Methods of risk assessment for muscular dystrophy gene therapy

Abstract

The disclosure provides for methods of assessing the risks (e.g., associated with a gene therapy for the treatment of DMD (e.g., delandistrogene moxeparvovec) comprising genotyping the DMD gene and analyzing the HLA type of a subject in need of the gene therapy (e.g., delandistrogene moxeparvovec).

Description

METHODS OF RISK ASSESSMENT FOR MUSCULAR DYSTROPHY GENE THERAPYFIELD OF THE DISCLOSURE

[0001] The present disclosure provides for methods for assessing the risks associated with administration of a gene therapy for the treatment of muscular dystrophy in a human subject in need thereof.BACKGROUND OF THE DISCLOSURE

[0002] Muscular dystrophy (MD) is a rare but highly debilitating class of genetic disorders. Duchenne muscular dystrophy (DMD), for example, is caused by a defect in the expression of the protein dystrophin. The gene encoding the protein dystrophin contains seventy-nine exons spread out over more than two-million nucleotides of DNA. Any mutation that alters the dystrophin gene reading frame (e.g., non-sense mutations introducing a stop codon, deletions removing one or more partial or entire exons, duplications of one or more exons), has the potential to disrupt the production of functional dystrophin, resulting in DMD.

[0003] Gene therapy offers a lasting means of treating a variety of pathologies (i.e., diseases, disorders, conditions, and syndromes) and adeno-associated viruses (AAV) are one of the most widely used gene therapy vectors. AAV vectors comprise a protein shell surrounding 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 is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Id. Its single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). Id. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. Id. The two cis-acting AAV ITRs are approximately 145 nucleotides in length with interrupted palindromic sequences that can fold into T shaped hairpin structures that function as primers during initiation of DNA replication.

[0004] AAV gene therapy is currently the most viable approach for treating DMD and BMD. For example, ELEVIDYS (delandistrogene moxeparvovec-rokl), an adeno-associated virus based gene therapy, is the first and only gene therapy approved by the FDA for the treatment of Duchenne muscular dystrophy (DMD) at this time. ELEVIDYS is supported by biologic and empirical evidences, in addition to the efficacy data from clinical studies. In a clinical trial, one subject treated with ELEVIDYS manifested a serious adverse effect.

[0005] Despite the low frequency, there is a strong need to avoid serious adverse effects in subjects administered gene therapies (e.g., delandistrogene moxeparvovec) for the treatment of DMD. Additionally, some subject that are considered not amenable for a specific gene therapy for the treatment of DMD (e.g., delandistrogene moxeparvovec) may indeed be amenable for the specific gene therapy for the treatment of DMD (e.g., delandistrogene moxeparvovec). Thus, there is a strong need for precise assessment of the risks associated with administration of a specific gene therapy for the treatment of DMD (e.g., delandistrogene moxeparvovec) to avoid both adverse effects and erroneous exclusion of subjects from treatment with a specific gene therapy for the treatment of DMD (e.g., delandistrogene moxeparvovec).BRIEF SUMMARY

[0006] The disclosure provides for methods of assessing the risk associated with administering a gene therapy for the treatment of DMD (e.g., delandistrogene moxeparvovec) to a subject in need thereof, comprising genotyping the DMD gene of the subject, and analyzing the T cells and / or analyzing the HLA type of the subject, before administering the gene therapy (e.g., delandistrogene moxeparvovec) to the subject.BRIEF DESCRIPTION OF DRAWINGS

[0007] Fig. 1 shows a schematic representation of the Dystrophin protein domains as compared to the Dystrophin gene exons, and to the Dystrophin protein domains comprised in delandistrogene moxeparvovec gene therapy vector. Fig. 1 further shows the localization of the peptide pools derived from delandistrogene moxeparvovec gene therapy vector (MDys) used in a IFN-y ELISpot assay.

[0008] Fig. 2 shows the outcome of an immune-mediated myositis (IMM) clinical event that occurred in a subject treated with delandistrogene moxeparvovec gene therapy vector.

[0009] Fig. 3 shows the results of an ELISpot analysis of 51 peptides in MDys Pool 1 grouped into 15 different pools to detect the specific peptides that were eliciting a T cell response in a subject treated with delandistrogene moxeparvovec gene therapy vector and having immune-mediated myositis (IMM). Fig. 3 further shows the localization peptides 38 and 39 relative to the Dystrophin protein and to the Dystrophin gene.

[0010] Fig. 4 shows the results of an ELISpot analysis for cellular immune response to micro-dystrophin of Pool 1-3 in a subject treated with delandistrogene moxeparvovec gene therapy vector and having immune-mediated myositis (IMM).

[0011] Fig. 5 shows the results of an in silico HLA epitope mapping based on HLA scores in a subject treated with delandistrogene moxeparvovec gene therapy vector and having immune-mediated myositis (IMM).

[0012] Fig. 6 shows a schematic summary of the ELISpot and in silico HLA epitope mapping findings.

[0013] Figs. 7A-7B show the outcome of an immune-mediated myositis (IMM) clinical event that occurred in two subjects (Case 1 and Case 2) treated with delandistrogene moxeparvovec gene therapy vector.

[0014] Fig. 8 shows a schematic representation of the Dystrophin protein domains as compared to the Dystrophin gene exons, and to the Dystrophin protein domains comprised in delandistrogene moxeparvovec gene therapy vector. Fig. 8 further shows the localization of the peptide pools derived from delandistrogene moxeparvovec gene therapy vector (MDys) used in a IFN-y ELISpot assay, and the Dystrophin protein deletions in the DMD gene in the two subjects (Case 1 and Case 2).

[0015] Figs. 9A-9B show the results of an ELISpot analysis for cellular immune response to micro-dystrophin of Pool 1-3 in the two subjects (Case 1 and Case 2) treated with delandistrogene moxeparvovec gene therapy vector and having immune-mediated myositis (IMM).

[0016] Figs. 10A-10B show the results of an ELISpot analysis of 51 peptides in MDys Pool 1 grouped into 15 different pools to detect the specific peptides that were eliciting a T cell response in the two subjects (Case 1 and Case 2) treated with delandistrogenemoxeparvovec gene therapy vector and having immune-mediated myositis (IMM). Figs. 10A-10B further show the localization peptides 38, 39 and 32 relative to the Dystrophin protein and to the Dystrophin gene.

[0017] Figs. 11A-11B show the results of an in silico HLA epitope mapping based on HLA scores in the two subjects (Case 1 and Case 2) treated with delandistrogene moxeparvovec gene therapy vector and having immune-mediated myositis (IMM).

[0018] Fig. 12 shows a schematic summary of the ELISpot and in silico HLA epitope mapping findings for Case 1 and Case 2.DETAILED DESCRIPTION OF THE DISCLOSURE

[0019] In some aspects, provided herein are methods of assessing the risk associated with administration of a gene therapy for the treatment of Muscular Dystrophy to a subject in need thereof. In some aspects, the Muscular Dystrophy is Duchene Muscular Dystrophy (DMD). In some aspects, the gene therapy results in the delivery of a nucleotide sequence encoding a functional Dystrophin protein, or a functional portion thereof (e.g., a mini- or micro-Dystrophin) to the cells of the subject. In some aspects, the nucleotide sequence encodes a functional full length Dystrophin protein. In some aspects, the nucleotide sequence encodes a functional portion of a Dystrophin protein (e.g., a mini- or microDystrophin). In some aspects, the mini- or micro-dystrophins is: DysAR4-R23 / ACTD (delandistrogene moxeparvovec gene therapy vector, |iDysH2), DysA17-48, DysAH2- R19, DysAH2-R15, DysAR2-23, AR2-15 / AR18-22 / ACTD (|lDys5), AR3-19 / AR20- 21 / ACTD, AR2-15 / AR18-19 / AR20-23 / ACTD, Dys3978, Dys3849, minidystrophin d3990, RGX-202, mDys5R (SGT-001), and DYSF. In some aspects, the micro-dystrophins is DysAR4-R23 / ACTD. In some aspects, the micro-dystrophins is |lDysH2. In some aspects, the gene therapy is an AAV vector-based gene therapy. In some aspects, the AAV vectorbased gene therapy is delandistrogene moxeparvovec.

[0020] In some aspects, the subject's genome carries one or more defective copies (i.e., comprising a mutation) of a Dystrophin gene. In some aspects, the one or more defective copies (i.e., comprising a mutation) of a Dystrophin gene encodes a non-functional or sub -functional (i.e., having reduced function compared to a Dystrophin protein encoded by a gene not comprising the mutation) Dystrophin protein. In some aspects, the one or more defective copies of the Dystrophin gene (e, g., encoding a non-functional or sub-functional Dystrophin protein) lacks one or more portions of the full length Dystrophin gene. In some aspects, the one or more defective copies of the Dystrophin gene (e.g., encoding the non-functional or sub -functional Dystrophin protein) lacks one or more exons of the full length Dystrophin gene, or one or more portions thereof. In some aspects, the one or more defective copies of the Dystrophin gene (e.g., encoding the nonfunctional or sub -functional Dystrophin protein) lacks exons 8 and / or 9 of the full length Dystrophin gene, or one or more portions thereof. In some aspects, the one or more defective copies of the Dystrophin gene (e.g., encoding the non-functional or subfunctional Dystrophin protein) lacks one or more portions of the Dystrophin gene encoding hinge 1 of the Dystrophin protein.

[0021] In some aspects, the one or more defective copies of the Dystrophin gene encodes a non-functional or sub -functional Dystrophin protein. In some aspects, the nonfunctional or sub-functional Dystrophin protein lacks one or more portions of the full length Dystrophin protein. In some aspects, the non-functional or sub -functional Dystrophin protein lacks one or more portions of the Dystrophin protein encoded by exons 8 and / or 9 of the Dystrophin gene, or by one or more portions thereof. In some aspects, the non-functional or sub -functional Dystrophin protein lacks one or more portions of the Dystrophin protein hinge region 1. In some aspects, the non-functional or sub -functional Dystrophin protein lacks the Dystrophin protein hinge region 1.

[0022] In some aspects, the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) comprises one or more portions that are not comprised in the non-functional or sub -functional Dystrophin protein encoded by the one or more defective copies of the Dystrophin gene (e.g., encoding the non-functional or sub -functional Dystrophin protein) comprised in the subject's genome.

[0023] In some aspects, the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) comprises one or more portions of the Dystrophin protein encoded by one or more portions of exons 8 and / or 9 of the full length Dystrophin gene, and the subject's genome lacks one or more portions of exons 8 and / or 9 of the full length Dystrophin gene.

[0024] In some aspects, the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) comprises one or more portions of the Dystrophin protein hinge region 1, and the subject's genome lacks one or more portions the full length Dystrophin gene encoding one or more portions of the Dystrophin protein hinge region 1.

[0025] In some aspects, the methods provided herein comprise analyzing the subject's genome to identify any mutation in one or more of the Dystrophin genes comprised in the subject's genome. In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if the subject's genome comprises one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if the subject's genome comprises one or more portions exons 8 and / or 9 of the full length Dystrophin gene. In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if the subject's genome comprises one or more portions of the full length Dystrophin gene encoding the Dystrophin protein hinge region 1. In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if the subject's genome comprises one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec AAV gene therapy vector.

[0026] In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if the subject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more portions of the Dystrophin gene comprised in the genome of the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if the subject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more portions of exons 8 and / or 9 of the full length Dystrophin gene. In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if the subject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more portions of the Dystrophin gene encoding the Dystrophin protein hinge region 1. In some aspects, the methods provided herein comprise analyzing the subject's genome to assess if thesubject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more portions of the Dystrophin gene comprised in the genome of a delandistrogene moxeparvovec AAV gene therapy vector.

[0027] In some aspects, the methods provided herein comprise analyzing the subject's T cells. In some aspects, the methods provided herein comprise analyzing the subject's T cells. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess the capability of such T cells to recognize certain antigens. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess the capability of such T cells to recognize certain antigens as non-self (i.e., recognized as foreign from the immune system). In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess their capability to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess their capability to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)), wherein the subject's genome does not comprise one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene, or portions thereof, wherein the genome of the subject lacks exons 8 and / or 9 of the full length Dystrophin gene, or one or more portions thereof. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides comprised in hinge region 1 of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess their capability to recognize one or more peptides comprised in hinge region 1 of the Dystrophin protein, wherein the genome of the subject does not comprise one or more portions of the full length Dystrophin gene encoding for hingeregion 1 of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector, wherein the subject's genome does not comprise one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector.

[0028] In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present certain antigens. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector that are not comprised in the genome of the subject. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene, or portions thereof, wherein the genome of the subject lacks exons 8 and / or 9 of the full length Dystrophin gene, or portions thereof. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assessthe capability of the HLA molecules encoded by such HLA alleles to present one or more peptides comprised in hinge region 1 of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides comprised in hinge region 1 of the Dystrophin protein, wherein the genome of the subject does not comprise one or more portions of the full length Dystrophin gene encoding for hinge region 1 of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules such encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector, wherein the subject's genome does not comprise one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector.

[0029] In some aspects, the methods provided herein comprise analyzing the subject's genome by any one of the means known in the published literature for genome analysis. For example, genome analysis may be conducted by any one of the platforms available for next generation sequencing. In some aspects, the methods provided herein comprise analyzing the subject's genome to assess the presence in the subject genome of one or more portion of the Dystrophin gene comprised in a gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). In some aspects, the methods provided herein comprise analyzing the subject's genome to assess the capability of the subject's genome to express one or more portion of the Dystrophin protein encoded by the one or more portions of the Dystrophin gene comprised in therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)).

[0030] In some aspects, the methods provided herein further comprise analyzing the subject's T cells. For example, analyzing the subject's T cells can comprise analyzing the capability of the subject's T cells to recognize one or more specific antigens. In some aspects, analyzing the subject's T cells can comprise analyzing the capability of thesubject's T cells to recognize one or more specific antigens as non-self. In some aspects, the methods provided herein comprise analyzing the capability of the subject's T cells to recognize one or more antigens (e.g., peptides) that can derive from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) administered to the subject. In some aspects, the antigens (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) administered to the subject are encoded by one or more portions of the Dystrophin gene that are comprised in the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy vector)) administered to the subject and are not comprised in the Dystrophin gene comprised in the subject's genome. In some aspects, the antigens (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy vector)) administered to the subject are encoded by one or more portion of exon 8 and / or exon 9 of the Dystrophin gene. In some aspects, the antigens (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy vector)) administered to the subject are derived from hinge 1 of the Dystrophin protein.

[0031] In some aspects, the methods provided herein further comprise analyzing the subject's human leukocyte antigen (HLA) alleles by any one of the means known in the published literature for the analysis of human leukocyte antigen (HLA) alleles. For example, analyzing the subject's human leukocyte antigen (HLA) alleles can comprise analyzing the capability of the subject's human leukocyte antigen (HLA) alleles to encode HLA molecules capable of presenting one or more specific antigens. In some aspects, the methods provided herein comprise analyzing the capability of the subject's human leukocyte antigen (HLA) alleles to encode HLA molecules capable of presenting one or more antigens (e.g., peptides) that can derive from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the genetherapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) administered to the subject. In some aspects, the antigens (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or microDystrophin) encoded by the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) administered to the subject are encoded by one or more portions of the Dystrophin gene that are comprised in the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) administered to the subject and are not comprised in the Dystrophin gene comprised in the subject's genome. In some aspects, the antigens (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) administered to the subject are encoded by one or more portion of exon 8 and / or exon 9 of the Dystrophin gene. In some aspects, the antigens (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or microDystrophin) encoded by the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) administered to the subject are derived from hinge 1 of the Dystrophin protein.

[0032] In some aspects, the methods provided herein comprise analyzing the subject's genome for the presence and / or for the capability to express one or more portion of the Dystrophin protein encoded by the one or more portions of the Dystrophin gene comprised in gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). If the subject's genome does not comprise a deletion of, or a mutation that otherwise prevents the expression from the genome of the subject of, one or more portions of the Dystrophin protein that is encoded by the genome of the gene therapy vector, the subject is deemed amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). If the subject's genome comprises a deletion of, or a mutation that otherwise prevents the expression from the genome of the subject of, one or more portions of the Dystrophin protein that is encoded by the genome of the gene therapy vector, the subject is deemed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)).

[0033] In some embodiments, a subject deemed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) can be further examined to confirm or reverse the results of the genotyping analysis. In some embodiments, the T cells of the subject can be analyzed to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). Wherein the analyzed T cells of the subject are deemed capable of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient is confirmed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector). Wherein the analyzed T cells of the subject are deemed not capable of, or to have a reduced capacity of, recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient can be deemed amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector).

[0034] In some embodiments, the subject's human leukocyte antigen (HLA) alleles are analyzed. Specifically, the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) is analyzed. Wherein the analyzed HLA alleles are deemed capable of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient is confirmed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector). Wherein the analyzed HLA alleles are deemed not capable of, or having a reduced capacity of, presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient can be deemed amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector).

[0035] In some aspects, the methods provided herein allow to assess the risk of administering a gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) encoding one or more portion of a functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) to a subject in need thereof. In some aspects, the subject suffers from a Muscular Dystrophy. In some aspects, the Muscular Dystrophy is Duchene Muscular Dystrophy. In some aspects, the methods provided herein allow to assess the risk of the subject to develop an immune response against one or more antigens (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) administered to the subject. In some aspects, the one or more antigen (e.g., peptides) derived from the functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) encoded by the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) administered to the subject are not encoded by the Dystrophin gene(s) comprised in the subject's genome. In some aspects, the immune response is a cell-mediated immune responses. In some aspects, the cell-mediated immune responses is a T cell-mediated immune responses. In some aspects, the immune response causes an inflammatory myopathy (i.e., myositis). In some aspects, the inflammatory myopathy is dermatomyositis (DM), polymyositis (PM), necrotizing myopathy (NM) and inclusion body myositis (IBM).

[0036] In some aspects, the methods provided herein allow assessment of the risk of administering a gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) encoding one or more portion of a functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) to a subject in need thereof, with better accuracy as compared to assessing the risk of administering the gene therapy to a subject in need thereof solely by analyzing the subjects genome for the presence (and expression) of one or more exons (or portions thereof) that are comprised in the administered gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)).

[0037] In some aspects, the methods provided herein allow to administer a gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) encodingone or more portion of a functional Dystrophin protein, or functional portion thereof (e.g., a mini- or micro-Dystrophin) to a subject in need thereof, where the subject would not have been administered the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)) solely by analyzing the subjects genome for the presence (and expression) of one or more exons (or portions thereof) that are comprised in the administered gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec gene therapy)).

[0038] In some aspects, the methods provided herein allow to safely administer delandistrogene moxeparvovec to patients with potentially higher-risk DMD mutations.

[0039] In some aspects, a method of assessing the risk associated with administration of delandistrogene moxeparvovec to a subject is disclosed, comprising analyzing the subject's human leukocyte antigen (HLA) alleles, wherein one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec are not comprised in the genome of the subject.

[0040] In some aspects, analyzing the subject's human leukocyte antigen (HLA) alleles comprises assessing the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec.

[0041] In some aspects, the T cells of the subject are not capable of, or have a reduced capacity of, recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec. In some aspects, delandistrogene moxeparvovec is administered to the subject.

[0042] In some aspects, the methods comprise administering an effective amount of delandistrogene moxeparvovec to a patient having a Dystrophin gene encoding nonfunctional or sub-functional Dystrophin protein, wherein the Dystrophin gene lacks one or more portions of exon 8 and / or 9. In some aspects, the methods provided herein comprise administering an effective amount of delandistrogene moxeparvovec to a patient having a Dystrophin gene encoding non-functional or sub -functional Dystrophin protein, wherein the Dystrophin gene lacks exon 8 and / or 9.

[0043] In some aspects, the methods provided herein comprise administering an effective amount of delandistrogene moxeparvovec to a patient having a Dystrophin gene encoding non-functional or sub -functional Dystrophin protein, wherein the Dystrophin genecomprises, but does not express one or more portions of exon 8 and / or 9. In some aspects, the methods provided herein comprise administering an effective amount of delandistrogene moxeparvovec to a patient having a Dystrophin gene encoding nonfunctional or sub-functional Dystrophin protein, wherein the Dystrophin gene comprises, but does not express exon 8 and / or 9.1. Definitions

[0044] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0045] It is to be noted that, as used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component; for example, "a nucleic acid sequence," is understood to represent one or more nucleic acid sequences, unless stated otherwise. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0046] Furthermore, "and / or", where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass 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).

[0047] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided.

[0048] As used herein, the term "about" refers to a value that is within 10% above or below the value being described.

[0049] The term "at least" prior to a value or series of values is understood to include the values adjacent to the term "at least," and all subsequent values (numbers, integers, or fractions) that could logically be included, as clear from context. For example, thenumber of nucleotides in a nucleic acid molecule must be an integer, e.g., "at least 18 nucleotides of a 21- nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).

[0050] As used herein, "no more than" or "less than" is understood as the value adjacent to the phrase and logical lower values (numbers, integers, or fractions), as logical from context, to zero. When "no more than" is present before a series of values or a range, it is understood that "no more than" can modify each of the value in the series or range.

[0051] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0052] The term "derived from," as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism.

[0053] "Nucleic acid," "polynucleotide," and "oligonucleotide," are used interchangeably in the present application. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA e.g., messenger RNAs (mRNAs), genomic DNAs, plasmid DNAs (pDNAs), or complementary DNAs (cDNAs)). The terms "nucleic acid," "polynucleotide," and "oligonucleotide," as used herein, are defined as it is generally understood by the person skilled in the art as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can also be referred to as nucleic acid molecules or oligomers. Polynucleotides can be made recombinantly, enzymatically, or synthetically, e.g., by solid-phase chemical synthesis followed by purification. When referring to a sequence of the polynucleotide or nucleic acid, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides.

[0054] As used herein, the term "nucleotide" refers to monomeric units of nucleic acid polymers (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). Naturallyoccurring nucleotides are composed of three subunit molecules: a nucleobase, a five- carbon sugar (ribose or deoxyribose), and phosphate group consisting of one to three phosphates. A nucleotide can also be a modified nucleotide, which is a nucleotide comprising a modified nucleobase, a modified sugar moiety, a modified backbone, or any combination thereof. As used herein, the term "nucleobases", also known as "nitrogenous bases" or "bases", refers to biological compounds that form nucleosides, which, in turn, are components of nucleotides. A "nucleoside" comprises a nucleobase and a sugar moiety. A "sugar moiety" may comprise ribose or deoxyribose, as present in naturally occurring nucleotides. As used herein, the term "backbone," and "backbone structure", refer to the connection between monomers of a nucleic acid. In naturally occurring oligonucleotides, the backbone comprises a 3 '-5 ' phosphodiester linkage connecting sugar moieties of the oligomer.

[0055] As used herein, the term "coding sequence" or a sequence "encoding" refers to a particular molecule which is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of RNA) into protein, in vitro or in vivo, when operably linked to an appropriate regulatory sequence, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Although a "stop codon" (e.g., TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

[0056] As used herein, the term "exon" refers to coding sections of a DNA molecule, or of an RNA molecule which is transcribed from a DNA molecule that are translated into protein. Exons can be separated by intervening sections of DNA that do not code for proteins, known as "introns". Therefore, the term "intron", as used herein, refers to a segment of nucleic acid that is transcribed and is present in the "pre-mRNA" but excised by the splicing machinery and therefore not present in the mature mRNA transcript. Following transcription, new, immature strands of messenger RNA, called "pre-mRNA", may contain both introns and exons. These pre-mRNA molecules go through amodification process in the nucleus called splicing during which the noncoding introns are cut out and only the coding exons remain in the "mature mRNA'. Splicing produces a mature messenger RNA molecule that is then translated into a protein. The term "first exon" refers to a coding sequence or sequence of nucleic acid that encodes a polypeptide or polypeptide region and the term "second exon" refers to a different second coding sequence or sequence of nucleic acid that encodes a second polypeptide region. Where the two exons are separated by an intervening intron in the pre-mRNA, the splicing machinery operates to remove the intervening intron and join the two exons in the mature mRNA.

[0057] The term "polyadenylation signal" refers to a nucleic acid sequence present in the RNA transcript that allows for the transcript, when in the presence of the enzyme polyadenyl transferase, to be polyadenylated.

[0058] The term "promoter," as used herein in, refers to a sequence sufficient to direct transcription, in a cell. A promoter is intended as a DNA region to which RNA polymerases bind and that directs the enzyme to transcribe an operably linked DNA sequence. A DNA sequence is operably linked to a promoter if the promoter is capable of directing transcription of that DNA sequence. Promoters for use in the invention include prokaryotic, eukaryotic (e.g., mammalian or yeast), and viral promoters, e.g., the CMV (mammalian cytomegalovirus) promoter, the CAG promoter (also known as CBA promoter; CMV early enhancer / chicken P actin promoter), the UbC (polyubiquitin C gene) promoter, or the CBh (an engineered CBA promoter in which the SV40 intron is replaced with a hybrid intron composed of a 5' donor splice site from the chicken P-actin 5' UTR and a 3' acceptor splice site from MVM). A promoter can be a "constitutive" promoter that is a promoter that, when operably linked to a polynucleotide encoding a gene product, results in the production of a gene product in the cell under most or all conditions of the cell. A promoter can be a "regulatable" promoter that is a promoter whose activity is affected by a cis or trans acting factor (e.g., an inducible promoter, such as an external signal or agent). The term "inducible" promoter means that when the promoter is operably linked to a polynucleotide encoding a specified gene product, it results in the production of a gene in the cell basically only when the inducer corresponding to the promoter is present in the cell. A promoter can be a "ubiquitous" promoter that is a promoter that is active in a wide range of cells, tissues and cell cycles,or a "tissue-specific" promoter, that is a promoter that has activity only or mostly in certain cell types, i.e., drives the expression of the operably linked nucleotide sequence only or mostly in certain cell types. A promoter can be a "bidirectional" promoter, which is a promoter that is an intergenic region between two divergent genes located on complementary strands of the DNA, and drives their coordinated transcription in opposite directions.

[0059] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence capable of regulating the expression of a nucleic acid sequence operably linked to said regulatory sequence, non-limiting examples of regulatory sequences are enhancers (a DNA sequence that increases the level of transcription of an operably linked gene), and silencers (a DNA sequence that decreases the level of transcription of an operably linked gene). The term "regulatory sequence" also refers to nucleic acid sequence in RNA transcripts capable of regulating, for example, the processing or the expression of said transcripts. Non-limiting examples of regulatory sequences that can be in RNA transcripts are nucleotide sequences that regulate localization or splicing of said RNA transcripts. The term "regulatory sequence" further refers to an amino acid sequence capable of regulating, for example, the localization (e.g., subcellular localization signals, such as nuclear localization signals), or the stability (e.g., degradation signals) of a protein.

[0060] The terms "operatively linked," "operatively inserted," "operatively positioned," "under control" means, with reference to two or more nucleic acid sequences, that the nucleic acid sequences are arranged in such a way that one of the two or more nucleic acid sequences can mediate a function that is exerted upon at least one of the other two or more nucleic acid sequences. For example, a regulatory nucleic acid sequence e.g., a promoter, an enhancer, or a silencer) can be "operatively linked," to a coding nucleic acid sequence, that is the regulatory nucleic acid sequence is in the correct location and orientation in relation to the coding nucleic acid sequence to control expression of the coding nucleic acid sequence (e.g., via control of RNA polymerase initiation). Wherein a regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer) is "operatively linked," to a coding region, the coding region is "under transcriptional control" of the regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer).

[0061] The term "operably linked" means that a nucleic acid sequence and a regulatory sequence(s) are arranged in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). The term "operably inserted" means that a nucleic acid sequence of interest is positioned adjacent a regulatory nucleic acid sequence which directs transcription and translation of the nucleic acid sequence of interest (i.e., facilitates the production of, e.g., a polypeptide encoded by a DNA of interest).

[0062] As used herein, the term "RNA" relates to a nucleic acid molecule that comprises ribonucleotide residues. In some aspects, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a b-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA. The term "mRNA," as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more peptide (e.g., oligopeptide, or polypeptide) or protein. The term "mRNA," as used herein includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. An mRNA molecule may also contain a 5' untranslated region (5'-UTR), and / or a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides.

[0063] The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA transcript or a protein. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knock-in, as well as both transient expression and stable expression. It may include, without limitation, transcription of the gene into messenger RNA (mRNA),and the translation of such mRNA into protein(s). Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA, or a non-coding RNA, produced by transcription of a gene, or a protein, which is translated from an mRNA transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., mRNAs which are processed, for example, by capping, splicing, and / or polyadenylation, or peptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like. Thus, term "protein expression" refers to the process by which a nucleic acid sequence undergoes transcription (DNA) translation (RNA) such that detectable levels of the amino acid sequence or protein are expressed.

[0064] As used herein, the terms "protein," "polypeptide," and "peptide," are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The amino acids may be natural or synthetic, and can contain chemical modifications such as disulfide bridges, substitution of radioisotopes, phosphorylation, substrate chelation e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide range of other modifications. A polypeptide may be attached to other molecules, for instance molecules required for function. Examples of molecules which may be attached to a polypeptide include, without limitation, cofactors, polynucleotides, lipids, metal ions, phosphate, etc. A polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids, which are not members of the standard twenty amino acids, may also be used. The standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V). These letters as used in terms of a " polypeptide sequence" or "amino acid sequence" are an alphabetical representation of a polypeptide molecule. A polypeptide may be naturally occurring, recombinant, or synthetic, or any combination of these. A polypeptide may also comprise a fragment of a naturallyoccurring polypeptide. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0065] A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides that do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. Nonlimiting examples of polypeptide include polypeptide fragments, denatured / unstructured polypeptide, polypeptide having a primary, a polypeptide, a polypeptide, or a polypeptide or aggregated structure, etc. A polypeptide may be a single molecule or may be a multi- molecular complex. There is expressly no requirement that a polypeptide must contain an intended function; a polypeptide can be functional, non-functional, function for unexpected / unintended purposes, or have unknown function.

[0066] As used herein, the term "domain" when referred to a polypeptide means a distinct functional and / or structural portion of a polypeptide. Usually domains are responsible for a particular function or interaction, contributing to the overall role of a polypeptide. Domains may exist in a variety of biological contexts, where similar domains can be found in polypeptide with different functions. For example, an "actin-binding domain" or "ABD" is to be understood as a portion of a polypeptide that is involved in the binding of the polypeptide with actin. For example, an ABD can comprise one or more amino acids that directly bind to actin.

[0067] As used herein, the term "truncated" when referred to a protein means a protein that is lacking one or more portion with the respect to the full length protein. A protein can lack, for example, an amino-terminal portion, a carboxy-terminal portion, any portion in between the two termini, or any combination thereof.

[0068] By "isolated" molecule (e.g., a protein or a nucleic acid molecule) is intended a molecule, which has been removed from its native environment. An isolated molecule includes molecules produced and maintained (e.g., upon purification) in vitro, or recombinant molecules maintained in host cells or purified (partially or substantially)from the host cell and maintained in solution. Non-limiting examples of isolated molecules include proteins or nucleic acid molecules extracted from a cell, produced synthetically (i.e., by chemical synthesis), produced by enzymatic digestion; DNA molecules produced by PCR, produced by in vitro retro-transcription (cDNA), by molecular cloning in a vector (e.g., a viral or a non-viral vector); RNA transcripts produced by in vitro transcription or produced in host cells (e.g., a heterologous host cells) and maintained in the host cell or purified (partially or substantially) from the host cell and maintained outside of the cell; proteins produced by in vitro translation or produced in host cells (e.g., a heterologous host cells) and maintained in the host cell or purified (partially or substantially) from the host cell and maintained outside of the cell.

[0069] The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may comprise specific functional sequences needed for insertion and / or expression of the desired DNA fragments. A "vector" can be any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. In some aspects, insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that may or not have complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes described inthe literature include: genes providing resistance to neomycin, ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, z.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters described in the literature include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), P-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.

[0070] As used herein, the term "adeno-associated vector" or "AAV vector" refers to a vector comprising one or more polynucleotides of interest (e.g., transgenes, such as micro-dystrophin) that are flanked by AAV terminal repeat sequences (ITRs). AAV is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York); Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). Such AAV vectors 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 rep and cap gene products. "AAV Cap" means AAV Cap proteins, VP1, VP2, and VP3 and analogs thereof. "AAV Rep" means AAV Rep proteins and analogs thereof. "Serotype," with respect to a vector or virus capsid, is defined by a distinct immunological profile based on the capsid protein sequences and capsid structure. Non-limiting examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6P1, AAV7, AAV8, AAV8P1, AAV9, AAVrhlO, AAVrhlOPl, AAVS10P4, AAVpol, AAV11, AAV12, AAV-DJ, AAV-DJ / 8, AAV-PHP.Eb, AAV-PHP.S, AAV-PHP.B, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAVS1, AAVS10, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMY03 (AAVS10P1), AAV9-RGD (e g., MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2A, MyoAAV 2E, MyoAAV 3A, MyoAAV 4A, MyoAAV 4C, and MyoAAV 4E).

[0071] As used herein, "flanked," with respect to a sequence that is flanked by other elements, indicates the presence of one or more the elements upstream and / or downstream, z.e., 5' and / or 3', relative to the sequence. The term "flanked" is not intendedto indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element. A sequence (e.g., a transgene) that is "flanked" by two other elements (e.g., ITRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences between.

[0072] As used herein, the terms "AAV virion," "AAV viral particle," or "AAV particle" refer to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. The particle can comprises a heterologous polynucleotide (z.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell). An AAV particle comprising a heterologous polynucleotide can be also referred to as a "recombinant AAV particle," "recombinant AAV vector," "rAAV particle," or "rAAV vector." As used herein, the term "minidystrophin" refers to a truncated dystrophin protein comprising more than four spectrin repeats.

[0073] As used herein, the term "micro-dystrophin" refers to a truncated dystrophin protein comprising four or fewer spectrin repeats.

[0074] Non-limiting examples of mini- and micro-dystrophins are: DysAR4-R23 / ACTD (delandistrogene moxeparvovec, |lDysH2), DysA17-48, DysAH2-R19, DysAH2-R15, DysAR2-23, AR2-15 / AR18-22 / ACTD (|lDys5), AR3-19 / AR20-21 / ACTD, AR2-15 / AR18- 19 / AR20-23 / ACTD, Dys3978, Dys3849, minidystrophin d3990, RGX-202, mDys5R (SGT-001), and DYSF.

[0075] As used herein, the term "pharmaceutical formulation" or "pharmaceutical composition" refers to an admixture comprising an effective amount of a therapeutically and / or prophylactic effective agent and at least one pharmaceutically acceptable excipient (e.g., carrier, diluent, stabilizer, or any combination thereof) or adjuvant. Examples of pharmaceutically acceptable excipients are, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, colorants, amino acids, stabilizers, bulking agents, surfactants, antimicrobials, preservatives, metal ions, chelators, cyclodextrin-based excipients, polyanions, polycations, salts, solubilizers, detergents, compatible solid or liquid fillers, encapsulating substances, or any combination thereof, which are suitable for administration to a subject. Specific examples of excipients include, without limitation,sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers, or any combination thereof. Examples of pharmaceutically acceptable carriers are, but are not limited to, lipids, polymers, polysaccharides, peptides, proteins, lipidoids, and any combination thereof. In some aspects, the lipid is selected from the group consisting of: cationic lipids, non-cationic lipids, steroid lipids, ionizable lipids, PEG-conjugated lipids, and any combination thereof. In some aspects, the fusion proteins, the expression cassettes, and the vectors disclosed herein are complexed, or packaged in a liposome, a nanoliposome, a lipid nanoparticle, a lipoplex, a micell, a nanomicell, a nanoemulsion, an oil-in-water emulsions, a PEG-conjugated lipid nanoparticle, a polymeric nanoparticle, a lipid-polymer hybrid nanoparticle, a polysaccharidic nanocarrier, an RNA / DNA-peptide nanoparticle, an RNA / DNA -peptide nanocomplex, a biomimetic nanovesicle, a lipidoid- RNA / DNA complex, a virus-like particle, dendrimer nanoparticle, a nanogel, a metallic nanoparticle, a gold nanoparticle (AuPNs), a magnetic nanoparticle, a theranostic nanoparticle, or any combination thereof. In some aspects, the combination of the fusion proteins, the expression cassettes, and the vectors disclosed herein with one or more of the carriers described herein facilitates, enhances or enables administration of the fusion proteins, the expression cassettes, and the vectors disclosed herein to the subject. In some aspects, the combination of a therapeutic agent with one or more of the carriers described herein facilitates, enhances or enables the delivery of the therapeutic agent to a target cell.

[0076] Pharmaceutically acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Pharmaceutical excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0077] As used herein, a "therapeutic agent" refers to a chemical compound, a peptide, a protein, a lipid, a carbohydrate, a nucleic acid, or any other molecule or compound capable of treating, ameliorating, or reducing the symptoms of a disease, a disorder, a condition, or a syndrome, or otherwise having a beneficial effect, upon administration to a subject suffering from the disease, disorder, condition, or syndrome.

[0078] As used herein, the term "administration" refers to the administration of a composition or substance to a subject or system. Administration to an animal subject (e.g., to a mammal) can be by any appropriate route. "Administering" refers to the physical introduction of a composition or substance, which may comprising a therapeutic agent, to a subject, using any of the various methods and delivery systems known to those skilled in the art. Examples of routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Administration can also be via a non-parenteral route, for example, orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, aborally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0079] As used herein, the terms "treat," "treated," and "treating" mean both therapeutic and prophylactic treatment or preventative measures wherein the object is to reverse, alleviate, ameliorate, lessen, inhibit, slow down progression, development, severity or recurrence of an undesired symptom, complication, condition, biochemical indicia of a disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the subject; or enhancement or improvement of condition, disorder, or disease. In some aspects, treatment includes eliciting a clinically significant response without excessive levels ofside effects. In some aspects, treatment includes prolonging survival as compared to expected survival if not receiving treatment. As used herein, the term "amelioration" or "ameliorating" refers to a lessening of severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or forestalling the onset, development or progression of a condition or disease for a period of time, including weeks, months, or years. As used herein, the term "prophylactic" (e.g., "prophylactic agent", "prophylactic treatment", "prophylactically effective amount"), refers to any complete or partial prevention of a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect and / or symptom attributable to the disease.

[0080] As used herein, the term "gene therapy" refers to the administration into an individual's cells and / or tissues of an exogenous molecule (e.g., a nucleic acid sequence (e.g., a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a gene product capable of interfering with the genomic sequence of the individual's cells and / or tissues)) to treat, reduce the symptoms of, or reduce the likelihood of a disease, disorder, syndrome, or condition. An exogenous molecule or sequence is understood to be molecule or sequence not normally occurring in the cell, tissue and / or individual to be treated. Both acquired and congenital diseases are amenable to gene therapy.

[0081] As used herein, the term "genotyping" refers to a process of determining the specific allelic composition of a cell and / or subject at one or more positions within the genome, e.g. by determining the nucleic acid sequence at that position. Genotyping refers to a nucleic acid analysis and / or analysis at the nucleic acid level. Numerous genotyping techniques are known to those skilled in the art

[0082] As used herein, the term "subject" refers to any organism to which a composition or a substance (e.g., a nucleotide molecule) can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal. A subject can seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be under care by a trained professional for a particular disease or condition. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; TheDictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0083] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0084] Various aspects of the invention are described in further detail in the following subsections.2. Methods of Gene Therapy Risk Assessment

[0085] The dystrophin gene is the largest known human gene, containing 79 exons and spanning > 2,200 kb, roughly 0.1% of the whole genome (Koenig M et al., “Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals,” Cell. 1987; 50:509- 517). Human Dystrophin is a 427 kDa cytoskeletal protein that localizes to the cytoplasmic face of the sarcolemma and is enriched at costameres (z.e., a component of striated muscle cells which connects the sarcomere of the muscle to the cell membrane (z.e. the sarcolemma)) in muscle fibers (Porter GA et al., “Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle,” J Cell Biol. 1992; 117:997-1005). Dystrophin protein has four main functional domains: an actin-binding amino-terminal domain (ABDI), a central rod domain, a cysteine-rich domain and a carboxy -terminus. Dystrophin’s central rod domain contains 24 spectrin repeats and harbors a second actin-binding motif (ABD2).

[0086] The 24 spectrin repeats are interrupted by four short proline-rich spacers, called "hinges."

[0087] The full length dystrophin proteins consists of, from the amino-terminus to the carboxy -terminus: an actin-binding domain 1 (ABDI), a central rod domain consisting of a first hinge (Hl), 3 spectrin-like repeats (R1 to R3), a second hinge (H2), 16 spectrin-like repeats (R4 to R19), a third hinge (H3), 5 spectrin-like repeats (R20 to R4), a fourth hinge (H4), a cysteine rich domain (CR), and a corboxy-terminus (Chamberlain et al.,“Microdystrophin Expression as a Surrogate Endpoint for Duchenne Muscular Dystrophy Clinical Trials,” Hum Gene Then 2023 May;34(9-10):404-415).

[0088] Gene therapies, (e.g., AAV gene therapies), to express human micro-dystrophins in a subject suffering from DMD, are being currently developed. Gene therapies for the treatment of DMD comprise administering to the subjects a gene therapy vector (e.g., an AAV gene therapy vector) comprising a vector genome encoding one or more portions of a functional Dystrophin protein, (e.g., a mini- or micro-Dystrophin). Non-limiting example of mini- or micro-Dystrophins that can be encoded by gene therapy vectors for the treatment of DMD are DysAR4-R23 / ACTD (delandistrogene moxeparvovec, |lDysH2), DysA17-48, DysAH2-R19, DysAH2-R15, DysAR2-23, AR2-15 / AR18- 22 / ACTD (|lDys5), AR3-19 / AR20-21 / ACTD, AR2-15 / AR18-19 / AR20-23 / ACTD, Dys3978, Dys3849, minidystrophin d3990, RGX-202, mDys5R (SGT-001), and DYSF. A vector genome comprised in a gene therapy vector (e.g., an AAV gene therapy vector) for the treatment of DMD can comprise one or more portions of a Dystrophin gene encoding a functional Dystrophin protein, or a functional portion thereof (e.g., a mini- or micro-Dystrophin). In some aspects, the micro-Dystrophin encoded by gene therapy vectors for the treatment of DMD is DysAR4-R23 / ACTD (delandistrogene moxeparvovec, |lDysH2). In some aspects, the micro-Dystrophin encoded by gene therapy vectors for the treatment of DMD is delandistrogene moxeparvovec.

[0089] In some aspects, a vector genome comprised in a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD can comprise one or more exons (or one or more portions thereof) of a Dystrophin gene encoding a functional Dystrophin protein, or a functional portion thereof (e.g., a mini- or micro-Dystrophin). In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can comprise one or more portions of any one of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, or any combination thereof, of a Dystrophin gene. In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can comprise one or more of any one of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, or any combination thereof, of a Dystrophin gene.

[0090] In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can comprise one or more portions of any one of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, or any combination thereof, of a Dystrophin gene. In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can comprise one or more of any one of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, or any combination thereof, of a Dystrophin gene.

[0091] In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can encode one or more portions of a functional Dystrophin protein, or a functional portion thereof (e.g., a mini- or microDystrophin). In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can encode one or more portions of a dystrophin protein acting binding domain, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, adystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrin-like repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrin-like repeat 23, a dystrophin protein spectrin-like repeat 24, a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4, a dystrophin protein cysteine-rich domain, a dystrophin protein carboxyl-terminus, or any combination thereof. In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can encode one or more of a dystrophin protein acting binding domain, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, a dystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrin-like repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrin-like repeat 23, a dystrophin protein spectrin-like repeat 24, a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4, a dystrophin protein cysteine-rich domain, a dystrophin protein carboxyl-terminus, or any combination thereof.

[0092] In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can encode one or more portions of a dystrophin protein acting binding domain, a dystrophin protein hinge region 1, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein hinge region 2, a dystrophin protein spectrin-like repeat 24, dystrophin protein hinge region 4, a dystrophin protein carboxyl-terminus, or any combination thereof. In some aspects, a vector genome comprised in a gene therapy vector (e.g., a AAV gene therapy vector) for the treatment of DMD can encode one or more of a dystrophin protein acting binding domain, a dystrophin protein hinge region 1, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein hinge region 2, a dystrophin protein spectrin-like repeat 24, dystrophin protein hinge region 4, a dystrophin protein carboxyl-terminus, or any combination thereof.

[0093] In some aspects, the genome of the subject suffering from DMD comprises one or more defective copies (i.e., comprising a mutation) of a Dystrophin gene encoding a nonfunctional or sub-functional (i.e., having reduced function compared to a Dystrophin protein encoded by a gene not comprising the mutation) Dystrophin protein. In some aspects, the Dystrophin gene comprised in the subject's genome lacks one or more portions of a Dystrophin gene. In some aspects, the Dystrophin gene comprised in the subject's genome lacks one or more portions of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, or any combination thereof. In some aspects, the Dystrophin gene comprised in the subject's genome lacks one or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28,exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, or any combination thereof.

[0094] In some aspects, the Dystrophin gene comprised in the subject's genome lacks one or more portions of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, or any combination thereof, of a Dystrophin gene. In some aspects, the Dystrophin gene comprised in the subject's genome lacks one or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, or any combination thereof, of a Dystrophin gene.

[0095] In some aspects, the Dystrophin gene comprised in the subject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more portions of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35, exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, or any combination thereof. In some aspects, the Dystrophin gene comprised in the subject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, exon 25, exon 26, exon 27, exon 28, exon 29, exon 30, exon 31, exon 32, exon 33, exon 34, exon 35,exon 36, exon 37, exon 38, exon 39, exon 40, exon 41, exon 42, exon 43, exon 44, exon 45, exon 46, exon 47, exon 48, exon 49, exon 50, exon 51, exon 52, exon 53, exon 54, exon 55, exon 56, exon 57, exon 58, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, exon 70, exon 71, exon 72, exon 73, exon 74, exon 75, exon 76, exon 77, exon 78, exon 79, or any combination thereof.

[0096] In some aspects, the Dystrophin gene comprised in the subject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more portions of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, or any combination thereof, of a Dystrophin gene. In some aspects, the Dystrophin gene comprised in the subject's genome comprises but does not express (e.g., due to the presence of a mutation) one or more of exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 59, exon 60, exon 61, exon 62, exon 63, exon 64, exon 65, exon 66, exon 67, exon 68, exon 69, or any combination thereof, of a Dystrophin gene.

[0097] In some aspects, the non-functional or sub -functional (i.e., having reduced function compared to a Dystrophin protein encoded by a gene not comprising the mutation) Dystrophin protein encoded by the Dystrophin gene(s) comprised in the subject's genome lacks one or more portion of a Dystrophin protein. In some aspects, the non-functional or sub -functional (i.e., having reduced function compared to a Dystrophin protein encoded by a gene not comprising the mutation) Dystrophin protein encoded by the Dystrophin gene(s) comprised in the subject's genome lacks one or more portion of a dystrophin protein acting binding domain, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, a dystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrin-like repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, adystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrin-like repeat 23, a dystrophin protein spectrin-like repeat 24, a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4, a dystrophin protein cysteine-rich domain, a dystrophin protein carboxyl-terminus, or any combination thereof. In some aspects, the non-functional or sub -functional (i.e., having reduced function compared to a Dystrophin protein encoded by a gene not comprising the mutation) Dystrophin protein encoded by the Dystrophin gene(s) comprised in the subject's genome lacks one or more of a dystrophin protein acting binding domain, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, a dystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrin-like repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrin-like repeat 23, a dystrophin protein spectrin-like repeat 24, a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4, a dystrophin protein cysteine-rich domain, a dystrophin protein carboxyl-terminus, or any combination thereof.

[0098] In some aspects, the non-functional or sub -functional (i.e., having reduced function compared to a Dystrophin protein encoded by a gene not comprising the mutation) Dystrophin protein encoded by the Dystrophin gene(s) comprised in the subject's genome lacks one or more portion of a dystrophin protein acting binding domain, a dystrophin protein hinge region 1, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, adystrophin protein hinge region 2, a dystrophin protein spectrin-like repeat 24, dystrophin protein hinge region 4, a dystrophin protein carboxyl-terminus, or any combination thereof. In some aspects, the non-functional or sub -functional (i.e., having reduced function compared to a Dystrophin protein encoded by a gene not comprising the mutation) Dystrophin protein encoded by the Dystrophin gene(s) comprised in the subject's genome lacks one or more of a dystrophin protein acting binding domain, a dystrophin protein hinge region 1, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein hinge region 2, a dystrophin protein spectrin-like repeat 24, dystrophin protein hinge region 4, a dystrophin protein carboxyl-terminus, or any combination thereof.

[0099] In some aspects, a vector genome comprised in a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD comprises one or more portions of exon 8 and / or exon 9 of a Dystrophin gene encoding a functional Dystrophin protein. In some aspects, a vector genome comprised in a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD encodes a micro-Dystrophin protein comprising: a dystrophin protein acting binding domain; dystrophin protein spectrin-like repeats 1, 2, 3, and 24; dystrophin protein hinge regions 1, 2, and 4; and a dystrophin protein cysteine- rich domain, or any combination thereof. In some aspects, a vector genome comprised in a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD encodes a micro-Dystrophin protein comprising a dystrophin protein hinge region 1. In some aspects, a vector genome comprised in a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD encodes a DysAR.4-R.23 / ACTD micro- Dystrophin protein. In some aspects, a vector genome comprised in a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD encodes a pDysH2 micro- Dystrophin protein. In some aspects, the gene therapy is delandistrogene moxeparvovec gene therapy.

[0100] In some aspects, the Dystrophin gene(s) comprised in the genome of the subject suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, lacks one or more portions of exon 8 and / or exon 9 of a Dystrophin gene. In some aspects, the Dystrophin gene(s) comprised in the genome of the subject suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, lacks exon 8 and / or exon9 of a Dystrophin gene. In some aspects, the genome of the subjects suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, encodes a Dystrophin protein lacking one or more portions of a dystrophin protein acting binding domain; dystrophin protein spectrin-like repeats 1, 2, 3, and 24; dystrophin protein hinge regions 1, 2, and 4; and a dystrophin protein cysteine- rich domain, or any combination thereof. In some aspects, the genome of the subjects suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, encodes a Dystrophin protein lacking one or more of a dystrophin protein acting binding domain; dystrophin protein spectrin-like repeats 1, 2, 3, and 24; dystrophin protein hinge regions 1, 2, and 4; and a dystrophin protein cysteine-rich domain, or any combination thereof. In some aspects, the genome of the subjects suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, encodes a Dystrophin protein lacking one or more portions of a dystrophin protein hinge region 1. In some aspects, the genome of the subjects suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, encodes a Dystrophin protein lacking a dystrophin protein hinge region 1.

[0101] In some aspects, a Dystrophin gene comprised in the genome of the subject suffering from DMD, to whom a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, comprises one or more portions of one or more exons (or portions thereof) of a Dystrophin gene comprised in the administered gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD.

[0102] In some aspects, a Dystrophin gene comprised in the genome of the subject suffering from DMD, to whom a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, comprises one or more exons of a Dystrophin gene comprised in the administered gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD.

[0103] In some aspects, a Dystrophin gene comprised in the genome of the subject suffering from DMD, to whom a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, comprises all the exons (or portions thereof) of a Dystrophin gene comprised in the administered gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD.

[0104] In some aspects, the Dystrophin gene(s) comprised in the genome of the subject suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, lacks one or more portions of one or more exons of a Dystrophin gene, and the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD comprises the one or more portions of the one or more exons of a Dystrophin gene that are not comprised in the genome of the subject suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered.

[0105] In some aspects, the Dystrophin gene(s) comprised in the genome of the subject suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered, lacks one or more exons of a Dystrophin gene, and the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD comprises the one or more exons of a Dystrophin gene that are not comprised in the genome of the subject suffering from DMD, to whom the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD is administered.

[0106] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), prior to administration of a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject. As used herein, the term "genotyping" refers to a process of determining the specific allelic composition of a cell and / or subject at one or more positions within the genome, e.g. by determining the nucleic acid sequence at that position. Genotyping refers to a nucleic acid analysis and / or analysis at the nucleic acid level. Numerous genotyping techniques are known to those skilled in the art. Non-limiting examples of genotyping techniques known to those skilled in the art are: Current methods of genotyping include restriction fragment length polymorphism identification (RFLPI) of genomic DNA, random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism detection (AFLPD), polymerase chain reaction (PCR), DNA sequencing, allele specific oligonucleotide (ASO) probes, and hybridization to DNA microarrays or beads.

[0107] In some aspects, the human dystrophin gene (DMD) of a subject is genotyped to characterize a mutation in the gene that would be especially amenable to treatment with a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD.

[0108] In some aspects, the human dystrophin gene (DMD') of a subject is genotyped to characterize a mutation in the gene that may contraindicate treatment with a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD (for example, due to an increased risk of severe immune response).

[0109] In some aspects, the subject is genotyped for at least one mutation in exons 1-79 of the DMD gene. In some aspects, the subject is genotyped for at least one mutation in exons 18-79 of the DMD gene. In some aspects, the subject is genotyped for at least one mutation in exons 1-17 of the DMD gene. In some aspects, the subject is genotyped for at least one mutation in exons 59-69 of the DMD gene. In some aspects, the subject is genotyped for at least one mutation in exons 1-17 and 59-69 of the DMD gene. In some aspects, the subject is genotyped for at least one mutation in exons 8-9 of the DMD gene. In some aspects, the at least one mutation is a frameshift deletion, a frameshift duplication, a premature stop, or other pathogenic variant. In some aspects, the at least one mutation results in the absence of expression of a functional human dystrophin protein, or in the expression of a sub -functional human dystrophin protein.

[0110] In some aspects, not detecting a specific mutation in the DMD gene in the subject's genome can identify that a subject may be administered a certain gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD. For example, the presence in the subject's genome of exons 9-13 of the DMD gene and / or of exons 8 and / or 9 of the DMD gene, can indicate that the subject may be administered a specific gene therapy (e.g., delandistrogene moxeparvovec gene therapy), wherein the gene therapy vector comprises exons 9-13 of the DMD gene and / or exons 8 and / or 9 of the DMD gene.[OHl] In some aspects, detecting at least one of mutation in the DMD gene in the subject's genome can identify that a subject may not be administered a specific gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD. For example, a deletion that fully includes exons 9-13 of the DMD gene and / or any deletion in exons 8 and / or 9 in the DMD gene, can indicate that the subject should be contraindicated from a specific gene therapy (e.g., delandistrogene moxeparvovec gene therapy), wherein the gene therapy vector comprises exons 9-13 of the DMD gene and / or exons 8 and / or 9 of the DMD gene.

[0112] Without wishing to be bound to any theory, it is believed that a subject is contraindicated from a specific gene therapy, wherein the genome of the subjectcomprises a mutation that prevents or decreases the expression of a portion of the Dystrophin protein that is encoded by the genome of the gene therapy vector. The mutation can be a deletion and / or any mutation that otherwise prevents the expression from the genome of the subject of the portion of the Dystrophin protein that is encoded by the genome of the gene therapy vector. For example, the genome of the subject may not comprise the portion of the Dystrophin gene comprised in the genome of the gene therapy vector, or the genome of the subject may comprise the portion of the Dystrophin gene comprised in the genome of the gene therapy vector but a mutation (e.g., a point mutation introducing a premature stop codon) in the genome of the subject may prevent the expression of the portion of the Dystrophin protein that is encoded by the genome of the gene therapy vector.

[0113] Surprisingly, the Inventors have found that the risk associated with administration of a specific gene therapy (e.g., delandistrogene moxeparvovec gene therapy) are also determined by the subject’s T cells capability to recognize certain antigens, and by the human leukocyte antigen (HL A) alleles of the subjects, specifically by the capability of the HLA molecules encoded by such HLA alleles to present certain antigens. For example, peptides derived from micro-dystrophin exons 8 and 9, comprised in a delandistrogene moxeparvovec gene therapy vector, can induce a T-cell response leading to an immune response in subjects with deletion of these exons. For example, the presence of T cells recognizing exons 8 or 9, and the peptides encoded therefrom, as nonself can lead to an immune response in subjects with deletion of these exons. The immune response can cause an inflammatory myopathy (i.e., myositis) in the subject. In some aspects, the myositis is dermatomyositis (DM), polymyositis (PM), necrotizing myopathy (NM), and inclusion body myositis (IBM).

[0114] T cells are a diverse and important group of lymphocytes that mature and undergo a positive and negative selection processes in the thymus. These cells play a vital role in both components of active immunity, including cell-mediated and to some extent humoral immunity. There are several types of T cells; the most common and well-known are the CD4+ T cells (helper T cells) and CD8+ T Cells (cytotoxic T cells, or killer T cells). T cells cannot recognize soluble, free antigens. T cells can only recognize protein-based, receptor-bound antigens. This recognition occurs via the use of the MHC (also known as HLA) 1 and 2 receptors, which along with the TCRs (T-cell receptors) bind the antigen inquestion and form a complex that allows the T cell to recognize the antigen. CD4+ T cells recognize MHC 2 bound antigens, while CD8+ T cells recognize MHC 1 bound antigens. Both CD4+ T cells and CD8+ T cells have the TCR (and the co-receptor CD3), but (as evidenced by their name) their other co-receptors vary. CD4+ T cells have CD4, whereas CD8+ T cells have CD8 as an added co-receptor. In some aspects, the methods provided herein comprise analyzing the subject's T cells.

[0115] In some aspects, the methods provided herein comprise analyzing the subject's T cells. In some aspects, the methods provided herein comprise analyzing the subject's T cell by any one of the means known in the art for the analysis of subject's T cell. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess the capability of such T cells to recognize certain antigens. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess the capability of such T cells to recognize certain antigens as non-self (i.e., recognized as foreign from the immune system). In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)), wherein the subject's genome does not comprise one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene, or portions thereof, wherein the genome of the subject lacks exons 8 and / or 9 of the full length Dystrophin gene, or one or more portions thereof. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides comprised in hinge region 1of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides comprised in hinge region 1 of the Dystrophin protein, wherein the genome of the subject does not comprise one or more portions of the full length Dystrophin gene encoding for hinge region 1 of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector. In some aspects, the methods provided herein comprise analyzing the subject's T cells to assess capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector, wherein the subject's genome does not comprise one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector.

[0116] The Major Histocompatibility complex (MHC) system, known as human leukocyte antigen (HLA) in humans, is located on the short arm of chromosome 6 (6p21.3) and contains the most polymorphic gene cluster of the entire human genome. Furthermore, the HLA consists of three regions which have been designated as class I, class II, and class III based on the structure and function of gene products. The main function of HLA class I gene products (HLA- A, -B, and -C) is to present endogenous peptides to responding CD8+T Cells while the class II coded molecules HLA-DR, -DP, and -DQ have restricted expression and process exogenous peptides for presentation to CD4+helper T Cells. The class III region, in turn, contains genes which encode for immune regulatory molecules, e.g., tumor necrosis factor (TNF), factors C3, C4, and C5 of complement and heat shock proteins. HLA class I molecules are expressed on the surface of almost all nucleated cells. Class II molecules are expressed only on B lymphocytes, antigen-presenting cells (monocytes, macrophages, and dendritic cells), and activated T lymphocytes. HLA genes are closely linked and the entire MHC is inherited as an HLA haplotype in a Mendelian fashion from each parent. The HLA system is known to be the most polymorphic in humans. The HLA polymorphism is not evenly spread throughout the molecule, but is clustered in the antigen-binding groove. Amino acid variations in several regions change the fine shape of the groove and thus alter the peptide-binding specificity of HLA molecules. The peptide-binding specificities of HLAmolecules are determined by a limited number of amino acid residues located in the peptide-binding pockets. Different HLA molecules show characteristic amino acid residue patterns in the bound peptide sequences. Conserved amino acid residues located at particular positions of the peptides act as peptide's anchoring residues in the peptide- binding groove.

[0117] In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles by any one of the means known in the art for the analysis of human leukocyte antigen (HLA) alleles. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present certain antigens. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector that are not comprised in the genome of the subject. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by exons 8 and / or 9 of the full length Dystrophin gene, or portions thereof, wherein the genome of the subject lacks exons 8 and / or 9 of the full length Dystrophin gene, or portions thereof. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides comprised in hinge region 1 of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen(HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides comprised in hinge region 1 of the Dystrophin protein, wherein the genome of the subject does not comprise one or more portions of the full length Dystrophin gene encoding for hinge region 1 of the Dystrophin protein. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector. In some aspects, the methods provided herein comprise analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules such encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector, wherein the subject's genome does not comprise one or more portions of the Dystrophin gene comprised in a delandistrogene moxeparvovec gene therapy vector.

[0118] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD) and analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD, prior to administration of the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD) and analyzing the subject’s human leukocyte antigen (HLA) alleles of the subject, prior to administration of a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject. In some aspects, analyzing the subject’s human leukocyte antigen (HLA) alleles comprises assessing the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD, prior to administration of a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject.

[0119] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD) and analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of theDystrophin gene comprised in delandistrogene moxeparvovec, prior to administration of delandistrogene moxeparvovec to the subject. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD) and analyzing the subject’s human leukocyte antigen (HLA) alleles of the subject, prior to administration of delandistrogene moxeparvovec to the subject. In some aspects, analyzing the subject’s human leukocyte antigen (HLA) alleles comprises assessing the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec, prior to administration of a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject.

[0120] In some aspects, knowledge of the subject’s T cells capability to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vectors (e.g., delandistrogene moxeparvovec)), in addition to the knowledge of the subject’s mutations (e.g., deletions) in the DMD gene, can help with assessing the risk of immune response associated with administration of a gene therapy vector (e.g., AAV gene therapy vectors (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to the subject.

[0121] In some aspects, knowledge of the subject’s human leukocyte antigen (HLA) alleles, in addition to the knowledge of the subject’s mutations (e.g., deletions) in the DMD gene, can help with assessing the risk of immune response associated with administration of a gene therapy vector (e.g., AAV gene therapy vectors (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to the subject. In some aspects, knowledge of the subject’s human leukocyte antigen (HLA) alleles, and of the capability of the HLA molecules encoded by such HLAs alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vectors (e.g., delandistrogene moxeparvovec)) for the treatment of DMD, can help with assessing the risk of immune response associated with administration of a gene therapy vector (e.g., AAV gene therapy vectors (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to the subject.

[0122] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD) and analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of theDystrophin gene comprised in delandistrogene moxeparvovec gene therapy vector, prior to administration of a delandistrogene moxeparvovec gene therapy to the subject. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD) and analyzing the subject’s human leukocyte antigen (HL A) alleles, prior to administration of a delandistrogene moxeparvovec gene therapy to the subject. In some aspects, analyzing the subject’s human leukocyte antigen (HLA) alleles comprises assessing the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec gene therapy vector, prior to administration of a delandistrogene moxeparvovec gene therapy to the subject.

[0123] In some aspects, knowledge of the subject’s T cells capability to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the delandistrogene moxeparvovec gene therapy vector, in addition to the knowledge of the subject’s mutations (e.g., deletions) in the DMD gene, can help with assessing the risk of immune response associated with administration of delandistrogene moxeparvovec gene therapy to the subject. In some aspects, knowledge of the subject’s human leukocyte antigen (HLA) alleles, in addition to the knowledge of the subject’s mutations (e.g., deletions) in the DMD gene, can help with assessing the risk of immune response associated with administration of delandistrogene moxeparvovec gene therapy to the subject. In some aspects, knowledge of the subject’s human leukocyte antigen (HLA) alleles, and of the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the delandistrogene moxeparvovec gene therapy vector, can help with assessing the risk of immune response associated with administration of delandistrogene moxeparvovec gene therapy to the subject.

[0124] Methods for analyzing human leukocyte antigen (HLA) alleles are known in the published literature. For example, processes used to perform HLA typing include serological cytotoxicity methods where samples of lymphocytes (taken from blood or spleen) are added to Terasaki plates. These plates hold individual wells that contain different specific antibodies (from either maternal sera or manufactured monoclonal antibodies). Another method used for HLA typing is flow cytometry, particularly when looking for specific alleles. Here fresh nucleated leucocytes are added to monoclonalantibodies that are labelled with a molecule that fluoresces. Cells with surface antigens that bind to the antibody become fluorescent. The flow cytometer detects the fluorescent cells by detecting the light emitted from them as they pass through a laser beam. A third process is used where very detailed typing is required. This process involves extracting the DNA from cells and amplifying the genes that encode for the HLA peptides using polymerase chain reaction techniques (e.g., qPCR), alternatively next generation sequencing methods can be used. The genes may be matched with known HLA nucleotide sequences found stored in several gene bank databases, including the IMGT / HLA database.

[0125] Methods for analyzing T cells capability to recognize a certain antigen are known to those skilled in the art. The enzyme-linked immunosorbent spot (ELISpot) assay is one of the most commonly used methods to measure antigen-specific T cells in both mice and humans. Some of the primary reasons for the popularity of the method are that ELISpot is highly quantitative, can measure a broad range of magnitudes of response and is capable of assessing critical cellular immune-related activities such as IFN-y secretion and granzyme B release. Among the various types of ELISpot, the IFN-y-based assay is the most common application of the technique. IFN-y is an abundant cytokine produced by Thl cells and is frequently used to track specific CD8+responses (see, e.g., Slota M, Lim JB, Dang Y, Disis ML, “ELISpot for measuring human immune responses to vaccines,” Expert Rev Vaccines. 2011 Mar;10(3):299-306. doi: 10.1586 / erv. l0.169. PMID: 21434798; PMCID: PMC3360522.).

[0126] The (IFN-y) ELISpot assay can be used to detect T cells directed at specific antigens, such as micro-dystrophin peptides. For example, the (IFN-y) ELISpot assay can be used to detect T cells directed at micro-dystrophin peptides that are encoded by a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD, prior to administering the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to a subject. In some aspects, the (IFN-y) ELISpot assay can be used to detect T cells directed at micro-dystrophin peptides that are encoded by a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD and are not encoded by the DMD gene comprised in the genome of the subject.

[0127] In some aspects, the (IFN-y) ELISpot assay can be used to detect T cells directed at micro-dystrophin peptides that are encoded by delandistrogene moxeparvovec gene therapy, prior to administering delandistrogene moxeparvovec gene therapy to a subject. In some aspects, the (IFN-y) ELISpot assay can be used to detect T cells directed at micro-dystrophin peptides that are encoded by delandistrogene moxeparvovec gene therapy and are not encoded by the DMD gene comprised in the genome of the subject.

[0128] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject are capable of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject are capable of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject, as detected in an ELISpot assay.

[0129] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD , wherein the T cells of the subject are capable of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject are capable of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec, as detected in an ELISpot assay.

[0130] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject produce more than 7.1 ng / ml, 7.2 ng / ml, 7.3 ng / ml, 7.4 ng / ml, 7.5 ng / ml, 8 ng / ml, 8.5 ng / ml, 9 ng / ml, 9.5 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, or 50 ng / ml of IFN-y per 400,000 cells when exposed to one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAVgene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject, as detected in an ELISpot assay.

[0131] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject produce more than 7.1 ng / ml, 7.2 ng / ml, 7.3 ng / ml, 7.4 ng / ml, 7.5 ng / ml, 8 ng / ml, 8.5 ng / ml, 9 ng / ml, 9.5 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, or 50 ng / ml of IFN-y per 400,000 cells when exposed to one or more peptides encoded by delandistrogene moxeparvovec, as detected in an ELISpot assay.

[0132] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject are not capable or have a reduce capability of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject are not capable or have a reduce capability of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vectors (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject, as detected in an ELISpot assay.

[0133] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject are not capable or have a reduce capability of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject are not capable or have a reduce capability of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec, as detected in an ELISpot assay.

[0134] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject produce less than 0.1 ng / ml, 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, 0.5 ng / ml, 1 ng / ml, 1.5 ng / ml, 2 ng / ml, 2.5 ng / ml, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.5 ng / ml, 6 ng / ml, 6.5 ng / ml, or 7 ng / mlof IFN-y per 400,000 cells when exposed to one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject, as detected in an ELISpot assay.

[0135] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the T cells of the subject produce less than 0.1 ng / ml, 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, 0.5 ng / ml, 1 ng / ml, 1.5 ng / ml, 2 ng / ml, 2.5 ng / ml, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 5.5 ng / ml, 6 ng / ml, 6.5 ng / ml, or 7 ng / ml of IFN-y per 400,000 cells when exposed to one or more peptides encoded by delandistrogene moxeparvovec, as detected in an ELISpot assay.

[0136] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), and analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec)). In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), and analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vectors (e.g., delandistrogene moxeparvovec)) by ELISpot assay (e.g., IFN-y ELISpot assay).

[0137] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD , and analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), and analyzing the subject's T cells to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec, by ELISpot assay (e.g., IFN-y ELISpot assay).

[0138] Peptide binding with either HLA-I or HLA-II molecules can be predicted in silico (in silico HLA epitope mapping). For example, the NetMHCpan server predicts binding of peptides to any MHC molecule of known sequence using artificial neural networks (ANNs). The method is trained on a combination of more than 850,000 quantitativeBinding Affinity (BA) and Mass-Spectrometry Eluted Ligands (EL) peptides. The BA data covers 170 MHC molecules from human (HLA-A, B, C, E), mouse (H-2), cattle (BoLA), primates (Patr, Mamu, Gogo), swine (SLA) and equine (Eqca). The EL data covers 177 MHC molecules from human (HLA-A, B, C, E), mouse (H-2), cattle (BoLA), primates (Patr, Mamu, Gogo), swine (SLA), equine (Eqca) and dog (DLA). Furthermore, the user can obtain predictions to any custom MHC class I molecule by uploading a full length MHC protein sequence. Predictions can be made for peptides of any length.

[0139] An in silico method can be used to predict binding of specific peptides to a subject's specific HLA alleles (i.e., to the HLA molecules encoded by the subject's specific HLA alleles). For example, the NetMHCpan server can be used to predict binding of specific peptides to a subject's specific HLA alleles (i.e., to the HLA molecules encoded by the subject's specific HLA alleles). In some aspects, the NetMHCpan server is used to predict binding of micro-dystrophin peptides that are encoded by a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD, prior to administering the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to a subject. In some aspects, the NetMHCpan server is used to predict binding of microdystrophin peptides that are encoded by delandistrogene moxeparvovec gene therapy to a subject's specific HLA alleles (i.e., to the HLA molecules encoded by the subject's specific HLA alleles), prior to administering delandistrogene moxeparvovec gene therapy to a subject.

[0140] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules capable of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules capable of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for thetreatment of DMD to be administered to the subject, as detected by in silico analysis (e.g., NetMHCpan).

[0141] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules capable of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules capable of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec, as detected by in silico analysis (e.g., NetMHCpan).

[0142] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD , wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules having an HLA-I epitope score of 20, 30, 40, 50, 60, 70, 80, 90, or more when tested in silico (e.g., NetMHCpan) against to one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject.

[0143] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules having an HLA-I epitope score of 20, 30, 40, 50, 60, 70, 80, 90, or more when tested in silico (e.g., NetMHCpan) against to one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec.

[0144] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules not capable or having a reduced capability of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLAmolecules not capable or having a reduced capability of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject, as detected by in silico analysis (e.g., NetMHCpan).

[0145] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules not capable or having a reduced capability of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules not capable or having a reduced capability of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec, as detected by in silico analysis (e.g., NetMHCpan).

[0146] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD , wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules having an HLA-I epitope score of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or less when tested in silico (e.g., NetMHCpan) against to one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD to be administered to the subject.

[0147] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), wherein the subject's human leukocyte antigen (HLA) alleles encode HLA molecules having an HLA-I epitope score of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or less when tested in silico (e.g., NetMHCpan) against to one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec.

[0148] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), and analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles topresent one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec)). In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), and analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLAs alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec)), by in silico analysis (e.g., NetMHCpan).

[0149] In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), and analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec. In some aspects, the methods disclosed herein comprise genotyping a subject’s Dystrophin gene (DMD), and analyzing the subject's human leukocyte antigen (HLA) alleles to assess the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec, by in silico analysis (e.g., NetMHCpan).

[0150] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) analyzing the subject's T cells (e.g., by ELISpot assay (e.g., IFN-y ELISpot assay)); iii) administering a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject.

[0151] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) analyzing the subject's T cells (e.g., by ELISpot assay (e.g., IFN-y ELISpot assay)); iii) not administering a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject.

[0152] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) analyzing the subject's T cells (e.g., by ELISpot assay (e.g., IFN-y ELISpot assay)); iii) administering a composition comprising delandistrogene moxeparvovec gene therapy to the subject.

[0153] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) analyzing the subject's Tcells (e.g., by ELISpot assay (e.g., IFN-y ELISpot assay)); iii) not administering a composition comprising delandistrogene moxeparvovec gene therapy to the subject.

[0154] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin DMD) gene of the subject prior to treatment; ii) analyzing the subject's human leukocyte antigen (HLA) alleles (e.g., in silico (e.g., NetMHCpan)); iii) administering a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject.

[0155] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) analyzing the subject's human leukocyte antigen (HLA) alleles (e.g., in silico (e.g., NetMHCpan)); iii) not administering a gene therapy vector (e.g., AAV gene therapy vectors) for the treatment of DMD to the subject.

[0156] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) analyzing the subject's human leukocyte antigen (HLA) alleles (e.g., in silico (e.g., NetMHCpan)); iii) administering a composition comprising delandistrogene moxeparvovec gene therapy to the subject.

[0157] In some aspects, the methods disclosed herein comprise: i) genotyping the human dystrophin (DMD) gene of the subject prior to treatment; ii) analyzing the subject's human leukocyte antigen (HLA) alleles (e.g., in silico (e.g., NetMHCpan)); iii) not administering a composition comprising delandistrogene moxeparvovec gene therapy to the subject.

[0158] In some aspects, the genotyping of the subject indicates that the subject is amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy). In some aspects, the genotyping of the subject indicates that the subject is not amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy).

[0159] In some aspects, the genotyping of the subject indicates that the subject is not amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogenemoxeparvovec gene therapy) and the analysis of the subject's T cells indicates that the subject is not amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy). In some aspects, the genotyping of the subject indicates that the subject is not amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) and the analysis of the subject's T cells indicates that the subject is amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy).

[0160] In some aspects, the genotyping of the subject indicates that the subject is not amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) and the analysis of the human leukocyte antigen (HLA) alleles of the subject indicates that the subject is not amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy). In some aspects, the genotyping of the subject indicates that the subject is not amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) and the analysis of the human leukocyte antigen (HLA) alleles of the subject indicates that the subject is amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy).

[0161] In some aspects, the methods provided herein comprise analyzing the subject's genome for the presence and / or for the capability to express one or more portion of the Dystrophin protein encoded by the one or more portions of the Dystrophin gene comprised in gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). If the subject's genome does not comprise a deletion of, or a mutation that otherwise prevents the expression from the genome of the subject of, one or more portions of the Dystrophin protein that is encoded by the genome of the gene therapy vector, the subject is deemed amenable for treatment with the gene therapy vector(e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). If the subject's genome comprises a deletion of, or a mutation that otherwise prevents the expression from the genome of the subject of, one or more portions of the Dystrophin protein that is encoded by the genome of the gene therapy vector, the subject is deemed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)).

[0162] In some embodiments, a subject deemed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) can be further examined to confirm or reverse the results of the genotyping analysis. In some embodiments, the T cells of the subject can be analyzed to assess the capability of such T cells to recognize one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)). Wherein the analyzed T cells of the subject are deemed capable of recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient is confirmed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector). Wherein the analyzed T cells of the subject are deemed not capable of, or to have a reduced capacity of, recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient can be deemed amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector).

[0163] In some embodiments, the subject's human leukocyte antigen (HLA) alleles are analyzed. Specifically, the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) is analyzed. Wherein the analyzed HLA alleles are deemed capable of presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient is confirmed not amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g.,delandistrogene moxeparvovec gene therapy vector). Wherein the analyzed HLA alleles are deemed not capable of, or having a reduced capacity of, presenting one or more peptides encoded by one or more portions of the Dystrophin gene comprised in the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector)) the patient can be deemed amenable for treatment with the gene therapy vector (e.g., AAV vector (e.g., delandistrogene moxeparvovec gene therapy vector).

[0164] In some aspects, the analysis of the subject's T cells allows safe administration of a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject that would be otherwise not considered amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) based on the genotyping of the subject.

[0165] In some aspects, the analysis of the human leukocyte antigen (HLA) alleles of the subject allows safe administration of a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject that would be otherwise not considered amenable to treatment with the gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) based on the genotyping of the subject.

[0166] In some aspects, the methods disclosed herein allow to safely administer gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with potentially higher-risk DMD gene mutations.

[0167] In some aspects, the methods disclosed herein allow to safely administer gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject that would have been otherwise excluded from the treatment based on their DMD genotyping.

[0168] In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subjectwith at least one mutation in exons 1-79 of the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one mutation in exons 18-79 of the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one mutation in exons 1-17 of the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one mutation in exons 59-69 of the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one mutation in exons 1-17 and / or 59-69 of the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one mutation in exons 8-9 of the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one deletion that fully includes exons 9-13 in the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one mutation that impedes or reduces the expression of exons 9-13 in the DMD gene. In some aspects, the methods disclosed herein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one deletion that fully includes exons 8 and / or 9 in the DATD gene. In some aspects, the methods disclosedherein allow to safely administer a gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy) to a subject with at least one mutation that impedes or reduces the expression of exons 8 and / or 9 in the DMD gene.

[0169] In some aspects, the methods disclosed herein allow to expand the population of subjects suffering with DMD that can be treated with a specific gene therapy vector (e.g., AAV gene therapy vector (e.g., delandistrogene moxeparvovec)) for the treatment of DMD (e.g., delandistrogene moxeparvovec gene therapy). In some aspects, the methods disclosed herein allow to expand the population of subjects suffering with DMD that can be treated with delandistrogene moxeparvovec gene therapy.ExamplesMethodsELISpot assay:

[0170] The IFN-y ELISpot assay was used to detect T cells directed at specific delandistrogene moxeparvovec micro-dystrophin (MDys) peptides. A combination of peptides from different regions of micro-dystrophin was selected to form three peptide Pools: MDys Pool 1, 2, or 3 (Fig. 1). The assay detected the specific peptide Pool that elicited a T-cell response in the subject. An analysis was performed at the following time points: baseline, Day 2, and Weeks 1, 2, 4, 10, 12, 24, 52, and 104.In silico HL A epitope mapping:

[0171] An in silico tool (NetMHCpan) was used to determine MDys peptide binding with either HLA-I (using 9-mer peptides) or HLA-II (using 15-mer peptides) molecules that correspond to the subject’s specific HLA allele combinations. HLA-I and HLA-II scores for exons 1-17 were determined by multiplying the number of predicted strong binder peptides by the number of alleles binding these peptides.Delandistrogene moxeparvovec

[0172] Delandistrogene moxeparvovec is an rAAVrh74 vector-based gene transfer therapy, designed to compensate for the absence of functional dystrophin in DMD bydelivering a transgene encoding delandistrogene moxeparvovec micro-dystrophin, an engineered protein that retains key functional domains of the wild-type protein.

[0173] As of January 2024, delandistrogene moxeparvovec is approved in the USA, UAE, and Qatar for the treatment of ambulatory pediatric patients aged 4 through 5 years with DMD with a confirmed mutation in the DMD gene.Example 1: IMM Subject outcome (Case 1)

[0174] A case of IMM that occurred in a 9-y ear-old subject with a deletion of exons 3-43 of the DMD gene, 35 days post-dosing of delandistrogene moxeparvovec gene therapy was analyzed. The subject underwent a series of six rounds of plasmapheresis (-days 39- 51) and was started on immunosuppressive treatment with tacrolimus on day 52 before discharge and was still on tacrolimus as of June 2023 (Fig. 2). At discharge (day 55), the subject did not need any respiratory support, and on Day 64-67 started walking independently. The subject recovered on Day 100 with sequalae (weakness). The patient was weaned off tacrolimus by day -980 without re-emergence of any symptoms of IMM.Example 2: Generation of MDys peptide pools to determine relative antigenic strength and Cellular immune response to micro-dystrophin

[0175] To determine the regions of the micro-dystrophin most effective at stimulating T- cell responses, we cleaved micro-dystrophin into small peptides, which we distributed into 3 Pools that were comprised of varying regions of the micro-dystrophin (MDys Pool 1, 2, and 3; Fig. 1). These Pools of peptides were then presented to T cells in the context of HL A, and we measured activation by IFN-y production using ELISpot. ELISpot analysis suggested that the IMM in Case 1 resulted from T cell-mediated responses directed against specific delandistrogene moxeparvovec micro-dystrophin peptides with elevated responses to peptides from MDys Pool 1 (Fig. 4).

[0176] Upon further analysis, the 51 peptides in MDys Pool 1 were grouped into 15 different pools (pools 1.1-1.15) to detect the specific peptides that were eliciting a T-cell response in the subject. ELISpot analysis suggested three peptide pools (pools 1.4, 1.12, and 1.13, in green in Fig. 3 right chart) that mounted a T-cell response, of which the common peptides were 38 and 39. Peptides 38 and 39 were identified to induce T-cell responses in ELISpot (Fig. 3).Example 3: Cellular immune response to micro-dystrophin - Summary

[0177] ELISpot analysis suggested that the IMM in Case 1 resulted from T cell-mediated responses directed against specific delandistrogene moxeparvovec micro-dystrophin peptides with elevated responses to peptides from MDys Pool 1 (Fig. 4 and Fig. 6). Further, ELISpot analysis of this subject with IMM (Case 1) identified T cells that recognize peptides 38 and 39 mapping to exons 8 and 9 (Fig. 3 and Fig. 6).Example 4: In silico HLA epitope mapping based on HLA scores

[0178] In silico analysis of HLA presentation in this subject (Case 1) suggested a high risk of immunogenicity for exons 8 and 9 of the DMD gene, which encodes a portion of the hinge 1 domain of dystrophin (Fig. 5). In silico epitope mapping identified peptides encoded by exons 8 and 9 with high propensities to be presented by the subject’s HLA-I and HLA-II molecules and drive an immune response (Fig. 6). The results also showed that HLA I or II does influence the counter-indication in conjunction with exons 8 and / or 9 mutation.Example 5: Investigation of antigenic features mediating IMM in two patients (Case 1 and Case 2) with DMD treated with delandistrogene moxeparvovec.

[0179] As shown in the preceding Examples, peptides derived from micro-dystrophin exons 8 and 9 may induce a T cell response leading to IMM in patients with deletion of these exons. However, not all patients with deletions in exons 8 and 9 develop IMM following gene therapy.

[0180] Additionally, in silico epitope mapping identified peptides encoded by exons 8 and 9 with high propensities to be presented by the subject’s HLA-I and HLA-II molecules and drive an immune response. Thus, knowledge of the patient’s HLA alleles, in addition to the patient’s deletion in the DMD gene, may help with assessing the risk of immune response to delandistrogene moxeparvovec micro dystrophin.

[0181] In individuals who have a portion of the DMD gene sequence deleted, there is a risk of the transgene being recognized as foreign and, in turn, eliciting an immune response (e.g., a T-cell response). Delandistrogene moxeparvovec may be contraindicated in patients with any deletion in exon 8 and / or exon 9 of the DMD gene.

[0182] One requisite of being detected by the immune system is the presentation of peptide fragments of the transgene by HLA-I or HLA-II (HLA is a complex of genes andproteins that encode and present antigenic peptides to T cells, enabling adaptive immune responses and tissue compatibility). However, not all patients in ENDEAVOR (SRP- 9001-103; NCT04626674) with deletions involving exons 1-17 and / or 59-71 who were treated with delandistrogene moxeparvovec gene therapy developed IMM.

[0183] Two cases of IMM were reported in ENDEAVOR (SRP-9001-103;NCT04626674), an open-label, multi-cohort Phase lb study assessing delandistrogene moxeparvovec in patients with DMD.

[0184] Case 1 occurred in a 9-year-old patient in Cohort 2 with a deletion of exons 3-43 of the DMD gene, 35 days post-dosing (for Case 1, see also Examples 1-4).

[0185] Case 2 occurred in a 7-year-old patient in Cohort 5 with a deletion of exons 8-9 of the DMD gene, 29 days post-dosing.

[0186] Both patients experienced muscle weakness and received immunosuppressive treatment, including high-dose corticosteroids and tacrolimus.

[0187] The results of the investigation of these two cases are presented below (for Case 1, also refer to Examples 1-4).

[0188] Case 1: The patient underwent six rounds of plasmapheresis and was started on tacrolimus before discharge and completed tacrolimus in January 2024. At discharge (Day 55), the patient did not need any respiratory support, and on Day 67 he regained the ability to walk independently. The patient recovered on Day 100 with sequalae (weakness) (Fig. 7A).

[0189] Case 2 : The patient was started on tacrolimus and IVIG and remains on both. At discharge (Day 35) strength had improved significantly, but per NSAA, was not at his pre-infusion level (Fig. 7B).

[0190] The IFN-y ELISpot assay, as described above, was used to detect T cells directed at specific delandistrogene moxeparvovec micro-dystrophin peptides. Briefly, to determine the regions of the micro-dystrophin most effective at stimulating T-cell responses a combination of peptides from regions of micro-dystrophin was selected to form three peptide Pools: MDys Pool 1, 2 or 3 (Fig. 8). We cleaved the micro-dystrophin into small peptides, and distributed the peptides into the 3 Pools, and then exposed T cells to each Pool of peptides and assessed their activation by measuring the amount of IFN-y produced using ELISpot. The assay detected the specific peptide Pools that elicited a T- cell response in the patients. An analysis was performed at the following time points:Case 1 - baseline, Day 2, and Weeks 1, 2, 4, 10, 12, 24, 52, and 104; Case 2 - baseline, Day 2, and Weeks 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 and 24. ELISpot analysis suggested that the IMM resulted from T cell-mediated responses directed against specific delandistrogene moxeparvovec micro-dystrophin peptides with elevated responses to peptides from MDys pool 1 in both Cases (Fig. 9A and Fig. 9B).

[0191] Upon further analysis, the 51 peptides in MDys Pool 1 were grouped into 15 different pools (pools 1.1-1.15) to detect the specific peptides that were eliciting a T-cell response in the patients. ELISpot analysis suggested three peptide pools in Case 1 (in green in Fig 10A) of which the common peptides were 38 and 39 (Fig 10A, see also Examples 1-4 for Case 1), and two peptide pools in Case 2 (in green in Fig 10A) of which the common peptide was 32 (Fig 10B), that mounted a T-cell response. Peptides 32, 38 and 39 were identified to induce T-cell responses in ELISpot (IFN-y secretion) and map to exons 8 and 9 of the DMD gene.

[0192] One prerequisite for immune detection is the presentation of peptide fragments derived from the transgene product, facilitated by human leukocyte antigen class I (HLA- I) or HLA-II. An in silico tool (NetMHCpan) was used to determine the propensity of each 9-mer peptide encoded by dystrophin exons 1-17 to bind each HLA-I molecule allele expressed by the patients. Based on the patients’ HLA genotypes, individual ELrank values displayed by NetMHCpan were used to calculate “epitope scores” for each dystrophin exon from 1-17. As low EL rank numbers correspond to higher affinities, ELranks of each 9-mer peptide / HLA allele combination were transformed as follows: [transformed score = -log2(EL_rank / zygosityA2)] and summed for each exon. The zygosity of the allele, whether homozygous or heterozygous, was accounted for in this transformation.

[0193] Any negative transformed scores were set to 0, establishing an upper EL rank threshold of 1.0 for heterozygous alleles and 4.0 for homozygous alleles.

[0194] In silico analysis of HLA presentation in the patients suggested a high risk of immunogenicity for peptides derived from exons 8 and 9 of the DMD gene (Fig. HA and Fig. 11B). This suggests that these epitopes have a higher potential to be presented at the cell surface to CD8+ T cells and in turn activate a cytotoxic response.

[0195] In summary, ELISpot analysis of the patients with IMM identified T cells that recognize peptides 32, 38 and 39 mapping to exons 8 and 9, and in silico epitope mappingidentified peptides encoded by exons 8 and 9 with high propensities to be presented by the patients’ HLA-I molecules and drive an immune response (Fig. 12).

[0196] In conclusion, the immunological investigation of IMM in patients with DMD treated with delandistrogene moxeparvovec gene therapy indicated that exons 8 and / or 9 appear to be highly immunogenic, however, not all patients with deletions in exons 8 and 9 develop IMM following gene therapy. These results are consistent with clinical trials of other investigational DMD gene therapies, suggesting that patients with deletions in regions of the DMD gene overlapping those expressed in a given micro dystrophin may be at an increased risk of an IMM event following gene therapy. It is hypothesized that a combination of the following factors poses the highest risk for IMM and led to IMM in these patients: deletion of exon 8 or 9; presence of T cells recognizing exons 8 or 9 as non-self; HLA type with strong HLA presentation of peptides mapping to exons 8 or 9. Work is currently underway to better understand these risk factors and to find ways to safely administer delandistrogene moxeparvovec to patients with potentially higher-risk DMD mutations.References1. Asher DR, et al. Expert Opin Biol Ther. 2020; 20:263-274;2. Zheng C and Baum BJ. Methods Mol Biol. 2008; 434:205-219;3. Mendell JR, et al. JAMA Neurol. 2020; 77: 1122-1131;4. US Food and Drug Administration. ELEVIDYS™ Highlights of prescribing information. https: / / www.fda.gov / media / 169679 / download. Published 2023 (Accessed September 2023);5. UAE Ministry of Health & Prevention, https: / / mohap.gov.ae / en / services / registered- medical-product directory (Accessed September 2023);6. ClinicalTrials.gov. NCT04626674 (Accessed September 2023);7. Zaidman CM, et al. Ann Neurol. 2023. Epub ahead of print, doi: 10.1002 / ana.26755.8. Qatar Ministry of Public Health Update, 27 September 2023. Roche data on file.All references disclosed herein are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method of assessing the risk associated with administration of delandistrogene moxeparvovec to a subject, comprising analyzing the subject's human leukocyte antigen (HLA) alleles, wherein one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec are not comprised in the genome of the subject.

2. The method of claim 1, wherein analyzing the subject's human leukocyte antigen (HLA) alleles comprises assessing the capability of the HLA molecules encoded by such HLA alleles to present one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec.

3. The method of claim 1 or 2, wherein the T cells of the subject are not capable of, or have a reduced capacity of, recognizing one or more peptides encoded by one or more portions of the Dystrophin gene comprised in delandistrogene moxeparvovec.

4. The method of claim 3, further comprising administering delandistrogene moxeparvovec to the subj ect.