claims 75 to 77, wherein the promoter comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to SEQ ID NO: 25 or 26.

79. The method according to any one of claims 75 to 77, wherein the promoter comprises the nucleic acid sequence shown in SEQ ID NO: 25 or 26.

80. The method of any one of claims 66 to 79, wherein the recombinant AAVrh74 viral vector further comprises an intron.

81. The method of claim 80, wherein the intron comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence shown in SEQ ID NOs: 27-29.

82. The method of any one of claims 80 or 81, wherein the intron comprises the nucleic acid sequence shown in SEQ ID NOs: 27-29.

83. The method of any one of claims 68 to 82, wherein the recombinant AAVrh74 viral vector further comprises a 3'UTR poly(A) tail sequence.

84. The method of claim 83, wherein the 3'UTR poly(A) tail sequence comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence shown in SEQ ID NO:

30.

85. The method of any one of claims 1 to 84, wherein the AAVrh74 viral vector comprises a nucleic acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to a nucleic acid sequence selected from the group consisting of the nucleic acid sequences shown in SEQ ID NOs: 20, 22 and 24.

86. The method of claims 57 to 85, wherein imrase is administered at a dose of 0.25 mg / kg.

87. The method of claim 85 or 86, wherein the recombinant AAVrh74 viral vector is delandistrogene moxeparvovec, bidridistrogene xeboparvovec, or patidistrogene bexoparvovec.

88. The method of claim 87, wherein the diandroestrogens moxpavovek, petriestrogens cospavovek or patieestrogens becospavovek are present in an amount of about 1.33×10 14 vg / kg dose.

89. The method of claims 57 to 88, wherein the administration of the imrase is intravenous.

90. The method according to claims 87 to 89, wherein said administration of said diandroestrogen moxpavovec, petri estrogen cospavovec or pati estrogen becospavovec is intravenous.

91. The method of any one of claims 87 to 90, wherein the recombinant AAVrh74 is the androestrogen moxepavik.

92. A kit for treating muscular dystrophy in a subject in need thereof, the kit comprising (i) an enzyme that cleaves IgG, (ii) a recombinant AAVrh74 viral vector, and (iii) instructions for administering the enzyme and / or the recombinant AAVrh74 viral vector to the subject according to any one of claims 1 to 91.

93. Use of a recombinant AAVrh74 viral vector for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof, wherein the subject has previously been administered an enzyme that cleaves IgG.

94. Use of a recombinant AAVrh74 viral vector in combination with an enzyme that cleaves IgG for the preparation of a medicament for treating muscular dystrophy in a subject in need thereof.

95. The use according to claim 94, wherein the recombinant AAVrh74 viral vector and the enzyme are administered simultaneously.

96. The use according to claim 94, wherein the recombinant AAVrh74 viral vector and the enzyme are administered sequentially.

97. The use according to claim 94 or 96, wherein the recombinant AAVrh74 viral vector is administered no later than 54 hours after the administration of the enzyme.

98. The use according to any one of claims 94 to 97, wherein the muscular dystrophy is selected from DMD and LGMD.

99. The use according to any one of claims 94 to 98, wherein the muscular dystrophy is DMD.

100. A composition comprising a recombinant AAVrh74 viral vector for use in treating muscular dystrophy in a subject in need thereof, wherein the subject has previously been administered an enzyme that cleaves IgG.

101. A composition comprising a recombinant AAVrh74 viral vector in combination with an enzyme that cleaves IgG, for use in treating muscular dystrophy in a subject in need thereof.

102. The composition for use of claim 101, wherein the recombinant AAVrh74 viral vector and the enzyme are administered simultaneously.

103. The composition for use of claim 101, wherein the recombinant AAVrh74 viral vector and the enzyme are administered sequentially.

104. The composition for use of claim 103, wherein the recombinant AAVrh74 viral vector is administered no later than 54 hours after the administration of the enzyme.

105. The composition for use of any one of claims 101 to 104, wherein the muscular dystrophy is selected from DMD and LGMD.

106. The composition for use of any one of claims 101 to 105, wherein the muscular dystrophy is DMD.

107. A method of removing anti-AAVrh74 antibodies to treat a subject in need thereof, the method comprising administering imrilase and measuring the titer of anti-rAAVrh74 antibodies in the subject after administration of imrilase.

108. The method of claim 107, wherein the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

109. The method of claim 107, wherein the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

110. The method of claim 107, wherein the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

111. The method of claim 110, wherein the subject's anti-AAVrh74 antibody titer is greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

112. The method of claim 108, wherein the subject has anti-AAVrh74 antibodies in a neutralizing cell assay.

113. The method of any one of claims 107 to 112, further comprising measuring the titer of anti-rAAVrh74 antibodies in the subject prior to administering imrase.

114. The method of claim 113, wherein the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

115. The method of claim 113, wherein the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

116. The method of claim 113, wherein the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

117. The method of claim 116, wherein the subject's anti-AAVrh74 antibody titer is greater than 1:400 in a total anti-AAVrh74 antibody ELISA assay.

118. The method of claim 114, wherein the subject has anti-AAVrh74 antibodies in a neutralizing cell assay.

119. The method of claim 116, further comprising administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is at or below 1:

400.

120. The method according to any one of claims 107 to 119, wherein imrilase is administered at a dose of 0.25 mg / kg.

121. The method of any one of claims 107 to 120, wherein the recombinant AAVrh74 viral vector is diastolic estrogen moxpavovek, petreline estrogen cospovovek or patiline estrogen becospovovek.

122. The method of any one of claims 107 to 121, wherein the recombinant AAVrh74 is the androestrogen moxepavik.

123. The method of claim 121 or 122, wherein the diandroestrogens moxpavovek, petriestrogens cospavovek or pattiestrogens becospavovek are present in an amount of about 1.33×10 14 vg / kg dose.

124. The method of claims 121 to 123, wherein the diandroestrogen moxepavik is present at about 1.33 x 10 14 vg / kg dose.

125. The method of any one of claims 107 to 124, wherein the administration of the imrilase is intravenous.

126. The method of any one of claims 107 to 125, wherein the administration of the diandroestrogen moxpavovec, petri estrogen cospavovec, or pati estrogen becospavovec is intravenous.

127. The method of claim 116, wherein the recombinant AAVrh74 viral vector is not administered to the subject if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is higher than 1:

400.

128. The method of claim 127, further comprising administering a second dose of imrilase to the subject.

129. The method of claim 128, wherein the second dose of imrilase is 0.25 mg / kg.

130. The method of claim 128 or 129, wherein the second dose of imrase is administered no more than 60 hours after the first dose.

131. The method of any one of claims 128 to 130, further comprising measuring the titer of anti-rAAVrh74 antibodies in the subject after the second dose of imrplase.

132. The method of claim 131, wherein the anti-AAVrh74 antibody is a neutralizing anti-AAVrh74 antibody.

133. The method of claim 131, wherein the anti-AAVrh74 antibody is a non-neutralizing anti-AAVrh74 antibody.

134. The method of claim 131, wherein the anti-AAVrh74 antibodies are total anti-AAVrh74 antibodies comprising both neutralizing antibodies and non-neutralizing antibodies.

135. The method of claim 134, further comprising administering a recombinant AAVrh74 viral vector if the titer of anti-rAAVrh74 antibodies measured in the subject in a total anti-AAVrh74 antibody ELISA assay is at or below 1:

400.

136. The method of claim 135, wherein the recombinant AAVrh74 viral vector is administered no more than 54 hours after the second dose of imrase.

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