Microdystrophin gene therapy constructs and uses thereof

Abstract

Provided is an invention based, in part, on novel gene constructs that encode a microdystrophin protein for use in gene therapy. The microdystrophin gene constructs and expression cassettes were engineered for improved therapy with respect to efficacy, potency and safety to the subject when expressed by a viral vector in muscle cells and / or CNS cells.

Description

0. SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 25, 2020, is named 38013_0009Pl_Sequence_Listing.txt and is 249,417 bytes in size. 1. FIELD OF THE INVENTION

[0002] The present invention relates to novel microdystrophins and gene therapy vectors, such as recombinant AAV vectors encoding the novel microdystrophins, as well as compositions and uses thereof and methods of treatment using the same. 2. BACKGROUND

[0003] A group of neuromuscular diseases called dystrophinopathies are caused by mutations in the DMD gene. Each dystrophinopathy has a distinct phenotype, with all patients suffering from muscle weakness and ultimately cardiomyopathy with ranging severity. Duchenne muscular dystrophy (DMD) is a severe, X-linked, progressive neuromuscular disease affecting approximately one in 3,600 to 9,200 live male births. The disorder is caused by frameshift mutations in the dystrophin gene abolishing the expression of the dystrophin protein. Due to the lack of the dystrophin protein, skeletal muscle, and ultimately heart and respiratory muscles (e.g., intercostal muscles and diaphragm), degenerate causing premature death. Progressive weakness and muscle atrophy begin in childhood. Affected individuals experience breathing difficulties, respiratory infections, and swallowing problems. Almost all DMD patients will develop cardiomyopathy. Pneumonia compounded by cardiac involvement is the most frequent cause of death, which frequently occurs before the third decade.

[0004] Becker muscular dystrophy (BMD) has less severe symptoms than DMD, but still leads to premature death. Compared to DMD, BMD is characterized by later-onset skeletal muscle weakness. Whereas DMD patients are wheelchair dependent before age 13, those with BMD lose ambulation and require a wheelchair after age 16. BMD patients also exhibit preservation of neck flexor muscle strength, unlike their counterparts with DMD. Despite milder skeletal muscle involvement, heart failure from DMD-associated dilated cardiomyopathy (DCM) is a common cause of morbidity and the most common cause of death in BMD, which occurs on average in the mid-40s.

[0005] Dystrophin is a cytoplasmic protein encoded by the DMD gene, and functions to link cytoskeletal actin filaments to membrane proteins. Normally, the dystrophin protein, located primarily in skeletal and cardiac muscles, with smaller amounts expressed in the brain, acts as a shock absorber during muscle fiber contraction by linking the actin of the contractile apparatus to the layer of connective tissue that surrounds each muscle fiber. In muscle, dystrophin is localized at the cytoplasmic face of the sarcolemma membrane.

[0006] The DMD gene is the largest known human gene. The most common mutations that cause DMD or BMD are large deletion mutations of one or more exons (60-70%), but duplication mutations (5-10%), and single nucleotide variants (including small deletions or insertions, single-base changes, and splice site changes accounting for approximately 25-35% of pathogenic variants in males with DMD and about 10-20% of males with BMD), can also cause pathogenic dystrophin variants. In DMD, mutations often lead to a frame shift resulting in a premature stop codon and a truncated, non-functional or unstable protein. Nonsense point mutations can also result in premature termination codons with the same result. While mutations causing DMD can affect any exon, exons 2-20 and 45-55 are common hotspots for large deletion and duplication mutations. In-frame deletions result in the less severe Becker muscular dystrophy (BMD), in which patients express a truncated, partially functional dystrophin.

[0007] Full-length dystrophin is a large (427 kDa) protein comprising a number of subdomains that contribute to its function. These subdomains include, in order from the amino-terminus toward the carboxy-terminus, the N-terminal actin-binding domain, a central so-called “rod” domain, a cysteine-rich domain and lastly a carboxy-terminal domain or region. The rod domain is comprised of 4 prolinerich hinge domains (abbreviated H), and 24 spectrin-like repeats (abbreviated R) in the following order: a first hinge domain (Hl), 3 spectrin-like repeats (RI, R2, R3), a second hinge domain (H2), 16 more spectrin-like repeats (R4, R5, R6, R7, R8, R9, RIO, Rll, R12, R13, R14, R15, RI6, RI7, RI8, R19), a third hinge domain (H3), 5 more spectrin-like repeats (R20, R21, R22, R23, R24), and a fourth hinge domain (H4) (including the WW domain). Following the rod domain are the cysteine-rich domain, and the COOH (C)-terminal (CT) domain.

[0008] With advances in use of adeno-associated virus (AAV) mediated gene therapy to potentially treat a variety of rare diseases, there has been hope and interest that AAV could be used to treat DMD, BMD and less severe dystrophinopathies. Due to limits on payload size of AAV vectors, attention has focused on creating micro- or mini- dystrophins, smaller versions of dystrophin that eliminate non-essential subdomains while maintaining at least some function of the full-length protein. AAV-mediated mini dystrophin gene therapy in mdx mice, an animal model for DMD, was reported as exhibiting efficient expression in muscle and improved muscle function (See, e.g., Wang et al., J. Orthop. Res. 27:421 (2009)).

[0009] Thus, there exists a need in the art for AAV vectors encoding micro- or mini- dystrophins that can be expressed at effective levels in transduced cells of subjects with DMD or BMD and preferably minimizing immune responses to the therapeutic protein. 3. SUMMARY OF THE INVENTION

[0010] Provided is an invention based, in part, on novel gene constructs that encode a microdystrophin protein for use in gene therapy. The microdystrophin gene constructs and expression cassettes were engineered for improved therapy with respect to efficacy, potency and safety to the subject when expressed by a viral vector in muscle cells and / or CNS cells. Based on in vivo therapeutic models, the microdystrophin gene therapies of the present disclosure showed measured improvements in grip strength, maximal and specific muscle force and / or reduction 2020392243   17 Sep 2026 in organ and muscle weight. Accordingly, provided are improved gene therapy vectors, for example, recombinant AAV vectors, such as recombinant AAV8 or AAV9 vectors, comprising these constructs for gene therapy expression of the microdystrophin proteins, and methods of using these gene therapy vectors in therapeutic methods and methods of making these gene therapy vectors as described herein. [0010a] Accordingly, in one aspect the present invention provides a recombinant adeno-associated virus (rAAV) particle comprising a) a nucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, or the reverse complement of the nucleotide sequence, and b) a capsid comprising a capsid protein. [0010b] In a further aspect, the present invention provides an rAAV particle comprising a) a nucleic acid comprising 5' to 3': a first AAV ITR sequence — a transcription regulatory element sequence —a nucleotide sequence encoding a microdystrophin protein — a polyadenylation sequence — a second AAV ITR sequence, wherein the microdystrophin protein comprises the amino acid sequence of SEQ ID NO: 1, wherein the transcription regulatory element is an SPc5-12 promoter or a transcriptionally active portion thereof, and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77. [0010c] In a further aspect, the present invention provides an rAAV particle comprising a) a nucleic acid comprising 5' to 3' (i) a first AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 73, (ii) an SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39, (iii) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, (iv) a polyadenylation nucleotide sequence of SEQ ID NO: 42, and (v) a second AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 74 and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77. [0010d] In a further aspect, the present invention provides a pharmaceutical composition comprising i) a rAAV particle comprising a) a nucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino 2020392243   17 Sep 2026 acid sequence of SEQ ID NO: 1, or the reverse complement of the nucleotide sequence, and b) a capsid comprising a capsid protein, and ii) a pharmaceutically acceptable carrier. [0010e]       In a further aspect, the present invention provides a pharmaceutical composition comprising: A.     1) an rAAV particle comprising a) a nucleic acid comprising 5' to 3': a first AAV ITR sequence — a transcription regulatory element sequence — a nucleotide sequence encoding a microdystrophin protein — a polyadenylation sequence — a second AAV ITR sequence, wherein the microdystrophin protein comprises the amino acid sequence of SEQ ID NO: 1, wherein the transcription regulatory element is an SPc5-12 promoter or a transcriptionally active portion thereof, and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77, and 2) a pharmaceutically acceptable carrier; or B.     1) an rAAV particle comprising a) a nucleic acid comprising 5' to 3' (i) a first AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 73, (ii) an SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39, (iii) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, (iv) a polyadenylation nucleotide sequence of SEQ ID NO: 42, and (v) a second AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 74 and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77 and 2) a pharmaceutically acceptable carrier. [0010f] In a further aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, or the reverse complement of the nucleotide sequence.

[0011] Provided are microdystrophin proteins and nucleic acid constructs encoding same that comprise the N-terminal actin binding domain and a subset of the hinge, rod and spectrin domains, followed by the cysteine-rich domain and, optionally, all or a portion, for example, a helix 1-containing portion, of the C-terminal domain. In particular embodiments, the microdystrophin has all or a portion of the C-terminal domain, or an a1-syntrophin and / or a-dystrobrevin 2020392243   17 Sep 2026 binding portion thereof. Microdystrophins having a C-terminal domain, or an al-syntrophin and / or a-dystrobrevin binding portion thereof, may have improved cardio-protective activity and / or result in improvement in or decrease / delay the progression of weakened cardiac muscle function.

[0012] Exemplary microdystrophins encoding constructs are illustrated in FIGS. 1A and 22. Embodiments described herein are a microdystrophin protein having from amino-terminus to the carboxy terminus:

[0013] ABD-H1-R1-R2-R3-H3-R24-H4-CR,

[0014] ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT

[0015] ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT, or

[0016] ABD-H1-R1-R2-R16-R17-R24-H4-CR,

[0017] wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R16 is a spectrin 16 region of dystrophin, R17 is a spectrin 17 region of dystrophin, R24 is a spectrin 24 region of dystrophin, CR is the cysteine-rich region of dystrophin or at least a portion thereof which binds P-dystroglycan, and CT is at least a portion of a C-terminal region of dystrophin, where the portion comprises a a1-syntrophin binding site and / or an a-dystrobrevin binding site. In certain embodiments, the CT domain has an amino acid sequence of SEQ ID NO: 35, 70, or 83. In certain embodiments, the H3 domain is the entire sequence of SEQ ID NO: 11. The CR domain may be the full length CR domain or a shortened CR domain, particularly a shortened CR domain which binds P-dystroglycan. In certain embodiments, the CR domain has an amino acid sequence of SEQ ID NO: 15 or 90. In certain embodiments, endogenous linker sequences link domains, for example, all or a 3 amino acid portion of the linker between R23 and R24 in the endogenous human dystrophin protein, link the H3 domain and the R24 domain. Alternatively, in some embodiments, H3 can be substituted with hinge 2 region of dystrophin (H2).

[0018] The microdystrophins provided herein exhibit dystrophin functions (see FIG. 13), such as (1) binding to one of, a combination of, or all of actin, P-dystroglycan, al-syntrophin, a-dystrobrevin, andnNOS (including nNOS binding indirectly via al-syntrophin); (2) promoting improved muscle function or slowing in the progression of reduction in muscle function in an animal model (for example, in the mdx mouse model described herein) or in human subjects; and / or (3) having a cardioprotective function or promoting improvement in cardiac muscle function or attenuation of cardiac dysfunction or slowing the progression of degeneration of cardiac function in animal models or human patients.

[0019] In particular embodiments, the microdystrophin has an amino acid sequence of SEQ ID NOs: 1, 2, 79, 91, 92, or 93.

[0020] Provided herein are nucleic acids encoding microdystrophins, including transgenes or gene cassettes for use in gene therapy. In embodiments, the microdystrophins are encoded by a nucleotide sequence of SEQ ID NOs: 20, 21, 81, 101, 102, or 103 or any nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 1, 2, 79, 91, 92, or 93. Exemplary constructs are illustrated in FIGS. 1A and 22. In certain embodiments, the constructs include an intron 5’ of the microdystrophin encoding sequence. In some embodiments, the intron is less than 100 nucleotides in length. In particular embodiments, the constructs include the human immunoglobulin heavy chain variable region (VH) 4 (VH4) intron and the intron is located 5’ of the microdystrophin encoding sequence. The presence of the VH4 intron may lead to improved expression of the microdystrophin in cells relative to expression from nucleic acid constructs not having the VH4 intron.

[0021] The trans genes provided herein contain promoters that drive expression of the microdystrophin in appropriate cell types, such as muscle cells (including skeletal muscle, cardiac muscle, and / or smooth muscle) and / or CNS cells. Reducing the size of transgenes used in gene therapy, such as with recombinant AAV vector therapy, may improve the efficacy and efficiency of the recombinant AAV vectors. Provided herein are transgenes in which the promoter is a musclespecific promoter, CNS specific promoter, or both. In certain embodiments, the promoter is a muscle-specific promoter that is less than 350 kb in length. In some embodiments, the promoter is an SPc5-12 promoter (SEQ ID NO: 39). Provided herein are transgenes in which the promoter is a truncated SPc5-12 promoter (SEQ ID NO: 40) that directs expression of the microdystrophin and is shorter than the SPc5-12 promoter as described more fully herein. In certain embodiments, the promoter is a CNS specific promoter.

[0022] Provided also are transgenes or gene cassettes in which the microdystrophin coding sequence has been codon optimized for increased expression. In addition or alternatively, the microdystrophin coding sequences and / or the transgene sequences may be depleted of CpG to reduce immunogenicity. In some embodiments, the microdystrophin transgene has fewer than two (2) CpG islands, or one (1) CpG island (in particular, as defined herein) and in certain embodiments has no CpG islands. The transgene with fewer than 2, 1 or has 0 CpG islands has reduced immunogenicity as measured by anti-drug antibody titer compared to microdystrophin constructs having more than 2 CpG islands.

[0023] Provided herein are nucleic acids comprising nucleotide sequences of SEQ ID NO: 53, 54, 55, 56, 82, 104, 105, or 106 which encode exemplary gene cassettes or transgenes.

[0024] The recombinant vector for delivering the transgenes described herein includes non-replicating recombinant adeno-associated virus vectors (rAAV), and may be of an AAV8 or AAV9 serotype or any other serotype appropriate for delivery of the microdystrophin coding sequences to muscle cells, including both skeletal muscle and cardiac muscle, and / or CNS cells which will express the microdystrophin and provide additional benefit to the patient, and / or deliver to muscle cells.

[0025] Also provided are pharmaceutical compositions comprising the recombinant vectors encoding the microdystrophins provided herein, including with a pharmaceutically acceptable excipient and methods of treatment for any dystrophinopathy, such as for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, as well as DMD or BMD female carriers, by administration of the gene therapy vectors described herein to a subject in need thereof. Provided are methods of treating, ameliorating the symptoms of or managing a dystrophinopathy, such as Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy by administration of an rAAV containing a transgene or gene cassette described herein, by administration to a subject in need thereof such that the microdystrophin is delivered to the muscle (including skeletal muscle, cardiac muscle, and / or smooth muscle) and / or the CNS. In particular embodiments, the rAAV is administered systemically.

[0026] Also provided are methods of manufacturing the viral vectors, particularly the AAV based viral vectors, and host cells for producing same. In specific embodiments, provided are methods of producing recombinant AAVs comprising culturing a host cell containing an artificial genome comprising a cis expression cassette flanked by AAV ITRs, wherein the cis expression cassette comprises a transgene encoding a therapeutic microdystrophin operably linked to expression control elements that will control expression of the transgene in human cells; a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and recovering recombinant AAV encapsidating the artificial genome from the cell culture.

[0027] The present inventions are illustrated by way of examples infra describing the construction and making of microdystrophin vectors and in vitro and in vivo assays demonstrating effectiveness. Exemplary Embodiments 1. A nucleic acid composition comprising a nucleic acid sequence encoding a microdystrophin protein wherein the microdystrophin protein comprises or consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, RI is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin or a [3-dystroglycan binding portion thereof, and CT is the C-terminal region of dystrophin or a portion of the C-terminal region comprising an al-syntrophin binding site or a dystrobrevin binding site. 2. The nucleic acid composition of embodiment 1 (1) comprising a nucleic acid sequence encoding the microdystrophin protein with an amino acid sequence of SEQ ID NO: 1 or 91, or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof encoding a therapeutically functional microdystrophin protein, or (2) comprising or consisting of a nucleic acid sequence of SEQ ID NO: 20 or 100 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof, wherein the nucleic acid sequence encodes a therapeutically functional microdystrophin protein. 3. The nucleic acid composition of embodiment 1 (1) comprising a nucleic acid sequence encoding the microdystrophin protein with an amino acid sequence of SEQ ID NO: 79 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof encoding a therapeutically functional microdystrophin protein, or (2) comprising or consisting of a nucleic acid sequence of SEQ ID NO: 81 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof, wherein the nucleic acid encodes a therapeutically functional microdystrophin protein. 4. A nucleic acid composition comprising a nucleic acid sequence comprising an intron (I) coupled to the 5’ end of a nucleic acid sequence encoding a microdystrophin protein, wherein the microdystrophin protein comprises or consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, RI is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is a cysteine-rich region of dystrophin. 5. The nucleic acid composition of embodiment 4 (1) comprising a nucleic acid sequence encoding the microdystrophin protein with an amino acid sequence of SEQ ID NO: 2 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof or (2) comprising or consisting of a nucleic acid sequence of SEQ ID NO: 21 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof, wherein the nucleic acid encodes a therapeutically functional dystrophin. 6. The nucleic acid composition of embodiments 1 to 3 further comprising an intron (I) coupled to the 5’ end of the nucleic acid sequence encoding the microdystrophin protein. 7. The nucleic acid composition of any of embodiments 4 to 6, wherein I is the human immunoglobin heavy chain variable region (VH) 4 intron (VH4) or the SV40 intron or the chimeric intron located 5’ of the microdystrophin encoding sequence. 8. The nucleic acid composition of embodiment 7, wherein the nucleic acid sequence encoding the VH4 intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 41 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases microdystrophin expression relative to a reference nucleic acid lacking the VH4 intron sequence; wherein the nucleic acid sequence encoding a chimeric intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 75 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases microdystrophin expression relative to a reference nucleic acid lacking the chimeric intron sequence; or wherein the nucleic acid sequence encoding a SV40 intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 76 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases microdystrophin expression relative to a reference nucleic lacking the chimeric intron sequence. 9. The nucleic acid composition of any of embodiments 1-3 or 6-8, wherein the nucleic acid sequence encoding the CT domain comprises or consists of the nucleic acid sequence of SEQ ID NO: 35 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to al-syntrophin, P-syntrophin, and / or dystrobrevin relative to a reference microdystrophin lacking the CT domain sequence; wherein the nucleic acid sequence encoding the CT domain comprises or consists of the nucleic acid sequence of SEQ ID NO: 70 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to al-syntrophin, P-syntrophin, and / or dystrobrevin relative to a reference microdystrophin lacking the CT domain sequence; or wherein the nucleic acid sequence encoding a minimal CT domain or consists of the nucleic acid sequence of SEQ ID NO: 80 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to al-syntrophin relative to a reference microdystrophin lacking the CT domain sequence. 10. The nucleic acid composition of embodiment 9 wherein the CT domain has an amino acid sequence of SEQ ID NO: 16 or 83 or comprises the amino acid sequence of SEQ ID NO: 84. 11. The nucleic acid composition of any of the foregoing embodiments, wherein the nucleic acid sequence encoding the CR domain comprises or consists of the nucleic acid sequence of SEQ ID NO: 34 or 69 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to P-dystroglycan relative to a reference microdystrophin lacking the CR domain sequence; wherein the nucleic acid sequence encoding the CR domain comprises or consists of the nucleic acid sequence of SEQ ID NO: 100 or 109 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to P-dystroglycan relative to a reference microdystrophin lacking the CR domain sequence. 12. The nucleic acid composition of embodiment 11, wherein the CR domain has an amino acid sequence of SEQ ID NO: 15 or 90. 13. The nucleic acid composition of any one of the foregoing embodiments, wherein the nucleic acid sequence encoding ABD consists of SEQ ID NO: 22 or 57 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22 or 57; the nucleic acid sequence encoding Hl consists of SEQ ID NO: 24 or 59 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 24 or 59; the nucleic acid sequence encoding RI consists of SEQ ID NO: 26 or 61 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26 or 61; the nucleic acid sequence encoding R2 consists of SEQ ID NO: 27 or 62 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27 or 62; the nucleic acid sequence encoding R3 consists of SEQ ID NO: 29 or 64 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 29 or 64; the nucleic acid sequence encoding H2 consists of SEQ ID NO: 38 or a sequence with at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 38; the nucleic acid sequence encoding H3 consists of SEQ ID NO: 30 or 65 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 30 or 65; the nucleic acid sequence encoding R24 consists of SEQ ID NO: 32 or 67 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32 or 67; the nucleic acid sequence encoding H4 consists of SEQ ID NO: 33 or 68 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33 or 68; the nucleic acid sequence encoding CR consists of SEQ ID NO: 34, 69, 100 or 109 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34, 69, 100 or 109; the nucleic acid sequence encoding CT, if present, consists of SEQ ID NO: 35, 70, or 80 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35, 70, or 80; and, optionally, the I nucleic acid sequence is a nucleic acid sequence of SEQ ID NO: 41 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 41 coupled at the 5’ end of the nucleic acid sequence encoding the microdystrophin. 14. The nucleic acid composition of any one of the foregoing embodiments, wherein the nucleic acid sequence that encodes ABD consists of SEQ ID NO: 22 or 57; the nucleic acid sequence that encodes Hl consists of SEQ ID NO: 24 or 59; the nucleic acid sequence that encodes RI consists of SEQ ID NO: 26 or 61; the nucleic acid sequence that encodes R2 consists of SEQ ID NO: 27 or 62; the nucleic acid sequence that encodes R3 consists of SEQ ID NO: 29 or 64; the nucleic acid sequence that encodes H2 consists of SEQ ID NO: 38; the nucleic acid sequence that encodes H3 consists of SEQ ID NO: 30 or 65; the nucleic acid sequence that encodes H4 consists of SEQ ID NO: 33 or 68; the nucleic acid sequence that encodes R24 consists of SEQ ID NO: 32 or 67; the nucleic acid sequence that encodes CR consists of SEQ ID NO: 34, 69, 100, or 109; I consists of SEQ ID NO: 41; and / or the nucleic acid sequence that encodes CT consists of SEQ ID NO: 35, 70 or 80. 15. The nucleic acid composition of any one of the foregoing embodiments, wherein the micro dystrophin protein comprises or consists of dystrophin sequences arranged from amino-terminus to the carboxy terminus: ABD-L1-H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR-CT or ABD-L1-H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR., wherein LI, L2, L3, and L4 are linkers. 16. The nucleic acid composition of any one of the foregoing embodiments, wherein the nucleic acid sequences encoding LI comprise or consist of SEQ ID NO: 23 or 58, L2 comprise or consist of SEQ ID NO: 25 or 60, L3 comprise or consist of SEQ ID NO: 28 or 63, and L4 comprise or consist of SEQ ID NO: 31, 36, 37, 66, 71 or 72. 17. A nucleic acid composition comprising a nucleic acid sequence encoding a microdystrophin protein, wherein the microdystrophin protein comprises or consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R16-R17-R24-H4-CR, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, RI is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, RI 6 is a spectrin 16 region of dystrophin, RI7 is a spectrin 17 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, and CR is a cysteine-rich region of dystrophin 18. The nucleic acid composition of embodiment 17 (1) comprising a nucleic acid sequence encoding the microdystrophin protein with an amino acid sequence of SEQ ID NO: 93 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof or (2) comprising or consisting of a nucleic acid sequence of SEQ ID NO: 103 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof, wherein the nucleic acid encodes a therapeutically functional microdystrophin. 19. The nucleic acid composition of embodiment 17 or 18, further comprising a nucleotide sequence encoding a CT domain that comprises a al-syntrophin binding site and / or a dystrobrevin binding site at the C-terminal end of the CR domain. 20. The nucleic acid composition of any one of embodiment 19 (1) comprising a nucleic acid sequence encoding the microdystrophin protein with an amino acid sequence of SEQ ID NO: 92 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof or (2) comprising or consisting of a nucleic acid sequence of SEQ ID NO: 102 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof, wherein the nucleic acid encodes a therapeutically functional microdystrophin. 21. The nucleic acid composition of embodiment 19 or 20, wherein the nucleic acid sequence encoding the CT domain comprises or consists of the nucleic acid sequence of SEQ ID NO: 35 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to al-syntrophin, P-syntrophin, and / or dystrobrevin relative to a reference microdystrophin lacking the CT domain sequence; wherein the nucleic acid sequence encoding the CT domain comprises or consists of the nucleic acid sequence of SEQ ID NO: 70 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to al-syntrophin, P-syntrophin, and / or dystrobrevin relative to a reference microdystrophin lacking the CT domain sequence; or wherein the nucleic acid sequence encoding a minimal CT domain or consists of the nucleic acid sequence of SEQ ID NO: 80 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases binding of the microdystrophin to al-syntrophin relative to a reference microdystrophin lacking the CT domain sequence. 22. The nucleic acid composition of any of embodiments 17 to 21, wherein the nucleic acid sequence encoding ABD consists of SEQ ID NO: 22 or 57 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22 or 57; the nucleic acid sequence encoding Hl consists of SEQ ID NO: 24 or 59 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 24 or 59; the nucleic acid sequence encoding RI consists of SEQ ID NO: 26 or 61 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26 or 61; the nucleic acid sequence encoding R2 consists of SEQ ID NO: 27 or 62 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27 or 62; the nucleic acid sequence encoding R16 consists of SEQ ID NO: 94 or 98 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 94 or 98; the nucleic acid sequence encoding RI7 consists of SEQ ID NO: 95 or 99 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 95 or 99; the nucleic acid sequence encoding R24 consists of SEQ ID NO: 32 or 67 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32 or 67; a nucleic acid sequence encoding H4 consists of SEQ ID NO: 33 or 68 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33 or 68; the nucleic acid sequence encoding CR consists of SEQ ID NO: 34, 69, 100 or 109 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34 or 69; the nucleic acid sequence encoding CT consists of SEQ ID NO: 35, 70, or 80 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35, 70, or 80 encoding a microdystrophin that has functional activity. 23. The nucleic acid composition of any one of embodiments 17 to 22, wherein the nucleic acid sequence that encodes ABD consists of SEQ ID NO: 22 or 57; the nucleic acid sequence that encodes Hl consists of SEQ ID NO: 24 or 59; the nucleic acid sequence that encodes RI consists of SEQ ID NO: 26 or 61; the nucleic acid sequence that encodes R2 consists of SEQ ID NO: 27 or 62; the nucleic acid sequence that encodes RI6 consists of SEQ ID NO: 94 or 98; the nucleic acid sequence that encodes RI7 consists of SEQ ID NO: 95 or 99; the nucleic acid sequence that encodes H4 consists of SEQ ID NO: 33 or 68; R24 consists of SEQ ID NO: 32 or 67; the nucleic acid sequence that encodes CR consists of SEQ ID NO: 34,69, 100 or 109; and, if present, the nucleic acid sequence that encodes CT consists of SEQ ID NO: 35, 70 or 80. 24. The nucleic acid composition of embodiments 17 to 23 further comprising an intron (I) coupled to the 5’ end of the nucleic acid sequence encoding the microdystrophin protein. 25. The nucleic acid composition of any of embodiment 24, wherein I is the human immunoglobin heavy chain variable region (VH) 4 intron (VH4) or the SV40 intron or the chimeric intron located 5’ of the microdystrophin encoding sequence. 26. The nucleic acid composition of embodiment 25, wherein the nucleic acid sequence encoding the VH4 intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 41 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases microdystrophin expression relative to a reference nucleic acid lacking the VH4 intron sequence; wherein the nucleic acid sequence encoding a chimeric intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 75 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases microdystrophin expression relative to a reference nucleic acid lacking the chimeric intron sequence; or wherein the nucleic acid sequence encoding a SV40 intron comprises or consists of the nucleic acid sequence of SEQ ID NO: 76 or a nucleic acid sequence at least 90%, 95% or 98% identical thereto or the reverse complement thereof and increases microdystrophin expression relative to a reference nucleic acid lacking the chimeric intron sequence. 27. The nucleic acid composition of any one of embodiments 17 to 26, wherein the microdystrophin protein comprises or consists of dystrophin sequences arranged from amino-terminus to the carboxy terminus: ABD-L1-H1-L2-R1-R2-L3-R16-L4.1 -RI 7-L4.2-R24-H4-CR-CT or ABD-L1 -Hl-L2-R1-R2-L3-R16-L4.1 -R17-L4.2-R24-H4-CR, wherein LI, L2, L3, L4.1and L4.2 are linkers. 28. The nucleic acid composition of embodiment 27, wherein the nucleic acid sequence encoding LI comprises or consists of SEQ ID NO: 23 or 58; the nucleic acid sequence encoding L2 comprises or consists of SEQ ID NO: 25 or 60; the nucleic acid sequence encoding L3 comprises or consists of SEQ ID NO: 28 or 63; the nucleic acid sequence encoding L4.1 comprises or consists of SEQ ID NO: 107 or 125; and the nucleic acid sequence encoding L4.2 comprises or consists of SEQ ID NO: 108 or 126. 29. The nucleic acid composition of any one of the foregoing embodiments, wherein the nucleic acid is a nucleic acid vector comprising a transcription regulatory element that promotes expression in muscle and / or CNS tissue operably linked to the nucleic acid sequence coding for the microdystrophin protein. 30. The nucleic acid composition of embodiment 29, wherein the transcription regulatory element comprises a muscle-specific promoter, optionally, skeletal, smooth, or / or cardiac muscle specific promoter. 31. The nucleic acid composition of embodiment 29 or 30, wherein the promoter is SPc5-12 or a transcriptionally active portion thereof. 32. The nucleic acid composition of embodiment 31, wherein the promoter consists of nucleic acid sequence of SEQ ID NO: 39 or 40. 33. The nucleic acid composition of embodiment 29, wherein the transcription regulatory element comprises a CNS-specific promoter. 34. The nucleic acid composition of embodiment 29, wherein the promoter is a CB7 promoter, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter (SEQ ID NO: 118), UB6 promoter, chicken beta-actin promoter, CAG promoter (SEQ ID NO: 116), RPE65 promoter, opsin promoter, TBG (Thyroxine-binding Globulin) promoter, APOA2 promoter, SERPINA1 (hAAT) promoter, MIR122 promoter, or an inducible promoter such as a hypoxia-inducible or rapamycin-inducible promoter. 35. The nucleic acid composition of embodiment 29 or 30, wherein the muscle-specific transcriptional regulatory element is one of a CK1 promoter, a CK4 promoter, a CK5 promoter, a CK6 promoter, a CK7 promoter, a CK8 promoter (SEQ ID NO: 115), a MCK promoter (or truncated form thereof) (SEQ ID NO: 121), a desmin promoter (SEQ ID NO: 119), a MHCK7 promoter (SEQ ID NO: 120), an enh358MCK promoter, a dMCK promoter, or a tMCK promoter. 36. The nucleic acid composition of any of the foregoing embodiments wherein the nucleotide sequence comprises a polyadenylation signal 3’ of the nucleotide sequence encoding the microdystrophin. 37.    The nucleic acid composition of embodiment 36, wherein the polyadenylation signal has a nucleotide sequence of SEQ ID NO: 42. 38. The nucleic acid composition of any one of the foregoing embodiments, wherein the nucleic acid comprises an AAV vector nucleotide sequence comprising from the 5’ to the 3’: (i) AAV ITR - transcription regulatory element-nucleic acid sequence encoding the microdystrophin domains arranged from N-terminus to C-terminus ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT-polyadenylation sequence - AAV ITR; (ii) AAV ITR-transcription regulatory element - nucleic acid sequence encoding the microdystrophin domains arranged from N-terminus to C-terminus ABD-H1-R1-R2-R3-H3-R24-H4-CR -polyadenylation sequence - AAV ITR; (iii) AAV ITR - transcription regulatory element-nucleic acid sequence encoding the microdystrophin domains arranged from N-terminus to C-terminus ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT-polyadenylation sequence - AAV ITR; or (iv) AAV ITR-transcription regulatory element-nucleic acid sequence encoding the microdystrophin domains arranged from N-terminus to C-terminus ABD-H1-R1-R2-R16-R17-R24-H4-CR -poly adenylation sequence - AAV ITR, wherein the AAV ITR is optionally AAV2 ITR. 39. The nucleic acid composition of any of the foregoing embodiments wherein the nucleotide sequence is codon optimized and / or depleted for CpG sequences. 40. The nucleic acid composition of any of the foregoing embodiments which has fewer than 2, or 1 CpG islands, or has no CpG islands. 41. The nucleic acid composition of embodiment 40, which exhibits reduced immunogenicity when administered to a human subject as measured by anti-drug antibody titer compared to a microdystrophin construct having more than 0 CpG islands. 42. The nucleic acid composition of any one of the foregoing embodiments comprising a nucleic acid sequence of SEQ ID NO: 53, 54, 55, 56, 82, 104, 105, or 106 43. The nucleic acid composition of any one of the foregoing embodiments comprising an AAV vector nucleotide sequence comprising an AAV ITR at the 5’ and 3’ ends of the nucleic acid sequence, wherein the AAV ITR is optionally AAV2 ITR. 44. The nucleic acid composition of embodiment 43, wherein the 5’ ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 73 and the 3’ ITR comprises or consists of the nucleotide sequence of SEQ ID NO: 74 45. A rAAV particle comprising an expression cassette comprising the nucleic acid composition of any one of the foregoing embodiments. 46. The rAAV particle of embodiment 45, which has a capsid protein from at least one AAV type selected from AAV type 1 (AAV 1), type 2 (AAV2), type 3 (AAV3), type 4 (AAV4), type 5 (AAV5), type 6 (AAV6), type 7 (AAV7), type 8 (AAV8), type rh8 (AAVrh8), type 9 (AAV9), type PHP.B (AAVPHP.B), type hu37 (AAV.hu37), type hu31 (AAV.hu31), type hu32 (AAV.hu32), type rhlO (AAVrhlO), type rh20 (AAVrh20), type rh39 (AAVrh39), and type rh74 (AAVrh74). 47. The rAAV particle of embodiment 45 or 46, wherein said capsid protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 77 (AAV8 capsid) or has an amino acid sequence of SEQ ID NO: 77. 48. The rAAV particle of embodiment 45 or 46, wherein said capsid protein has an amino acid sequence that is at least 95% identical to SEQ ID NO 78 (AAV9 capsid) or has an amino acid sequence of SEQ ID NO: 78. 49. A pharmaceutical composition comprising a therapeutically effective amount of an rAAV particle of any one of embodiments 45 to 48 and a pharmaceutically acceptable carrier. 50. A method of delivering a transgene to a cell, said method comprising contacting said cell with the rAAV particle of any one of embodiments 45 to 49, wherein said cell is contacted with the vector. 51. A pharmaceutical composition for treating a dystrophinopathy in a human subject in need thereof, comprising a therapeutically effective amount of an rAAV particle of any one of embodiments 45 to 49, optionally wherein said rAAV particle is formulated for administration to the circulation, muscle tissue, or CNS of said subject said subject. 52. A method of treating a dystrophinopathy in a human subject in need thereof, comprising: administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of a rAAV particle of any one of embodiments 45 to 49, so that a depot is formed in the muscle of said subject that releases a microdystrophin protein. 53. A method of preventing transmission of a dystrophinopathy to progeny of a human subject in need thereof, comprising: administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of a rAAV particle of any one of embodiments 45 to 49, such that the nucleic acid encoding the microdystrophin is incorporated into the germline of said subject. 54. The pharmaceutical composition or the method of embodiments 51 to 53, wherein the dystrophinopathy is DMD, BMD, X-linked dilated cardiomyopathy or the subject is a female carrier of DMD or BMD. 55. The pharmaceutical composition or the method of embodiments 51 to 54, wherein the composition is administered with at least a second agent effective for treating the dystrophinopathy. 56. The pharmaceutical composition or the method of embodiment 55, wherein the second agent is selected from the group consisting of an antisense oligonucleotide that causes exon skipping of the DMD gene, an anti-myostatin antibody, an agent that promotes ribosomal read-through of nonsense mutations, an agent that suppresses premature stop codons, an anabolic steroid and a corticosteroid. 57. The pharmaceutical composition or the method of any one of embodiments 51 to 56, wherein said administration improves the patient’s grip strength was improved, increases the maximal and specific muscle force and / or reduced organ and muscle weight. 58. The pharmaceutical composition or method of any one of embodiments 51 to 57, wherein administration of the rAAV particle improves or maintains cardiac function or slows the decline of cardiac function. 59. The pharmaceutical composition or method of any one of embodiments 51 to 58, wherein administration of the rAAV particle increases muscle mass or strength or maintains muscle mass or strength or reduces the likelihood of loss of muscle mass or strength. 60. A microdystrophin protein comprising or consisting of dystrophin domains arranged from the amino-terminus to the carboxy terminus ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, RI is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, CR is a cysteine-rich region of dystrophin, and CT is at least a portion of a C-terminal region of dystrophin comprising an al-syntrophin binding site, P-syntrophin binding site, and / or dystrobrevin site. 61. The microdystrophin protein of embodiment 60 comprising or consisting of an amino acid sequence of SEQ ID NOs: 1, 79, or 91. 62. The microdystrophin protein of embodiment 60 or 61, wherein the CT domain is a truncated CT domain which comprises an al-syntrophin binding site. 63. The microdystrophin protein of any one of embodiments 60 to 62 wherein the CT domain comprises or consist of the amino acid sequence of SEQ ID NO: 16 or 83 or comprises the amino acid sequence of SEQ ID NO: 84. 64. The microdystrophin protein of any one of embodiments 60 to 63, wherein CR domain comprises P-dystroglycan binding site. 65. The microdystrophin protein of any one of embodiments 60 to 64 wherein the CR domain comprises or consists of the amino acid sequence of SEQ ID NO: 15 or 90. 66. The microdystrophin protein of any one embodiments 60 to 66, wherein ABD consists of SEQ ID NO: 3 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3; Hl consists of SEQ ID NO: 5 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5; RI consists of SEQ ID NO: 7 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7; R2 consists of SEQ ID NO: 8 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8; H3 consists of SEQ ID NO: 11 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11; R24 consists of SEQ ID NO: 13 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13; H4 consists of SEQ ID NO: 14 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14; CR consists of SEQ ID NO: 15 or 90 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 or 90; and CT consists of SEQ ID NOs: 16 or 83 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 or 83. 67. The microdystrophin protein of any one of embodiments 60 to 66, wherein ABD consists of SEQ ID NO: 3, Hl consists of SEQ ID NO: 5; RI consists of SEQ ID NO: 7; R2 consists of SEQ ID NO: 8; R3 consists of SEQ ID NO: 10; H3 consists of SEQ ID NO: 11; R24 consists of SEQ ID NO: 13; H4 consists of SEQ ID NO: 14; CR consists of SEQ ID NO: 15 or 90; or CT consists of SEQ ID NO: 16 or 83. 68. The microdystrophin protein of any one of embodiments 60 to 67, comprising dystrophin domains arranged from the amino-terminus to the carboxy terminus: ABD-L1-H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR-CT, wherein LI, L2, L3, and L4 are linkers. 69. The microdystrophin protein of embodiment 68, wherein the amino acid sequences of LI, L2, L3, and L4 consist of SEQ ID NOs: 4, 6, 9, and 12, respectively. 70. A microdystrophin protein comprising or consisting of dystrophin domains arranged from the amino-terminus to the carboxy terminus ABD-H1-R1-R2-R16-R17-R24-H4-CR, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, RI is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, RI6 is a spectrin 16 region of dystrophin, RI7 is a spectrin 17 region of dystrophin, R24 is a spectrin 24 region of dystrophin, and CR is a cysteine-rich region of dystrophin. 71. The microdystrophin protein of embodiment 70 comprising or consisting of the amino acid sequence of SEQ ID NO: 93. 72. The microdystrophin protein of embodiment 70 comprising or consisting of dystrophin domains arranged from the amino-terminus to the carboxy terminus ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT wherein CT is at least a portion of a C-terminal region of dystrophin comprising an al-syntrophin binding site or a dystrobrevin binding site. 73. The microdystrophin protein of embodiment 72 wherein the CT domain comprises or consist of the amino acid sequence of SEQ ID NO: 16 or 83 or comprises the amino acid sequence of SEQ ID NO: 84. 74. The microdystrophin protein of embodiment 72 or 73 comprising or consisting of the amino acid sequence of SEQ ID NOS: 92. 75. The microdystrophin protein of any one of embodiments 70 to 74, wherein H4 domain comprises P-dystroglycan binding site. 76. The microdystrophin protein of any one embodiments 70 to 75, wherein ABD consists of SEQ ID NO: 3 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3; Hl consists of SEQ ID NO: 5 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5; RI consists of SEQ ID NO: 7 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7; R2 consists of SEQ ID NO: 8 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8; R16 consists of SEQ ID NO: 86 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 86; RI7 consists of SEQ ID NO: 87 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 87; R24 consists of SEQ ID NO: 13 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13; H4 consists of SEQ ID NO: 14 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14; and CR consists of SEQ ID NO: 15 or 90 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 or 90; 77. The microdystrophin protein of any of embodiments 70 to 76 comprising or consisting of a CT domain at the C terminus of the CR domain wherein the CT consists of SEQ ID NOs: 16 or 83 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 or 83. 78. The microdystrophin protein of any one of embodiments 70 to 77, wherein ABD consists of SEQ ID NO: 3, Hl consists of SEQ ID NO: 5; RI consists of SEQ ID NO: 7; R2 consists of SEQ ID NO: 8; R16 consists of SEQ ID NO: 86; R17 consists of SEQ ID NO: 87; R24 consists of SEQ ID NO: 13; H4 consists of SEQ ID NO: 14; and CR consists of SEQ ID NO: 15 or 90; and / or CT consists of SEQ ID NO: 16 or 83. 79. The microdystrophin protein of any one of embodiments 70 to 78, wherein the CT consists of SEQ ID NO: 16 or 83. 80. The microdystrophin protein of any one of embodiments 70 to 80, comprising dystrophin domains arranged from the amino-terminus to the carboxy terminus: ABD-L1-H1-L2-R1-R2-L3-R16-L4.1-R17-L4.2-R24-H4-CR-CT or ABD-L1-H1-L2-R1-R2-L3-R16-L4.1-R17-L4.2-R24-H4-CR, wherein LI, L2, L3, L4.1 and L4.2 are linkers. 81. The microdystrophin protein of embodiment 80, wherein the amino acid sequences of LI, L2, L3, L4.1 and L4.2 consist of SEQ ID NOs: 4, 6, 9, 110, and 89, respectively. 82. A method of treating a dystrophinopathy in a human subject in need thereof, comprising delivering to the circulation, muscle tissue and / or cerebrospinal fluid of said human subj ect, a therapeutically effective amount of a microdystrophin protein according to any one of embodiments 60 to 81. 83. A pharmaceutical composition for treatment of a dystrophinopathy in a human subject comprising a therapeutically effective amount of a microdystrophin protein according to any one of embodiments 60 to 81 formulated for delivery to the circulation, muscle tissue and / or cerebrospinal fluid of said human subject. 84. The method or pharmaceutical composition of embodiment 82 or 83, wherein the dystrophinopathy is DMD, BMD or X-linked dilated cardiomyopathy. 85. The method or pharmaceutical composition of any one of embodiments 82 to 84, wherein the CT domain comprises an al-syntrophin binding site, a P-syntrophin binding site, and / or a dystrobrevin binding site. 86. The method or pharmaceutical composition of embodiment 85, wherein the CT domain is a truncated CT domain comprising an al-syntrophin binding site. 87. The method or pharmaceutical composition of any one of embodiments 82 to 86, wherein H4 comprises P-dystroglycan binding site. 88. A method of producing recombinant AAVs comprising: (a) culturing a host cell containing: (i) an artificial genome comprising a cis expression cassette, wherein the cis expression cassette comprises a nucleic acid composition of any one of embodiments 38 to 44; (ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; (iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and (b) recovering recombinant AAV encapsidating the artificial genome from the cell culture. 89. A host cell comprising: a. an artificial genome comprising a cis expression cassette, wherein the cis expression cassette comprises a nucleic acid composition of any one of embodiments 38 to 44; b. a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; and c. sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins. 4. BRIEF DESCRIPTION OF THE FIGURES

[0028] FIGS. 1A-C. FIG. 1A illustrate vector gene expression cassettes and microdystrophin constructs for use in a Cis-plasmid for gene therapy. DNA length for each component and complete transgene are listed for each construct. SPc5-12: synthetic muscle-specific promoter; Mini-SPc: truncated synthetic muscle-specific promoter; CT1.5: truncated / minimal CT domain; VH4: human immunoglobin heavy chain variable region intron; ABD: actin binding domain; H: hinge; R: rod; CR: cysteine rich domain; CT: C-terminal domain; smPA: small poly A; ABD: Actin Binding Domain 1 (ABDI). FIGS. 1B-C depict protein bands detected by Western Blot (antibody (lc7) against dystrophin) showing relative size of microdystrophin proteins expressed from plasmids RGX-DYS1, RGX-DYS3 and RGX-DYS5.

[0029] FIGS. 2A-F depict fluorescent microscopy of differentiated C2C12 cells three days post-infection with reporter AAV vectors AAV8-GFP (A-C) and AAV8-VH4-GFP (D-F) at various dosage (indicated above the images: 5 x 10e5 vg / cell (A, D), 1 x 10e5 vg / cell (B, E), 0.2 x 10e5 vg / cell (C, F)). Scale bar: 200 pM. vg: vector genomes.

[0030] FIG. 3 shows mean fluorescence intensity (units) of transduced C2C12 cells measured three days post infection with AAV8-GFP and AAV8-VH4-GFP vectors at three different dosages: 5 x 10e5 vg / cell, 1 x 10e5 vg / cell, and 0.2 x 10e5 vg / cell.

[0031] FIGS. 4A-C depict fluorescent microscopy of differentiated C2C12 cells six days post infection with AAV8-CAG-GFP. Images A-C were taken daily using an EVOS™ microscope with transmitted light and GFP channels under the same magnification: A, microscopic image set to the GFP channel; B, brightfield (or phase contrast) to observe the confluence of cells; C, merged image of A and B to observe the number of infected cells to be approximately 50%.

[0032] FIGS. 5A-H depicts in vitro potency testing of microdystrophin vector (RGX-DYS1-03, E-H) as compared to the reference control (RGX-DYS-RS, A-D) by immunofluorescent staining of dystrophin protein. There were three replicates for each dosage (indicated above respective images): lel2 vg / ml (A, E), 4el 1 vg / ml (B, F), 1.6el 1 vg / ml (C, G), and 6.4el0 vg / ml (D, H).

[0033] FIG. 6 provides infectivity data in mouse muscle cell line C2C12 cells for each vector, as a measure of vector potency. Normalized data (vector copy number / reference control) for each vector batch RGX-DYS1-01, RGX-DYS1-02, RGX-DYS2-01, RGX-DYS3-01, RGX-DYS3-02, RGX-DYS4-01, and RGX-DYS1-RS are shown. An internal control vector based on an earlier batch of DYS1 (RGX-DYS1-RS) was considered as reference standard (1.0).

[0034] FIG. 7 provides microdystrophin data in mouse muscle cell line C2C12 cells for each vector from different production batches each using the same process (RGX-DYS1-01, RGX-DYS1-02, RGX-DYS2-01, RGX-DYS3-01, RGX-DYS3-02, RGX-DYS4-01, and RGX-DYS1-RS), as a measure of mRNA expression. Two different vector dosages were used to infect C2C12 cells (le5 vg / cell and 5e4 vg / cell). mRNA expression level of each batch was calculated as the fold change (delta CT) in qPCR between primer / probe for microdystrophin and for endogenous control mouse GAPDH from the same cDNA sample. The graph shows fold increase and RGX-DYS1-RS was considered a 100% reference standard and set to 1.

[0035] FIG. 8 shows weekly changes in body weight (g). Data are presented as mean ± SEM. n = 12 for mdx RGX-DYS1 group; n= 13 for mdx vehicle group; n = 14 for BL10 vehicle group.

[0036] FIGS. 9A-B depicts mouse muscle and organ weight measurements (normalized to body weight, g / kg). Quadriceps and soleus weights are shown in FIG. 9A, and triceps and TA weights are shown in FIG. 9B. Data are presented as mean ± SEM. n = 12 for mdx RGX-DYS1 group; n= 13 for mdx vehicle group; n = 14 for BL10 vehicle group. ***P<0.001 (One-way ANOVA); ###P<0.001 (t-test).

[0037] FIG. 10 depicts grip strength measurement (KGF / kg). *-One way ANOVA (***P < 0.001); #- t-test (### p < 0.001). The forearm muscle grip force was normalized for each mouse by muscle weight, n = 12 for mdx RGX-DYS1 group; n= 13 for mdx vehicle group; n = 14 for BL10 vehicle group.

[0038] FIG. 11 illustrates in vitro muscle force contractile force analysis at week-6 post treatment revealed significant improvement of the muscle force in RGX-DYS1-treated mdx mice compared to mdx mice treated with vehicle. Maximal force (mN) and specific force (kN / m2) are shown. ***, p< 0.001 by one-way ANOVA. ###, p < 0.001 via t-test. n = 12 of mdx RGX-DYS1 group; n= 13 for mdx vehicle group; n = 14 for BL10 vehicle group.

[0039] FIG. 12 Vector copy numbers (vg / diploid genome) in skeletal muscle, cardiac muscle, and liver by ddPCR method. The Naica Crystal Digital PCR system from Stilla Technologies was used, n = 13 for each treated tissue. The numbers listed are average ± Stdev. Vector copy number was calculated as 2X microdystrophin transgene copy number / endogenous control mouse glucagon copy number. The uninjected mdx liver samples (n=13) were used as negative control samples. TA, tibialis anterior muscle; EDL, extensor digitorum longus.

[0040] FIG. 13 Illustration of the sarcolemma showing interaction between a wild-type dystrophin or a microdystrophin containing dystrobrevin and al- and pi-syntrophin binding sites, e.g. RGX-DYS1, and the dystrophin-associated protein complex (DAPC) with the actin cytoskeleton. It is envisioned that RGX-DYS1 having dystrobrevin, al-syntrophin, and pi-syntrophin binding sites, will partly recruit and anchor nNOS to the sarcolemma through al-syntrophin.

[0041] FIG. 14 Immunofluorescent staining on gastrocnemius muscle from mdx RGX-DYS1, mdx control, and WT control groups. Cryo-sections were stained with anti-a-dystrobrevin, anti-P-dystroglycan, anti-nNos, anti-dystrophin (anti-dys), and anti-a-syntrophin. The secondary antibody was labelled with CY3 and all sections were counterstained with DAPI before mounting.

[0042] FIG. 15: Western blot against dystrophin extracted from AAV-p-dystrophin vector-injected gastrocnemius muscle tissues. Lanes 1 through 4 = protein samples from AAV8-RGX-DYS1-injected mdx mice, Lanes 5 through 8 = protein samples from AAV8-RGX-DYS5 injected mdx mice, and Lanes 9 through 12 = protein samples from AAV8-RGX-DYS3 injected mdx mice, al-actin serves as the loading control in each lane. Mdx (Lane 13) indicated an un-injected mdx mice. For dystrophin blot, mouse anti-dystrophin monoclonal antibody was used (1:100 dilution). For anti-alphal-actin blot, polyclonal antibody was used at a dilution factor of 1:10,000, and the secondary (anti-rabbit) antibody was used at 1:20,000.

[0043] FIGS. 16A-C: Quantification of p-dystrophin bands by western blot (Panel A), AAV-p-Dys vector copy numbers by ddPCR (Panel B), and quantification of p-dystrophin bands normalized by AAV-p-Dys vector copy numbers (Panel C). * p< 0.05; ** P < 0.01; ***P<0001.

[0044] FIGS. 17A-B: mRNA expression of p-dystrophin and wild-type (WT) dystrophin in skeletal muscles (gastrocnemius). Total RNA was extracted from the skeletal muscles and cDNA synthesized. The copies numbers of p-dystrophin, WT-dystrophin, and endogenous control Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were measured using digital PCR (Naica Crystal Digital PCR system, Stilla technologies). A. Relative p- or WT-dystrophin mRNA expression normalized by GAPDH. The ratio of WT-dystrophin to GAPDH in B6-WT skeletal muscle was considered as 1. B. Relative p- or WT-dystrophin mRNA expression in a single cell, p- or WT-dystrophin mRNA expression copy numbers were normalized by GAPDH and genome copy numbers per cell.

[0045] FIG. 18. Gastrocnemius muscle extracted from mdx mice, tissue sections prepared and immunofluorescently (IF) stained against dystrophin and dystrophin associated protein complexes including dystrobrevin, [3-dystroglycan, and syntrophin. Mice were treated as described: B16 (untreated wild-type mice); RGX-DYS1 (mouse ID 3553, and mouse ID 3588); RGX-DYS3 (mouse ID 5, and mouse ID 7); and RGX-DYS5 (mouse ID 9, and mouse ID 11). Objective lens: 40x.

[0046] FIGS. 19A-C: Syntrophin expression in skeletal muscles. A. Gastrocnemius muscle extracted from mdx mice, tissue sections prepared and immunofluorescently (IF) stained against syntrophin. Mice were treated as described: B16 (untreated wild-type mice); RGX-DYS1 (mouse ID 3553, and mouse ID 3588); RGX-DYS3 (mouse ID 5, and mouse ID 7); and RGX-DYS5 (mouse ID 9, and mouse ID 11). Objective lens: 40x. B. Western blot against syntrophin from muscle tissue lysate. C. Quantification of western blot bands. *, p< 0.05; ***, p < 0.0001. D. Western blot against syntrophin from total muscle membrane protein. E. Quantification of western blot bands.

[0047] FIGS. 20A-C: nNOS expression in skeletal muscles. A. Immunofluorescent staining against nNOS. B. Western blot against nNOS. C. Quantification of western blot bands.

[0048] FIGS. 21A-E: Transduction of satellite cells and amelioration of cell regeneration by AAV vector encoding p-dystrophin gene. A-B. RNAScope Images of RGX-DYS1-treated mdx mice (panel A) and untreated mdx mice (panel B) revealing co-expression of p-dystrophin (red) and pax7 satellite cells (green). The RNAscope multiplex fluorescent analysis of AAV transgene and Pax7 mRNA expression service was performed at Advanced Cell Diagnostics Inc (Newark, CA). C. Percentage of AAV-DMD transduced satellite cells. D. Total satellite cell counting in RNAscope images. E. Pax7 mRNA expression in skeletal muscles from different groups revealed by ddPCR. The primes and probe against p-dystrophin was the same as previously described. The ratio of pax7 to GAPDH in B6-WT skeletal muscle was considered as 1. **, p< 0.01; ***, p < 0.001; ****, p<0.0001 as compared to the untreated mdx mice.

[0049] FIG. 22: Illustration of additional modified p-dystrophin constructs. CR short: Cysteine-rich domain is 150 bp shorter than in wild-type dystrophin. R16 / R17: dystrophin spectrin-like repeats 16 and 17.

[0050] FIGS. 23A-C: In vitro infection of C2C12 myotubes with different versions of AAV8-p-dystrophin constructs. C2C12 myoblast cells were induced in differentiation media, then infected with AAV vectors. The cells were harvested five days after infection for western blot or mRNA expression. 1: Negative control; 2: RGX-DYS8; 3: RGX-DYS7; 4: RGX-DYS6; 5: RGX-DYS3; 6: RGX-DYS5; 7: RGX-DYS1; 8: RGX-DYS1; 9: RGX-DYS1; 10: RGX-DYS1; 11: RGX-DYS1. A. Western blot analysis of p-dystrophin expression from C2C12 cells. B. Quantification of western blot analysis. C. Detection of p-dystrophin mRNA expression by ddPCR. 5. DETAILED DESCRIPTION

[0051] Provided are microdystrophin protein, for example, as shown in FIG. 1A and FIG. 22 and nucleic acid compositions and rAAV vectors encoding the same as well as pharmaceutical compositions and treatment methods related thereto. 5.1. Definitions

[0052] The term “AAV” or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses. The AAV can be an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and / or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene. An example of the latter includes a rAAV having a capsid protein having a modified sequence and / or a peptide insertion into the amino acid sequence of the naturally-occurring capsid.

[0053] The term “rAAV” refers to a “recombinant AAV.” In some embodiments, a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.

[0054] The term “rep-cap helper plasmid” refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and / or the cap gene sequences.

[0055] The term “cap gene” refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus. For AAV, the capsid protein may be VP1, VP2, or VP3.

[0056] The term “rep gene” refers to the nucleic acid sequences that encode the non-structural protein needed for replication and production of virus.

[0057] The terms “nucleic acids” and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA / RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes. The nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.

[0058] Amino acid residues as disclosed herein can be modified by conservative substitutions to maintain, or substantially maintain, overall polypeptide structure and / or function. As used herein, “conservative amino acid substitution” indicates that: hydrophobic amino acids (i.e., Ala, Cys, Gly, Pro, Met, Vai, he, and Leu) can be substituted with other hydrophobic amino acids; hydrophobic amino acids with bulky side chains (i.e., Phe, Tyr, and Trp) can be substituted with other hydrophobic amino acids with bulky side chains; amino acids with positively charged side chains (i.e., Arg, His, and Lys) can be substituted with other amino acids with positively charged side chains; amino acids with negatively charged side chains (i.e., Asp and Glu) can be substituted with other amino acids with negatively charged side chains; and amino acids with polar uncharged side chains (i.e., Ser, Thr, Asn, and Gin) can be substituted with other amino acids with polar uncharged side chains.

[0059] The terms “subject”, “host”, and “patient” are used interchangeably. A subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), most preferably a human.

[0060] The term “therapeutically functional microdystrophin” means that the microdystrophin exhibits therapeutic efficacy in one or more of the assays for therapeutic utility described in Section 5.4 herein or in assessment of methods of treatment described in Section 5.5 herein.

[0061] The terms “subject”, “host”, and “patient” are used interchangeably. A subject is preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), most preferably a human.

[0062] The terms “therapeutic agent” refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. A “therapeutically effective amount” refers to the amount of agent, (e.g., an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom. Further, a therapeutically effective amount with respect to an agent of the invention means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.

[0063] The term “prophylactic agent” refers to any agent which can be used in the prevention, reducing the likelihood of, delay, or slowing down of the progression of a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. A “prophylactically effective amount” refers to the amount of the prophylactic agent (e.g., an amount of product expressed by the transgene) that provides at least one prophylactic benefit in the prevention or delay of the target disease or disorder, when administered to a subject predisposed thereto. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent, reduce the likelihood of, or delay the occurrence of the target disease or disorder; or slow the progression of the target disease or disorder; the amount sufficient to delay or minimize the onset of the target disease or disorder; or the amount sufficient to prevent or delay the recurrence or spread thereof. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the exacerbation of symptoms of a target disease or disorder. Further, a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or when in combination with other agents, that provides at least one prophylactic benefit in the prevention or delay of the disease or disorder.

[0064] A prophylactic agent of the invention can be administered to a subject “pre-disposed” to a target disease or disorder. A subject that is “pre-disposed” to a disease or disorder is one that shows symptoms associated with the development of the disease or disorder, or that has a genetic makeup, environmental exposure, or other risk factor for such a disease or disorder, but where the symptoms are not yet at the level to be diagnosed as the disease or disorder. For example, a patient with a family history of a disease associated with a missing gene (to be provided by a transgene) may qualify as one predisposed thereto. Further, a patient with a dormant tumor that persists after removal of a primary tumor may qualify as one predisposed to recurrence of a tumor.

[0065] The term “CpG islands” means those distinctive regions of the genome that contain the dinucleotide CpG (e.g. C (cytosine) base followed immediately by a G (guanine) base (a CpG)) at high frequency, thus the G+C content of CpG islands is significantly higher than that of non-island DNA. CpG islands can be identified by analysis of nucleotide length, nucleotide composition, and frequency of CpG dinucleotides. CpG island content in any particular nucleotide sequence or genome may be measured using the following criteria: island size greater than 100, GC 2020392243   17 Sep 2026 Percent greater than 50.0 %, and ratio greater than 0.6 of observed number of CG dinucleotides to the expected number on the basis of the number of Gs and Cs in the segment (Obs / Exp greater than 0.6). Obs / Exp CpG = Number of CpG * N / (Number of C * Number of G)

[0066] where N = length of sequence.

[0067] Various software tools are available for such calculations, such as world-wide-web.urogene.org / cgi-bin / methprimer / methprimer.cgi,          world-wide- web.cpgislands.usc.edu / ,                                           world-wide- web.ebi.ac.uk / Tools / emboss / cpgplot / index.html     and           world-wide- web.bioinformatics.org / sms2 / cpg_islands.html. (See also Gardiner-Garden and Frommer, J Mol Biol. 1987 Jul 20;196(2):261-82; Li LC and Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002 Nov;18(11):1427-31.). In one embodiment the algorithm to identify CpG islands is found at www.urogene.org / cgi-bin / methprimer / methprimer.cgi. [0067a] Where any or all of the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components. [0067b] A reference herein to a patent document or any other matter identified as prior art, is not to be taken as an admission that the document or other matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims. 5.2. Microdystrophin Transgenes 5.2.1 Microdystrophin

[0068] Embodiments described herein comprise a microdystrophin protein having from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR (e.g., SEQ ID NO: 2) or ABD1-H1-R1-R2-R16-R17-R24-H4-CR (SEQ ID NO: 93), wherein ABD is an actin-binding domain of dystrophin, H1 is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2020392243   17 Sep 2026 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R16 is a spectrin 16 region of dystrophin, R17 is a spectrin 17 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is a hinge 4 region of dystrophin, CR is a cysteine-rich region of dystrophin.

[0069] As explained above, the microdystrophins in accordance with the present disclosure comprise ABD-H1-R1-R2-R3-R24-H4 or ABD-H1-R1-R2-R16-R17-R24-H4. The NH2 terminus and a region in the rod domain of dystrophin bind directly to but do not cross-link cytoskeletal actin. The rod domain of wild type dystrophin is composed of 24 repeating units that are similar to the triple helical repeats of spectrin. This repeating unit accounts for the majority of the dystrophin protein and is thought to give the molecule a flexible rod-like structure similar to P-spectrin. These a-helical coiled-coil repeats are interrupted by four proline-rich hinge regions. At the end of the 24th repeat is the fourth hinge region that is immediately followed by the WW domain [Blake, D. et al, Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle. Physiol. Rev. 82: 291329, 2002], Microdystrophins disclosed herein do not include R4 to R23, or, alternatively, do not include R3 (or, in some embodiments R4) to RI5 and RI 8 to R23 (that is, such that the microdystrophin includes RI6 and RI7, but may not, in certain embodiments, include R3), and only include 2 or 3 of the 4 hinge regions or portions thereof. Embodiments may contain dystrophin spectrin-like repeats 16 and 17 which are understood to anchor nNOS to the sarcolemma. In some embodiments, no new amino acid residues or linkers are introduced into the microdystrophin.

[0070] In some embodiments, microdystrophin comprises H3 (e.g, SEQ ID NOS: 1, 2, or 79). In embodiments, H3 can be a full endogenous H3 domain from N-terminal to C-terminal, e.g., SEQ ID NO: 11. Stated another way, some microdystrophin embodiments do not contain a fragment of the H3 domain but contain the entire H3 domain. In some embodiments, the C-terminal amino acid of the R3 domain is coupled directly (or covalently bonded to) the N-terminal amino acid of the H3 domain. In some embodiments, the C-terminal amino acid of the R3 domain coupled to the N-terminal amino acid of the H3 domain is Q. In some embodiments, the 5' amino acid of the H3 domain coupled to the R3 domain is Q.

[0071] In other embodiments, microdystrophin comprises H2 instead of H3. H2 can be the full endogenous H2 domain (SEQ ID NO: 19). Such microdystrophin protein embodiments have from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H2-R24-H4-CR. In some embodiments, the C-terminal amino acid of the R3 domain coupled to the N-terminal amino acid of the hinge domain is Q. In other embodiments, the N-terminal amino acid of the H2 domain coupled to the R3 domain is P. In certain embodiments, the C-terminal amino acid of the R3 domain is directly coupled to the N-terminal amino acid of the hinge domain, wherein the N-terminal amino acid of the hinge domain is P or Q. In still other embodiments, the C-terminal amino acid of the R3 domain is directly coupled to the N-terminal amino acid of the H2 domain, wherein the N-terminal amino acid of the H2 domain is P.

[0072] Without being bound by any one theory, a full hinge domain may be appropriate in any microdystrophin construct in order to convey full activity upon the derived microdystrophin protein. Hinge segments of dystrophin have been recognized as being proline-rich in nature and may therefore confer flexibility to the protein product (Koenig and Kunkel, 265(6):4560-4566, 1990). Any deletion of a portion of the hinge, especially removal of one or more proline residues, may reduce its flexibility and therefore reduce its efficacy by hindering its interaction with other proteins in the DAP complex.

[0073] Microdystrophins disclosed herein comprise the wild-type dystrophin H4 sequence (which contains the WW domain) to and including the CR domain (which contains the ZZ domain, represented by a single underline (UniProtKB-P11532 aa 3307-3354) in SEQ ID NO: 15). The WW domain is a protein-binding module found in several signaling and regulatory molecules. The WW domain binds to proline-rich substrates in an analogous manner to the src homology-3 (SH3) domain. This region mediates the interaction between [3-dystroglycan and dystrophin, since the cytoplasmic domain of [3-dystroglycan is proline rich. The WW domain is in the Hinge 4 (H4 region). The CR domain contains two EF-hand motifs that are similar to those in a-actinin and that could bind intracellular Ca2+. The ZZ domain contains a number of conserved cysteine residues that are predicted to form the coordination sites for divalent metal cations such as Zn2+. The ZZ domain is similar to many types of zinc finger and is found both in nuclear and cytoplasmic proteins. The ZZ domain of dystrophin binds to calmodulin in a Ca2+-dependent manner. Thus, the ZZ domain may represent a functional calmodulin-binding site and may have implications for calmodulin binding to other dystrophin-related proteins.

[0074] Certain embodiments comprise a truncated portion of the CR domain, which comprises the ZZ domain. For example, the microdystrophin protein comprises from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR(short)-CT (e.g., SEQ ID NO: 91, see RGX-DYS6 in FIG. 22). In certain embodiments, the CR domain, for example, has an amino acid sequence of SEQ ID NO: 90.

[0075] To overcome the packaging limitation that is typical of AAV vectors, many of the microdystrophin genes developed for clinical use are lacking the CT domain. Several researchers have indicated that the DAPC does not even require the C-terminal domain in order to assemble or that the C-terminus is non-essential [Crawford, et al., J Cell Biol, 2000, 150(6): 1399-1409; and Ramos, J.N, et al. Molecular Therapy 2019, 27(3):1-13]. The CT domain of dystrophin protein could nevertheless provide beneficial effects on cardiomyopathy. A special interaction between the CT domain of dystrophin and [3-dystroglycan in cardiac muscle has been shown, where a direct molecular interaction exists at the plasma membrane interface, indicating a direct role for the CT domain in anchoring DAP complexes in the cardiomyocyte membrane [Stevenson, S., et al., Spatial relationship of the C-terminal domains of dystrophin and beta-dystroglycan in cardiac muscle support a direct molecular interaction at the plasma membrane interface. Circ Res, 1998. 82(1): p. 82-93], Dystrophin genotype-cardiac phenotype corrections in a study of 274 Duchenne and Becker muscular dystrophy patients revealed the presence of N-terminal actin binding domain (ABDI) and CR domain plus CT domain had a decreased risk of cardiomyopathy, further pointing to a beneficial cardio-protective effect for the CT domain of dystrophin protein [Tandon, A., et al., Dystrophin genotype-cardiac phenotype correlations in Duchenne and Becker muscular dystrophies using cardiac magnetic resonance imaging. Am J Cardiol, 2015. 115(7): p. 967-71], Additionally, overexpression of a microdystrophin gene containing helix 1 of the coiled-coil motif of the CT domain in skeletal muscle of mdx mice increased the recruitment al-syntrophin and a-dystrobrevin, which are members of DAP complex, serving as modular adaptors for signaling proteins recruited to the sarcolemmamembrane [Koo, T., et al., Delivery of AAV2 / 9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of al-syntrophin and a- dystrobrevin in skeletal muscles of mdx mice. Hum Gene Ther, 2011. 22(11): p. 1379-88], Overexpression of the longer version of microdystrophin also improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice as compared with the shorter version [Koo, T., et al. 2011, supra].

[0076] It has been shown that significantly reduced cardiac function persists in DMD patients. Treatments that restore neuronal nitric oxide synthase (nNOS) function are thought to be beneficial by improving cardiac function, as such leading to significant improvement of the systolic BP, fraction shortening and ejection fraction and in turn a reduction in cardiac fibrosis. Progression of cardiac fibrosis is indicated as patients first exhibit left ventricle (LV) dilation and hypertrophy, which progresses to a stage known as dilated cardiomyopathy (DCM).

[0077] The CT domain of dystrophin contains two polypeptide stretches that are predicted to form a-helical coiled coils similar to those in the rod domain (see Hl indicated by single underlining and H2 indicated by double underlining in SEQ ID 16 in Table 1 below). Each coiled coil has a conserved repeating heptad (a,b,c,d,e,f,g)„ similar to those found in leucine zippers where leucine predominates at the “d” position. This domain has been named the CC (coiled coil) domain. The CC region of dystrophin forms the binding site for dystrobrevin and may modulate the interaction between al-syntrophin and other dystrophin-associated proteins.

[0078] Both syntrophin isoforms, al-syntrophin and pi-syntrophin are thought to interact directly with dystrophin through more than one binding site in dystrophin exons 73 and 74 (Yang et al, JBC 270(10):4975-8 (1995)). al- and pi-syntrophin bind separately to the dystrophin C-terminal domain, and the binding site for al-syntrophin resides at least within the amino acid residues 3447 to 3481, while that for pi-syntrophin resides within the amino acid residues 3495 to 3535 (Table 1, SEQ ID NO: 16, italic). Alphal- (al-) syntrophin and alpha-syntrophin are used interchangeably throughout.

[0079] Helix 1 (see Hl indicated as single underlined sequence within SEQ ID NO: 16 in Table 1 below) of the coiled-coil motif in the C-terminal (CT) domain of the microdystrophin gene cassettes may be advantageous for cardiomyocyte protection, and otherwise stabilizing dystrophin-associated (glyco)protein (DAP) complexes (DAPCs). The DAPC may participate in important signaling roles as well as a structural role. Certainly, there have been indications of altered nitric oxide (NO) production, and possible alterations in other functions caused by the destabilization and loss of the complex.

[0080] Unexpectedly, certain microdystrophin constructs disclosed herein were found to bind to and recruit nNOS, as well as alpha-syntrophin, alpha-dystrobrevin and beta-dystroglycan. Binding to nNOS, in the context of a microdystrophin construct including a C-terminal domain of dystrophin binding to nNOS, means that the microdystrophin construct expressed in muscle tissue was determined by immunostaining with appropriate antibodies to identify each of alpha-syntrophin, alpha-dystrobrevin, and nNOS in or near the sarcolemma in a section of the transduced muscle tissue. See Example 5 and 7 in Sections 6.5 and 6.7, infra. In certain embodiments, the microdystrophin protein has a C-terminal domain that “increases binding” to al-syntrophin, P-syntrophin and / or dystrobrevin compared to a comparable microdystrophin that does not contain the C-terminal domain (but has the same amino acid sequence otherwise, that is a “reference microdystrophin protein”), meaning that the DAPC is stabilized or anchored to the sarcolemma, to a greater extent than a reference microdystrophin that does not have the C-terminal domain (but has the same amino acid sequence otherwise as the microdystrophin), as determined by greater levels of one or more DAPC components in the muscle membrane by immunostaining of muscle sections or western blot analysis of muscle tissue lysates or muscle membrane preparations for one of more DAPC components, including al-syntrophin, P-syntrophin, a-dystrobrevin, P-dystroglycan or nNOS in mdx mouse muscle treated with the microdystrophin having the C-terminal domain, as compared to the mdx mouse muscle treated with the reference microdystrophin protein (having the same sequence and dystrophin components except not having the C-terminal domain) (see Sections 6.5 and 6.7 infra).

[0081] In some embodiments, the microdystrophin construct including a C-terminal domain of dystrophin comprises a syntrophin binding site and / or a dystrobrevin binding site in the C-terminal domain. In some embodiments, the C- terminal domain comprising an al-syntrophin binding site is a truncated C-terminal domain. In certain embodiments, the amino acid sequence of the truncated C-terminal domain is SEQ ID NO: 83. In certain embodiments, the truncated C-terminal domain comprises the amino acid sequence MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ (al-syntrophin binding site) (SEQ ID NO: 84). In certain embodiments, the truncated C-terminal domain comprises an al-syntrophin binding site, wherein the binding site has amino acid sequence MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ (SEQ ID NO: 84) but does not have a pi-syntrophin or dystrobrevin binding site.

[0082] The microdystrophin constructs of the present disclosure may further prevent progressive ventricular fibrosis, as measured by the reduction in myocardial macrophage concentrations, the reduction of the expression of adhesion molecules, and / or normalized electrocardiogram (ECG) readouts, for example end systolic volume (left ventricle), end diastolic volume, stroke volume, ejection fraction, heart rate, or cardiac output, following administration of the microdystrophin constructs. End systolic volume and other cardiac readouts can also be measured using MRI (magnetic resonance tomography), cardiac CT (computed tomography) or SPECT (single photon emission computed tomography). Cardiac function improvements following administration of the microdystrophin constructs of the invention may also be tested in a DBA / 2J-mdx mouse model.

[0083] Accordingly, embodiments described herein can further comprise all or a portion of the CT domain comprising the Helix 1 of the coiled-coil motif. For example, the microdystrophin protein comprises from amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT (e g., SEQ ID NO: 1, 79 or 91) or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT (e g., SEQ ID NO: 92). In some embodiments, CT is at least a portion of a C-terminal domain of dystrophin comprising a al-syntrophin binding site and / or a dystrobrevin binding site as illustrated in FIG. 14. In certain embodiments, the CT domain comprises an al-syntrophin binding site and does not have a pi-syntrophin or dystrobrevin binding site, for example it has an amino acid sequence of SEQ ID NO: 83, which function in part to recruit and anchor nNOS to the sarcolemma through al- syntrophin. In some embodiments, the CT comprises the amino acid sequence of SEQIDNO: 16 or 83.

[0084] Microdystrophin embodiments can further comprise linkers (LI, L2, L3, L4, L4.1 and / or L4.2) or portions thereof connected the domains as shown as follows: ABD1-L1-H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR-CT (e.g., SEQ ID NO: 1, 79, or 91), ABD1-L1-H1-L2-R1-R2-L3-R3-H3-L4-R24-H4-CR (e.g., SEQ ID NO: 2), ABD1-L1-H1-L2-R1-R2-L3-R16-L4.1-R17-L4.2-R24-H4-CR (e g., SEQ ID NO: 92), or ABD1-L1-H1-L2-R1-R2-L3-R16-L4.1-R17-L4.2-R24-H4-CR-CT (e.g., SEQ ID NO: 93). LI can be an endogenous linker LI (e.g., SEQ ID NO: 4) that can couple ABDI to HL L2 can be an endogenous linker L2 (e.g., SEQ ID NO: 6) that can couple Hl to RI. L3 can be an endogenous linker L3 (e.g., SEQ ID NO: 9) that can couple R2 to R3 or RI6.

[0085] L4 can also be an endogenous linker that can couple H3 and R24. In some embodiments, L4 is 3 amino acids, e.g. TLE (SEQ ID NO: 12) that precede R24 in the native dystrophin sequence. In other embodiments, L4 can be the 4 amino acids that precede R24 in the native dystrophin sequence (SEQ ID NO: 17) or the 2 amino acids that precede R24 (SEQ ID NO: 18). In other embodiments, there is no linker, L4 or otherwise, in between H3 and R24. On the 5’ end of H3, as mentioned above, no linker is present, but rather R3 is directly coupled to H3, or alternatively H2.

[0086] L4.1 can be an endogenous linker that can couple R16 and R17. In some embodiments, L4.1 is 2 amino acids, e.g. SV (SEQ ID NO: 110) that precede R17 in the native dystrophin sequence. In other embodiments, L4.2 can be an endogenous linker or part of an endogenous linker that can couple RI 7 and R24. In some embodiments, L4.2 is 4 amino acids, e.g. Q that follows R17 and TLE (SEQ ID NO: 12) that precede R24 (SEQ ID NO: 89).

[0087] The above described components of microdystrophin other domains not specifically described can have the amino acid sequences as provided in Table 1 below. The amino acid sequences for the domains provided herein correspond to the dystrophin isoform of UniProtKB-P11532 (DMD HUMAN), which is herein incorporated by reference. Other embodiments can comprise the domains from naturally-occurring functional dystrophin isoforms known in the art, such as UniProtKB-A0A075B6G3 (A0A075B6G3HUMAN), (incorporated by reference herein) wherein, for example, R24 has an R substituted for the Q at amino acid 3 of SEQIDNO: 13. Table 1: Microdystrop bin segment amino acid sequences Structure SEQ ID Sequence ABDI 3 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENL FSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKA LRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQV KNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFT TSWSDGLALNALIHSHRPDLFDWNSWCQQSATQRLEHAF NIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLP LI 4 QQVSIEAIQEVE Hl 5 MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSP KPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPED L2 6 KSFGSSLME RI 7 SEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWK DQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDE ETEVQEQMNLLNSRWECLRVASMEKQSNLHR R2 8 VLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLK RQVQQ HKVL Q E D L EQ E Q VRVN S L T HMVVWD ESSGDHATA ALEEQLKVLGDRWANICRWTEDRWVLLQD L3 9 IL R3 10 LKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLS SLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQ KTEAWLDNFARCWDNLVQKLEKSTAQISQ H3 11 QPDLAPGLTTIGASPTQTVTLVTQPWTKETAISKLEMPS SLMLEVP L4 12 TLE RI 6 86 EISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDL FKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVER VKLQEALSQLDFQWEKVNKMYKDRQGRFDR L4.1 110 SV RI 7 87 EKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKW YLKELQDGIGQRQTWRTLNATGEEIIQQSSKTDASILQE KLGSLNLRWQEVCKQLSDRKKRLEE R16-R17 88 EISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDL FKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVER VKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHY DIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDG IGQRQTWRTLNATGEEIIQQSSKTDASILQEKLGSLNLR WQEVCKQLSDRKKRLEE L4. 1 linker connecting RI6 and RI7 is underlined. L4.2 89 QTLE Structure SEQ ID Sequence R24 13 RLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDH LEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNL STLEDLNTRWKLLQVAVEDRVRQLHE H4 14 AHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQT TCWDHPKMTELYQSLADLNNVRFSAYRTAMKL WW domain is represented by a single underline (UniProtKB-P 11532 aa 3055-3088) Cysteine-rich domain (CR) 15 RRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIIN CLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTG RIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQR RLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANN KPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAK CNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKM HYPMVEYC ZZ domain is represented by a single underline (UniProtKB-P 11532 aa 3307-3354) CR short 90 AKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRV AKGHKMHYPMVEYC C-terminal Domain (CT) 16 T P T T S GE DVRD FAKVL KNKF RT KRY FAKH P RMGYL PVQTV LEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHY ASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLN Q D S P L S Q P R S PAQILIS L E SEERGELERILADLEEENRNL QAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPR Coiled-coil motif Hl is represented by a single underline; motif H2 is represented by a double underline; dystrobrevin-binding side is in italics. Minimal / trunc ated C-terminal Domain (CT1.5) 83 T P T T S GE DVRD FAKVL KNKF RT KRY FAKH P RMGYL PVQTV LEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHY AS RLAEMENSWGS YLNDSISPNESIDDEHLLIQHYCQSLN QDSPLSQPRSPAQILISLES al-syntrophin-binding site is in italics. L4 17 ETLE L4 18 LE H2 19 PSLTQTTVMETVTTVTTREQILVKHAQEELPPPPPQKKRQ ITVD Minimal alpha-syntrophin binding site 84 MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ

[0088] The present disclosure also contemplates variants of these sequences so long as the function of each domain and linker is substantially maintained and / or the therapeutic efficacy of microdystrophin comprising such variants is substantially maintained. Functional activity includes (1) binding to one of, a combination of, or all of actin, P-dystroglycan, al-syntrophin, a-dystrobrevin, and nNOS; (2) improved muscle function in an animal model (for example, in the mdx mouse model described herein) or in human subjects; and / or (3) cardioprotective or improvement in cardiac muscle function in animal models or human patients. In particular, microdystrophin can comprise ABD consisting of SEQ ID NO: 3 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3; Hl consisting of SEQ ID NO: 5 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5; RI consisting of SEQ ID NO: 7 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7; R2 consisting of SEQ ID NO: 8 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8; H2 consisting of SEQ ID NO: 19 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19; H3 consisting of SEQ ID NO: 11 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11; R24 consisting of SEQ ID NO: 13 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13; H4 consisting of SEQ ID NO: 14 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14; CR consisting of SEQ ID NO: 15 or 90 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 or 90; CT consisting of SEQ ID NO: 16 or 83 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 or 83, or CT comprising SEQ ID NO: 84. An alternative embodiment is the same as the foregoing except that the H3 domain is replaced by the H2 domain that consists of SEQ ID NO: 19 or a sequence with at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 19, likewise encoding a microdystrophin that has functional activity. In addition to the foregoing, microdystrophin can comprise linkers in the locations described above that comprise or consist of sequences as follows: LI consisting of SEQ ID NO: 4 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4; L2 consisting of SEQ ID NO: 6 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6; L3 consisting of SEQ ID NO: 9 or an amino acid sequence with at least 50% identity to SEQ ID NO: 9 or a variant with conservative substitutions for both L3 residues; and L4 consisting of SEQ ID NO: 12, 17, or 18 or an amino acid sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 12, 17, or 18.

[0089] In particular embodiments, microdystrophin can comprise ABD consisting of SEQ ID NO: 3 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3; Hl consisting of SEQ ID NO: 5 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5; RI consisting of SEQ ID NO: 7 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7; R2 consisting of SEQ ID NO: 8 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8; R16 consisting of SEQ ID NO: 86 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 86; RI7 consisting of SEQ ID NO: 87 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 87; R24 consisting of SEQ ID NO: 13 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13; H4 consisting of SEQ ID NO: 14 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14; CR consisting of SEQ ID NO: 15 or 90 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 or 90; CT consisting of SEQ ID NO: 16 or 83 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 or 83, or CT comprising SEQ ID NO: 84. In addition to the foregoing, microdystrophin can comprise linkers in the locations described above that comprise or consist of sequences as follows: LI consisting of SEQ ID NO: 4 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4; L2 consisting of SEQ ID NO: 6 or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6; L3 consisting of SEQ ID NO: 9 or an amino acid sequence with at least 50% identity to SEQ ID NO: 9 or a variant with conservative substitutions for both L3 residues; L4.1 consisting of SEQ ID NO: 110 or an amino acid sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 110.; and L4.2 consisting of SEQ ID NO: 89 or an amino acid sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 89.

[0090] Table 2 provides the amino acid sequences of the microdystrophin embodiments in accordance with the present disclosure. It is also contemplated that other embodiments are substituted variant of microdystrophin as defined by SEQ ID NOs: 1, 2, 79, 91, 92, or 93. For example, conservative substitutions can be made to SEQ ID NOs: 1, 2, 79, 91, 92, or 93 and substantially maintain its functional activity. In embodiments, microdystrophin may have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 79, 91, 92, or 93 and maintain functional microdystrophin activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed in Section 5.4, infra. Table 2: Amino acid sequences of RGX-DYS proteins Structure SEQ ID NO: Amino Acid Sequence DYS1, DYS2, and DYS4 1 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFS DLQDGRRL LDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLV NIGSTDIV DGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLS WVRQSTRN YPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSWCQQSA TQRLEHAF NIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQ VSIEAIQE VEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSP KPRFKSYA YTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLD RYQTALEE VLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGYMMDLTAH QGRVGNIL QLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQ SNLHRVLM DLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQ HKVLQEDL EQEQVRVNSLTHMWWDESSGDHATAALEEQLKVLGDRWAN ICRWTEDR WVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKD QNEMLSSL QKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKTEA WLDNFARC WDNLVQKLEKSTAQISQQPDLAPGLTTIGASPTQTVTLVTQP WTKETAI SKLEMPSSLMLEVPTLERLQELQEATDELDLKLRQAEVIKGS WQPVGDLL IDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQL SPYNLSTL EDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQG PWERAISP NKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTA MKLRRLQK Structure SEQ ID NO: Amino Acid Sequence ALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYD RLEQEHNN LVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKA HLEDKYRY LFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNI EPSVRSCF QFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKH QAKCNICK ECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEY CTPTTSGE DVRD FAKVL KNK FRT KRY FAKH P RMGYL PVQTVL E GDNMET P VTLINFWP VDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSI SPNESIDD EHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERI LADLEEEN RNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPR DYS3 2 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFS DLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVL QNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKN IMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLA LNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVE MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKP RFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLME SEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQ FHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEV QEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQ VRVN S L T HMVWVD ESSGDHATAALEEQLKVLGD RW AN ICRW TEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTT GFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLST LKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQQPDLA PGLTTIGASPTQTVTLVTQPWTKETAISKLEMPSSLMLEVP TLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQD HLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLS TLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSV QGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLN NVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQN DQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLL NVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASS TGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCF QFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKH QAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHK MHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMG YLPVQTVLEGDNMET DYS5 79 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFS DLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVL Structure SEQ ID NO: Amino Acid Sequence QNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKN IMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLA LNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVE MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKP RFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLME SEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQ FHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEV QEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQ VRVN S L T HMVVWD E S S GDHAT AAL E E Q L KVL GD RW AN IC RW TEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTT GFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLST LKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQQPDLA PGLTTIGASPTQTVTLVTQPWTKETAISKLEMPSSLMLEVP TLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQD HLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLS TLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSV QGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLN NVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQN DQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLL NVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASS TGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCF QFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKH QAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHK MHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMG YLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHS RIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQ SLNQDSPLSQPRSPAQILISLES DYS6 91 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFS DLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVL QNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKN IMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLA LNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVE MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKP RFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLME SEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQ FHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEV QEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQ VRVN S L T HMVWVD ESSGDHATAALEEQLKVLGD RW AN ICRW TEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTT GFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLST LKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQQPDLA PGLTTIGASPTQTVTLVTQPWTKETAISKLEMPSSLMLEVP TLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQD HLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLS Structure SEQ ID NO: Amino Acid Sequence TLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSV QGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLN NVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQN DQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLL NVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASS TGFCDQRRLGLLLHDSIQIPRQLGEVASFGGAKHQAKCNICK ECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEY CTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVL EGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASR LAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPL SQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRL KQQHEHKGLSPLPSPPEMMPTSPQSPR DYS7 92 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFS DLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVL QNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKN IMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLA LNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVE MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKP RFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLME SEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQ FHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEV QEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQ VRVN S LTHMVWVDE S S GDHATAAL EEQL KVL GDRWAN ICRW TEDRWVLLQDILEISYVPSTYLTEITHVSQALLEVEQLLNAP DLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAAL QSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEK WRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKE LQDGIGQRQTWRTLNATGEEIIQQSSKTDASILQEKLGSLN LRWQEVCKQLSDRKKRLEEQTLERLQELQEATDELDLKLRQA EVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHV NDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQ LHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQ TTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCL DLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQ EHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIIS LCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQ LGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEP QSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKH FNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFA KVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINF WPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLND SISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLE S DSY8 93 MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFS DLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVL QNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKN Structure SEQ ID NO: Amino Acid Sequence IMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLA LNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIE KLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIEAIQEVE MLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKP RFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLME SEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQ FHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEV QEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKELND WLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQ VRVN S LTHMVWVDE S S GDHATAAL EEQL KVL GDRWAN ICRW TEDRWVLLQDILEISYVPSTYLTEITHVSQALLEVEQLLNAP DLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAAL QSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEK WRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKE LQDGIGQRQTWRTLNATGEEIIQQSSKTDASILQEKLGSLN LRWQEVCKQLSDRKKRLEEQTLERLQELQEATDELDLKLRQA EVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHV NDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQ LHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQ TTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCL DLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQ EHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIIS LCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQ LGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEP QSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKH FNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFA KVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMET 5.2.2 Nucleic Acid Compositions encoding Microdystrophin

[0091] Another aspect of the present disclosure are nucleic acids comprising a nucleotide sequence encoding a microdystrophin as described herein. Such nucleic acids comprise nucleotide sequences that encode the microdystrophin that has the domains arranged N-terminal to C-terminal as follows: ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT, ABD1-H1-R1-R2-R3-H3-R24-H4-CR, ABD1-H1-R1-R2-R16-R17-R24-H4-CR-CT, or ABD1-H1-R1-R2-R16-R17-R24-H4-CR. The nucleotide sequence can be any nucleotide sequence that encodes the domains. The nucleotide sequence may be codon optimized and / or depleted of CpG islands for expression in the appropriate context. In particular embodiments, the nucleotide sequences encode a microdystrophin having an amino acid sequence of SEQ ID NO: 1, 2, 79, 91, 92, or 93. The nucleotide sequence can be any sequence that encodes the microdystrophin, including the microdystrophin of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 79, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93, which nucleotide sequence may vary due to the degeneracy of the code. Tables 3 and 4 provide exemplary nucleotide sequences that encode the DMD domains. Table 3 provides the wild type DMD nucleotide sequence for the component and Table 4 provides the nucleotide sequence for the DMD component used in the constructs herein, including sequences that have been codon optimized and / or CpG depleted of CpG islands as follows: Table 3: Dystrophin segment nucleotide sequences________________________ Structure SEQ ID Nucleic Acid Sequence ABDI 22 ATGCTTTGGTGGGAAGAAGTAGAGGACTGTTATGAAAGAGA AGAT GT T CAAAAGAAAACAT T CACAAAAT GGGTAAAT GCAC AATTTTCTAAGTTTGGGAAGCAGCATATTGAGAACCTCTTC AGTGACCTACAGGATGGGAGGCGCCTCCTAGACCTCCTCGA AGGCCTGACAGGGCAAAAACTGCCAAAAGAAAAAGGATCCA CAAGAGTTCATGCCCTGAACAATGTCAACAAGGCACTGCGG GTT T T GCAGAACAATAAT GT T GATT TAGT GAATAT T GGAAG TACTGACATCGTAGATGGAAATCATAAACTGACTCTTGGTT TGATTTGGAATATAATCCTCCACTGGCAGGTCAAAAATGTA ATGAAAAATAT CAT GGGT GGAT T GCAACAAACCAACAGT GA AAAGATTCTCCTGAGCTGGGTCCGACAATCAACTCGTAATT ATCCACAGGTTAATGTAATCAACTTCACCACCAGCTGGTCT GATGGCCTGGCTTTGAATGCTCTCATCCATAGTCATAGGCC AGACCTATTTGACTGGAATAGTGTGGTTTGCCAGCAGTCAG CCACACAACGACTGGAACATGCATTCAACATCGCCAGATAT CAAT TAGGCATAGAGAAAC TACTCGATCCT GAAGAT GT T GA TACCACCTATCCAGATAAGAAGTCCATCTTAATGTACATCA CATCACTCTTCCAAGTTTTGCCT LI 23 CAACAAGTGAGCAT T GAAGCCAT CCAGGAAGT GGAA Hl 24 ATGTTGCCAAGGCCACCTAAAGTGACTAAAGAAGAACATTT T CAGT TACAT CAT CAAAT GCAC TAT T C T CAACAGAT CAC GG TCAGTCTAGCACAGGGATATGAGAGAACTTCTTCCCCTAAG CCTCGATTCAAGAGCTATGCCTACACACAGGCTGCTTATGT CACCACCTCTGACCCTACACGGAGCCCATTTCCTTCACAGC ATTTGGAAGCTCCTGAAGAC L2 25 AAGT CAT TT GGCAGT T CAT T GAT GGAG RI 26 AGTGAAGTAAACCTGGACCGTTATCAAACAGCTTTAGAAGA AGTATTATCGTGGCTTCTTTCTGCTGAGGACACATTGCAAG CACAAGGAGAGAT T T CTAAT GAT GT GGAAGTGGT GAAAGAC CAGT T T CATACT CAT GAGGGGTACAT GAT GGAT T T GACAGC CCATCAGGGCCGGGTTGGTAATATTCTACAATTGGGAAGTA AGCTGATTG GAACAGGAAAAT TAT CAGAAGAT GAAGAAAC T Structure SEQ ID Nucleic Acid Sequence GAAGTACAAGAGCAGATGAATCTCCTAAATTCAAGATGGGA ATGCCTCAGGGTAGCTAGCATGGAAAAACAAAGCAATTTAC ATAGA R2 27 GTTTTAATGGATCTCCAGAATCAGAAACTGAAAGAGTTGAA TGACTGGCTAACAAAAACAGAAGAAAGAACAAGGAAAATGG AGGAAGAGC CT C T T GGAC C T GAT CT T GAAGAC C TAAAAC GC CAAGTACAACAACATAAGGTGCTTCAAGAAGATCTAGAACA AGAACAAGTCAGGGTCAATTCTCTCACTCACATGGTGGTGG TAGTTGATGAATCTAGTGGAGATCACGCAACTGCTGCTTTG GAAGAACAACTTAAGGTATTGGGAGATCGATGGGCAAACAT CTGTAGATGGACAGAAGACCGCTGGGTTCTTTTACAAGAC L3 28 ATCCTT R3 29 CTCAAATGGCAACGTCTTACTGAAGAACAGTGCCTTTTTAG T GCAT GGCT TT CAGAAAAAGAAGAT GCAGT GAACAAGAT T C ACACAACTGGCTTTAAAGATCAAAATGAAATGTTATCAAGT CTTCAAAAACTGGCCGTTTTAAAAGCGGATCTAGAAAAGAA AAAGCAATC CAT GGGCAAACT GTAT T CACT CAAACAAGAT C TTCTTTCAACACTGAAGAATAAGTCAGTGACCCAGAAGACG GAAGCATGGCTGGATAACTTTGCCCGGTGTTGGGATAATTT AGT C CAAAAAC T T GAAAAGAGTACAGCACAGAT T T CACAG RI 6 94 gaaatttcttatgtgccttctacttatttgactgaaatcac tcatgtctcacaagccctattagaagtggaacaacttctca atgctcctgacctctgtgctaaggactttgaagatctcttt aagcaagaggagtctctgaagaatataaaagatagtctaca acaaagctcaggtcggattgacattattcatagcaagaaga cagcagcattgcaaagtgcaacgcctgtggaaagggtgaag ctacaggaagctctctcccagcttgatttccaatgggaaaa agttaacaaaatgtacaaggaccgacaagggcgatttgaca ga L4.1 107 TCTGTT RI 7 95 gagaaatggcggcgttttcattatgatataaagatatttaa tcagtggctaacagaagctgaacagtttctcagaaagacac aaattcctgagaattgggaacatgctaaatacaaatggtat cttaaggaactccaggatggcattgggcagcggcaaactgt tgtcagaacattgaatgcaactggggaagaaataattcagc aatcctcaaaaacagatgccagtattctacaggaaaaattg ggaagcctgaatctgcggtggcaggaggtctgcaaacagct gtcagacagaaaaaagaggctagaa R16-R17 96 gaaatttcttatgtgccttctacttatttgactgaaatcac tcatgtctcacaagccctattagaagtggaacaacttctca atgctcctgacctctgtgctaaggactttgaagatctcttt aagcaagaggagtctctgaagaatataaaagatagtctaca acaaagctcaggtcggattgacattattcatagcaagaaga cagcagcattgcaaagtgcaacgcctgtggaaagggtgaag ctacaggaagctctctcccagcttgatttccaatgggaaaa agttaacaaaatgtacaaggaccgacaagggcgatttgaca gaTCTGTTgagaaatggcggcgttttcattatgatataaag atatttaatcagtggctaacagaagctgaacagtttctcag Structure SEQ ID Nucleic Acid Sequence aaagacacaaattcctgagaattgggaacatgctaaataca aatggtatcttaaggaactccaggatggcattgggcagcgg caaactgttgtcagaacattgaatgcaactggggaagaaat aattcagcaatcctcaaaaacagatgccagtattctacagg aaaaattgggaagcctgaatctgcggtggcaggaggtctgc aaacagctgtcagacagaaaaaagaggctagaa L4.2 108 CAAACCCTTGAA H3 30 CAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCTC TCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTA CTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCC TTGATGTTGGAGGTACCT L4 31 ACCCTTGAA R24 32 AGACTCCAACTTCAAGAGGCCACGGATGAGCTGGACCTCAA GCTGCGCCAAGCTGAGGTGATCAAGGGATCCTGGCAGCCCG TGGGCGATCTCCTCATTGACTCTCTCCAAGATCACCTCGAG AAAGTCAAGGCACTTCGAGGAGAAATTGCGCCTCTGAAAGA GAACGTGAGCCACGTCAATGACCTTGCTCGCCAGCTTACCA CTTTGGGCATTCAGCTCTCACCGTATAACCTCAGCACTCTG GAAGACCTGAACACCAGATGGAAGCTTCTGCAGGTGGCCGT CGAGGACCGAGTCAGGCAGCTGCATGAA H4 33 GCCCACAGGGACTTTGGTCCAGCATCTCAGCACTTTCTTTC CACGTCTGTCCAGGGTCCCTGGGAGAGAGCCATCTCGCCAA ACAAAGTGCCCTACTATATCAACCACGAGACTCAAACAACT TGCTGGGACCATCCCAAAATGACAGAGCTCTACCAGTCTTT AGCTGACCTGAATAATGTCAGATTCTCAGCTTATAGGACTG CCATGAAACTC Cysteine-rich domain (CR) 34 CGAAGACTGCAGAAGGCCCTTTGCTTGGATCTCTTGAGCCT GTCAGCTGCATGTGATGCCTTGGACCAGCACAACCTCAAGC AAAATGACCAGCCCATGGATATCCTGCAGATTATTAATTGT TTGACCACTATTTATGACCGCCTGGAGCAAGAGCACAACAA TTTGGTCAACGTCCCTCTCTGCGTGGATATGTGTCTGAACT GGCTGCTGAATGTTTATGATACGGGACGAACAGGGAGGATC CGTGTCCTGTCTTTTAAAACTGGCATCATTTCCCTGTGTAA AGCACATTTGGAAGACAAGTACAGATACCTTTTCAAGCAAG TGGCAAGTTCAACAGGATTTTGTGACCAGCGCAGGCTGGGC CTCCTTCTGCATGATTCTATCCAAATTCCAAGACAGTTGGG TGAAGTTGCATCCTTTGGGGGCAGTAACATTGAGCCAAGTG TCCGGAGCTGCTTCCAATTTGCTAATAATAAGCCAGAGATC GAAGCGGCCCTCTTCCTAGACTGGATGAGACTGGAACCCCA GTCCATGGTGTGGCTGCCCGTCCTGCACAGAGTGGCTGCTG CAGAAACTGCCAAGCATCAGGCCAAATGTAACATCTGCAAA GAGT GT CCAAT CAT T GGAT T CAGGTACAGGAGT CTAAAGCA CTTTAATTATGACATCTGCCAAAGCTGCTTTTTTTCTGGTC GAGTTGCAAAAGGCCATAAAATGCACTATCCCATGGTGGAA TATTGC CR short 109 gccaagcatcaggccaaatgtaacatctgcaaagagtgtcc aatcattggattcaggtacaggagtctaaagcactttaatt Structure SEQ ID Nucleic Acid Sequence atgacatctgccaaagctgctttttttctggtcgagttgca aaaggccataaaatgcactatcccatggtggaatattgc C-terminal (CT) Domain 35 ACTCCGACTACATCAGGAGAAGATGTTCGAGACTTTGCCAA GGTACTAAAAAACAAATTTCGAACCAAAAGGTATTTTGCGA AGCATCCCCGAATGGGCTACCTGCCAGTGCAGACTGTCTTA GAGGGGGACAACATGGAAACTCCCGTTACTCTGATCAACTT CTGGCCAGTAGATTCTGCGCCTGCCTCGTCCCCTCAGCTTT GAGAC GAT GATAC T CAT T CAC GCAT T GAACAT TATGCTAGC AGGCTAGCAGAAATGGAAAACAGCAATGGATCTTATCTAAA T GATAGCAT CTCTCCTAATGAGAGCATAGATGAT GAACAT T TGTTAATCCAGCATTACTGCCAAAGTTTGAACCAGGACTCC CCCCTGAGCCAGCCTCGTAGTCCTGCCCAGATCTTGATTTC CTTAGAGAGTGAGGAAAGAGGGGAGCTAGAGAGAATCCTAG CAGAT CT TGAGGAAGAAAACAGGAAT CT GCAAGCAGAATAT GACCGTCTAAAGCAGCAGCACGAACATAAAGGCCTGTCCCC ACTGCCGTCCCCTCCTGAAATGATGCCCACCTCTCCCCAGA GTCCCCGG L4 36 GAGACCCTTGAA L4 37 CTTGAA H2 38 C CAT CAC TAACACAGACAAC T GTAAT GGAAACAGTAAC TAG GGTGACCACAAGGGAACAGATCCTGGTAAAGCATGCTCAAG AGGAAC T T C CAC CAC CAC C T C C C CAAAAGAAGAGGCAGAT T ACTGTGGAT Table 4: RGX-DYS segment nucleotide sequences Structure SEQ ID Nucleic Acid Sequence ABD 57 ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGG AAGAT GT GCAGAAGAAAACCT T CACCAAAT GGGT CAAT GC CCAGT T CAGCAAGT T T GGCAAGCAGCACAT TGAGAACCT G TTCAGTGACCTGCAGGATGGCAGAAGGCTGCTGGATCTGC TGGAAGGCCTGACAGGCCAGAAGCTGCCTAAAGAGAAGGG CAGCACAAGAGT GCAT GCCCT GAACAAT GT GAACAAGGCC CTGAGAGTGCTGCAGAACAACAATGTGGACCTGGTCAATA TTGGCAGCACAGACATTGTGGATGGCAACCACAAGCTGAC CCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAAGTG AAGAAT GTGAT GAAGAACAT CAT GGCT GGCCT GCAGCAGA CCAACTCTGAGAAGATCCTGCTGAGCTGGGTCAGACAGAG CAC CAGAAACTAC C C T CAAGT GAAT GT GAT CAAC T T CAC C ACCTCTTGGAGTGATGGACTGGCCCTGAATGCCCTGATCC ACAGCCACAGACCTGACCTGTTTGACTGGAACTCTGTTGT GTGCCAGCAGTCTGCCACACAGAGACTGGAACATGCCTTC AACATTGCCAGATACCAGCTGGGAATTGAGAAACTGCTGG ACC C T GAGGAT GT GGACAC CAC C TAT C C T GACAAGAAAT C CATCCTCAT GTACATCACCAGCCTGTTCCAGGTGCTGCCC LI 58 CAGCAAGTGTCCATTGAGGCCATTCAAGAGGTTGAG Structure SEQ ID Nucleic Acid Sequence Hl 59 ATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAACACT TCCAGCTGCACCACCAGATGCACTACTCTCAGCAGATCAC AGTGTCTCTGGCCCAGGGATATGAGAGAACAAGCAGCCCC AAGCCTAGGTTCAAGAGCTATGCCTACACACAGGCTGCCT ATGTGACCACATCTGACCC CACAAGAAGC C CAT T T C CAAG CCAGCATCTGGAAGCCCCTGAGGAC L2 60 AAGAGCTTTGGCAGCAGCCTGATGGAA RI 61 TCTGAAGTGAACCTGGATAGATACCAGACAGCCCTGGAAG AAGTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCA GGCTCAGGGTGAAATCAGCAATGATGTGGAAGTGGTCAAG GACCAGTTTCACACCCATGAGGGCTACATGATGGACCTGA CAGCCCACCAGGGCAGAGTGGGAAATATCCTGCAGCTGGG CTCCAAGCTGATTGGCACAGGCAAGCTGTCTGAGGATGAA GAGACAGAGGT GCAAGAGCAGAT GAACCT GCT GAACAGCA GATGGGAGTGTCTGAGAGTGGCCAGCATGGAAAAGCAGAG CAACCTGCACAGA R2 62 GTGCTCATGGACCTGCAGAATCAGAAACTGAAAGAACTGA ATGACTGGCTGACCAAGACAGAAGAAAGGACTAGGAAGAT GGAAGAGGAACCTCTGGGACCAGACCTGGAAGATCTGAAA AGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTTG AGCAAGAGCAAGTCAGAGTGAACAGCCTGACACACATGGT GGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGCT GCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGGG CCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGCT CCAGGAC L3 63 ATTCTG R3 64 C T GAAGT GG CAGAGAC T GACAGAGGAACAGT GC C T GT T T T CTGCCTGGCTCTCTGAGAAAGAGGATGCTGTCAACAAGAT CCATACCACAGGCT T CAAGGAT CAGAAT GAGAT GCT CAGC TCCCTGCAGAAACTGGCTGTGCTGAAGGCTGACCTGGAAA AGAAAAAGCAGTCCATGGGCAAGCTCTACAGCCTGAAGCA GGACCTGCTGTCTACCCTGAAGAACAAGTCTGTGACCCAG AAAACTGAGGCCTGGCTGGACAACTTTGCTAGATGCTGGG ACAACCTGGTGCAGAAGCTGGAAAAGTCTACAGCCCAGAT CAGCCAG H3 65 CAACCTGATCTTGCCCCTGGCCTGACCACAATTGGAGCCT CTCCAACACAGACTGTGACCCTGGTTACCCAGCCAGTGGT CACCAAAGAGACAGCCATCAGCAAACTGGAAATGCCCAGC TCTCTGATGCTGGAAGTCCCC L4 66 ACACTGGAA RI 6 97 GAGAT CAGC TAT GT GCCCAGCACCTACCT GACAGAGAT CA CCCATGTGTCTCAGGCCCTGCTGGAAGTGGAACAGCTGCT GAATGCCCCTGACCTGTGTGCCAAGGACTTTGAGGACCTG TTCAAGCAAGAGGAAAGCCTGAAGAACATCAAGGACAGCC TGCAGCAGTCCTC T GGCAGAAT T GACAT CAT C CACAGCAA GAAAACAGCTGCCCTGCAGTCTGCCACACCTGTGGAAAGA GTGAAGCTGCAAGAGGCCCTGAGCCAGCTGGACTTCCAGT Structure SEQ ID Nucleic Acid Sequence GGGAGAAAGTGAACAAGATGTACAAGGACAGGCAGGGCAG ATTTGATAGA L4.1 125 AGTGTG RI 7 98 GAAAAGTGGAGAAGGTTCCACTATGAGATCAAGATCTTCA ACCAGTGGCTGACAGAGGCTGAGCAGTTCCTGAGAAAGAC ACAGATCCCTGAGAACTGGGAGCATGCCAAGTACAAGTGG TATCTGAAAGAACTGCAGGATGGCATTGGCCAGAGACAGA CAGTTGTCAGAACCCTGAATGCCACAGGGGAAGAGATCAT CCAGCAGAGCAGCAAGACAGATGCCAGCATCCTGCAAGAG AAGCTGGGCAGCCTGAACCTGAGATGGCAAGAAGTGTGCA AGCAGCTGTCTGACAGAAAGAAGAGGCTGGAAGAA R16-R17 99 GAGAT CAGC TAT GT GCCCAGCACCTACCT GACAGAGAT CA CCCATGTGTCTCAGGCCCTGCTGGAAGTGGAACAGCTGCT GAATGCCCCTGACCTGTGTGCCAAGGACTTTGAGGACCTG TTCAAGCAAGAGGAAAGCCTGAAGAACATCAAGGACAGCC T GCAGCAGT CCT CT GGCAGAAT T GACAT CATCCACAGCAA GAAAACAGCTGCCCTGCAGTCTGCCACACCTGTGGAAAGA GTGAAGCTGCAAGAGGCCCTGAGCCAGCTGGACTTCCAGT GGGAGAAAGTGAACAAGATGTACAAGGACAGGCAGGGCAG ATTTGATAGAAGTGTGGAAAAGTGGAGAAGGTTCCACTAT GACAT CAAGAT CT T CAACCAGT GGCT GACAGAGGCT GAGC AGTTCCTGAGAAAGACACAGATCCCTGAGAACTGGGAGCA TGCCAAGTACAAGTGGTATCTGAAAGAACTGCAGGATGGC ATTGGCCAGAGACAGACAGTTGTCAGAACCCTGAATGCCA CAGGGGAAGAGAT CAT CCAGCAGAGCAGCAAGACAGAT GC CAGCATCCTGCAAGAGAAGCTGGGCAGCCTGAACCTGAGA T GGCAAGAAGT GT GCAAGCAGCT GT CT GACAGAAAGAAGA GGCTGGAAGAA L4.2 126 CAGACACTGGAA R24 67 AGGCTGCAAGAACTTCAAGAGGCCACAGATGAGCTGGACC TGAAGCTGAGACAGGCTGAAGTGATCAAAGGCAGCTGGCA GCCAGTTGGGGACCTGCTCATTGATAGCCTGCAGGACCAT CTGGAAAAAGTGAAAGCCCTGAGGGGAGAGATTGCCCCTC TGAAAGAAAATGTGTCCCATGTGAATGACCTGGCCAGACA GCTGACCACACTGGGAATCCAGCTGAGCCCCTACAACCTG AGCACCCTTGAGGACCTGAACACCAGGTGGAAGCTCCTCC AGGTGGCAGTGGAAGATAGAGTCAGGCAGCTGCATGAG H4 68 GCCCACAGAGATTTTGGACCAGCCAGCCAGCACTTTCTGT CTACCTCTGTGCAAGGCCCCTGGGAGAGAGCTATCTCTCC TAACAAGGTGCCCTACTACATCAACCATGAGACACAGACC ACCTGTTGGGATCACCCCAAGATGACAGAGCTGTACCAGA GTCTGGCAGACCTCAACAATGTCAGATTCAGTGCCTACAG GACTGCCATGAAGCTC Cysteine-rich domain (CR) 69 AGAAGGCTCCAGAAAGCTCTGTGCCTGGACCTGCTTTCCC TGAGTGCAGCTTGTGATGCCCTGGACCAGCACAATCTGAA GCAGAATGACCAGCCTATGGACATCCTCCAGATCATCAAC TGCCTCACCACCATCTATGATAGGCTGGAACAAGAGCACA ACAATCTGGTCAATGTGCCCCTGTGTGTGGACATGTGCCT Structure SEQ ID Nucleic Acid Sequence GAATT GGCT GCT GAAT GT GTAT GACACAGGCAGAACAGGC AGGATCAGAGTCCTGTCCTTCAAGACAGGCATCATCTCCC TGTGCAAAGCCCACTTGGAGGACAAGTACAGATACCTGTT CAAGCAAGTGGCCTCCAGCACAGGCTTTTGTGACCAGAGA AGGCTGGGCCTGCTCCTGCATGACAGCATTCAGATCCCTA GACAGCTGGGAGAAGTGGCTTCCTTTGGAGGCAGCAATAT TGAGCCATCAGTCAGGTCCTGTTTTCAGTTTGCCAACAAC AAGCCTGAGATTGAGGCTGCCCTGTTCCTGGACTGGATGA GACTTGAGCCTCAGAGCATGGTCTGGCTGCCTGTGCTTCA TAGAGTGGCTGCTGCTGAGACTGCCAAGCACCAGGCCAAG TGCAACATCTGCAAAGAGTGCCCCATCATTGGCTTCAGAT ACAGATCCCTGAAGCACTTCAACTATGATATCTGCCAGAG CTGCTTCTTTAGTGGCAGGGTTGCCAAGGGCCACAAAATG CACTACCCCATGGTGGAATACTGC CR short (DYS6) 100 GCCAAGCACCAGGCCAAGTGCAACATCTGCAAAGAGTGCC CCATCATTGGCTT CAGATACAGAT GGCT GAAGCAC T T CAA CTATGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGGGTT GCCAAGGGCCACAAAATGCACTACCCCATGGTGGAATACT GC C-terminal (CT) Domain (DYS1, DYS2, DYS4, DYS6) 70 ACCCCAACAACCTCTGGGGAAGATGTTAGAGACTTTGCCA AGGT GC T GAAAAACAAGT T CAGGAC CAAGAGATAC T T T GC TAAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTG CTTGAGGGTGACAACATGGAAACCCCTGTGACACTGATCA ATTTCTGGCCAGTGGACTCTGCCCCTGCCTCAAGTCCACA GCTGTCCCATGATGACACCCACAGCAGAATTGAGCACTAT GCCTCCAGACTGGCAGAGATGGAAAACAGCAATGGCAGCT ACCT GAATGATAGCAT CAGCCCCAAT GAGAGCAT T GAT GA TGAGCATCTGCTGATCCAGCACTACTGTCAGTCCCTGAAC CAGGACTCTCCACTGAGCCAGCCTAGAAGCCCTGCTCAGA TCCTGATCAGCCTTGAGTCTGAGGAAAGGGGAGAGCTGGA AAGAATCCTGGCAGATCTTGAGGAAGAGAACAGAAACCTG CAGGCAGAGTAT GACAGGCT CAAACAGCAGCAT GAGCACA AGGGACTGAGCCCTCTGCCTTCTCCTCCTGAAATGATGCC CACCTCTCCACAGTCTCCAAGGTGATGA(stop codons underlined) Minimal C-terminal (CT1.5) Domain (DYS5, DYS7) 80 ACCCCAACAACCTCTGGGGAAGATGTTAGAGACTTTGCCA AGGT GC T GAAAAACAAGT T CAGGAC CAAGAGATAC T T T GC TAAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTG CTTGAGGGTGACAACATGGAAACCCCTGTGACACTGATCA ATTTCTGGCCAGTGGACTCTGCCCCTGCCTCAAGTCCACA GCTGTCCCATGATGACACCCACAGCAGAATTGAGCACTAT GCCTCCAGACTGGCAGAGATGGAAAACAGCAATGGCAGCT ACCT GAATGATAGCAT CAGCCCCAAT GAGAGCAT T GAT GA TGAGCATCTGCTGATCCAGCACTACTGTCAGTCCCTGAAC CAGGACTCTCCACTGAGCCAGCCTAGAAGCCCTGCTCAGA TCCTGATCAGCCTTGAGTCTTGATGA (stop codons underlined) L4 71 GAAACACTGGAA or GAGACACTGGAA Structure SEQ ID Nucleic Acid Sequence L4 72 CTGGAA

[0092] In some embodiments, such compositions comprise a nucleic acid sequence encoding ABDI that consists of SEQ ID NO: 22 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22; a nucleic acid sequence encoding Hl that consists of SEQ ID NO: 24 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 24; a nucleic acid sequence encoding RI that consists of SEQ ID NO: 26 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26; a nucleic acid sequence encoding R2 that consists of SEQ ID NO: 27 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27; a nucleic acid sequence encoding R3 that consists of SEQ ID NO: 29 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 29; a nucleic acid sequence encoding H3 that consists of SEQ ID NO: 30 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 30; a nucleic acid sequence encoding R24 that consists of SEQ ID NO: 32 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32; a nucleic acid sequence encoding H4 that consists of SEQ ID NO: 33 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33; a nucleic acid sequence encoding CR that consists of SEQ ID NO: 34 or 109 or or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34 or 109; and / or a nucleic acid sequence encoding CT that consists of SEQ ID NO: 35 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35, encoding a microdystrophin that has functional activity. An alternative embodiment is the same as the foregoing except that the H3 nucleic acid sequence is replaced by a nucleic acid encoding H2 that consists of SEQ ID NO: 38 or a sequence with at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 38, likewise encoding a microdystrophin that has functional activity.

[0093] In some embodiments, such compositions comprise a nucleic acid sequence encoding ABDI that consists of SEQ ID NO: 22 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22 and encodes for the ABDI domain of SEQ ID NO: 3; a nucleic acid sequence encoding Hl that consists of SEQ ID NO: 24 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 24 and encodes for the Hl domain of SEQ ID NO: 5; a nucleic acid sequence encoding RI that consists of SEQ ID NO: 26 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26 and encodes for the RI domain of SEQ ID NO: 7; a nucleic acid sequence encoding R2 that consists of SEQ ID NO: 27 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27 and encodes for the R2 domain of SEQ ID NO: 8; a nucleic acid sequence encoding R3 that consists of SEQ ID NO: 29 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 29 and encodes for the R3 domain of SEQ ID NO: 10; a nucleic acid sequence encoding H3 that consists of SEQ ID NO: 30 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 30 and encodes for the H3 domain of SEQ ID NO: 11; a nucleic acid sequence encoding R24 that consists of SEQ ID NO: 32 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32 and encodes for the R24 domain of SEQ ID NO: 13; a nucleic acid sequence encoding H4 that consists of SEQ ID NO: 33 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33 and encodes for the H4 domain of SEQ ID NO: 14; a nucleic acid sequence encoding CR that consists of SEQ ID NO: 34 or 109 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34 or 109 and encodes for the CR domain of SEQ ID NO: 15 or 90; and / or a nucleic acid sequence encoding CT that consists of SEQ ID NO: 35 or 80 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35 or 80 and encodes for the CT domain of SEQ ID NO: 16 or 83. An alternative embodiment is the same as the foregoing except that the H3 nucleic acid sequence is replaced by a nucleic acid encoding H2 that consists of SEQ ID NO: 38 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 38 and encodes the H2 domain of SEQ ID NO: 19.

[0094] In addition to the foregoing, the nucleic acid compositions can optionally comprise nucleotide sequences encoding linkers in the locations described above that comprise or consist of sequences as follows: a nucleic acid sequence encoding LI consisting of SEQ ID NO: 23 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23 (e.g. encoding the LI domain of SEQ ID NO: 4); a nucleic acid sequence encoding L2 consisting of SEQ ID NO: 25 or sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 (e.g. encoding the L2 domain of SEQ ID NO: 6); a nucleic acid sequence encoding L3 consisting of SEQ ID NO: 28 or a sequence with at least 50% identity to SEQ ID NO: 28, encoding the L3 domain of SEQ ID NO: 9 or a variant with conservative substitutions for both L3 residues; and a nucleic acid sequence encoding L4 consisting of SEQ ID NO: 31, 36, or 37 or a sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 31, 36, or 37 (e.g. encoding the L4 domain of SEQ ID NO: 12, 17, or 18 or a variant with conservative substitutions for any of the L4 residues).

[0095] In some embodiments, such compositions comprise a nucleic acid sequence encoding ABDI that consists of SEQ ID NO: 22 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22; a nucleic acid sequence encoding Hl that consists of SEQ ID NO: 24 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 24; a nucleic acid sequence encoding RI that consists of SEQ ID NO: 26 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26; a nucleic acid sequence encoding R2 that consists of SEQ ID NO: 27 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27; a nucleic acid sequence encoding R16 that consists of SEQ ID NO: 94 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 94; a nucleic acid sequence encoding RI7 that consists of SEQ ID NO: 95 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 95; a nucleic acid sequence encoding R24 that consists of SEQ ID NO: 32 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32; a nucleic acid sequence encoding H4 that consists of SEQ ID NO: 33 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33; a nucleic acid sequence encoding CR that consists of SEQ ID NO: 34 or 109 or or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34 or 109; and / or a nucleic acid sequence encoding CT that consists of SEQ ID NO: 35 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35, encoding a microdystrophin that has functional activity. An alternative embodiment is the same as the foregoing except that the H3 nucleic acid sequence is replaced by a nucleic acid encoding H2 that consists of SEQ ID NO: 38 or a sequence with at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 38, likewise encoding a microdystrophin that has functional activity.

[0096] In some embodiments, such compositions comprise a nucleic acid sequence encoding ABDI that consists of SEQ ID NO: 22 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 22 and encodes for the ABDI domain of SEQ ID NO: 3; a nucleic acid sequence encoding Hl that consists of SEQ ID NO: 24 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 24 and encodes for the Hl domain of SEQ ID NO: 5; a nucleic acid sequence encoding RI that consists of SEQ ID NO: 26 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 26 and encodes for the RI domain of SEQ ID NO: 7; a nucleic acid sequence encoding R2 that consists of SEQ ID NO: 27 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 27 and encodes for the R2 domain of SEQ ID NO: 8; a nucleic acid sequence encoding RI6 that consists of SEQ ID NO: 94 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 94 and encodes for the R16 domain of SEQ ID NO: 86; a nucleic acid sequence encoding RI7 that consists of SEQ ID NO: 95 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 95 and encodes for the RI7 domain of SEQ ID NO: 87; a nucleic acid sequence encoding R24 that consists of SEQ ID NO: 32 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 32 and encodes for the R24 domain of SEQ ID NO: 13; a nucleic acid sequence encoding H4 that consists of SEQ ID NO: 33 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 33 and encodes for the H4 domain of SEQ ID NO: 14; a nucleic acid sequence encoding CR that consists of SEQ ID NO: 34 or 109 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 34 or 109 and encodes for the CR domain of SEQ ID NO: 15 or 90; and / or a nucleic acid sequence encoding CT that consists of SEQ ID NO: 35 or 80 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 35 or 80 and encodes for the CT domain of SEQ ID NO: 16 or 83. An alternative embodiment is the same as the foregoing except that the H3 nucleic acid sequence is replaced by a nucleic acid encoding H2 that consists of SEQ ID NO: 38 or a sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity to SEQ ID NO: 38 and encodes the H2 domain of SEQ ID NO: 19.

[0097] In addition to the foregoing, the nucleic acid compositions can optionally comprise nucleotide sequences encoding linkers in the locations described above that comprise or consist of sequences as follows: a nucleic acid sequence encoding LI consisting of SEQ ID NO: 23 or a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23 (e.g. encoding the LI domain of SEQ ID NO: 4); a nucleic acid sequence encoding L2 consisting of SEQ ID NO: 25 or sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 (e.g. encoding the L2 domain of SEQ ID NO: 6); a nucleic acid sequence encoding L3 consisting of SEQ ID NO: 28 or a sequence with at least 50% identity to SEQ ID NO: 28, encoding the L3 domain of SEQ ID NO: 9 or a variant with conservative substitutions for both L3 residues; a nucleic acid sequence encoding L4.1 consisting of SEQ ID NO: 125 or a sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 125 (e.g. encoding the L4.1 domain of SEQ ID NO: 110 or a variant with conservative substitutions for any of the L4.1 residues); and a nucleic acid sequence encoding L4.2 consisting of SEQ ID NO: 126 or a sequence with at least 50%, at least 75% sequence identity to SEQ ID NO: 126 (e.g. encoding the L4.2 domain of SEQ ID NO: 89 or a variant with conservative substitutions for any of the L4.2 residues).

[0098] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding the microdystrophin having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 79, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93. In embodiments, the nucleic acid comprises a nucleotide sequence which is SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 81, SEQ ID NO: 101, SEQ ID NO: 102, or SEQ ID NO: 103 (encoding the microdystrophins of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 79, SEQ ID NO: 91, SEQ ID NO: 92, and SEQ ID NO: 93, respectively). In various embodiments, the nucleotide sequence encoding a microdystrophin may have at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 20, 21, 83, 101, 102, or 103 (Table 5) or the reverse complement thereof and encode a therapeutically effective microdystrophin. Table 5: RGX-DYS Construct nucleotide sequences Structure SEQID Nucleic Acid Sequence DYS1, DYS2, and DYS4 20 ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAA GATGTGCA GAAGAAAACCTTCACCAAATGGGTCAATGCCCAGTTCAGCAA GTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGGCA GAAGGCTG CTGGATCTGCTGGAAGGCCTGACAGGCCAGAAGCTGCCTAAA GAGAAGGG CAGCACAAGAGTGCATGCCCTGAACAATGTGAACAAGGCCCT GAGAGTGC T G CAGAACAACAAT GT GGAC C T GGT CAATAT T GGCAGCACAG ACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACATC ATCCTGCA CTGGCAAGTGAAGAATGTGATGAAGAACATCATGGCTGGCCT GCAGCAGA CCAACTCTGAGAAGATCCTGCTGAGCTGGGTCAGACAGAGCA CCAGAAAC TACCCTCAAGTGAATGTGATCAACTTCACCACCTCTTGGAGT GATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTT TGACTGGA ACTCTGTTGTGTGCCAGCAGTCTGCCACACAGAGACTGGAAC ATGCCTTC AACATTGCCAGATACCAGCTGGGAATTGAGAAACTGCTGGAC CCTGAGGA TGTGGACACCACCTATCCTGACAAGAAATCCATCCTCATGTA CATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGCCA TTCAAGAG GTTGAGATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAA CACTTCCA GCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTGTC TCTGGCCC Structure SEQID Nucleic Acid Sequence AGGGATATGAGAGAACAAGCAGCCCCAAGCCTAGGTTCAAGA GCTATGCC TACACACAGGC T GC C TAT GT GAG GAGAT C T GAG C C CACAAGA AGCCCATT TCCAAGCCAGCATCTGGAAGCCCCTGAGGACAAGAGCTTTGG CAGCAGCC TGATGGAATCTGAAGTGAACCTGGATAGATACCAGACAGCCC TGGAAGAA GTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCT CAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCACAC CCATGAGG GCTACATGATGGACCTGACAGCCCACCAGGGCAGAGTGGGAA ATATCCTG CAGCTGGGCTCCAAGCTGATTGGCACAGGCAAGCTGTCTGAG GATGAAGA GACAGAGGTGCAAGAGCAGATGAACCTGCTGAACAGCAGATG GGAGTGTC TGAGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAGAG TGCTCATG GACCTGCAGAATCAGAAACTGAAAGAACTGAATGACTGGCTG ACCAAGAC AGAAGAAAGGAC TAGGAAGAT GGAAGAGGAAC CTCTGGGACC AGACCTGG AAGATCTGAAAAGACAGGTGCAGCAGCATAAGGTGCTGCAAG AGGACCTT GAGCAAGAGCAAGT CAGAGT GAACAGCCT GACACACAT GGT G GTGGTTGT GGATGAGTCCTCTGGGGATCATGCCACAGCTGCTCTGGAAGA ACAGCTGA AGGTGCTGGGAGACAGATGGGCCAACATCTGTAGGTGGACAG AGGATAGA TGGGTGCTGCTCCAGGACATTCTGCTGAAGTGGCAGAGACTG ACAGAGGA ACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGATGC TGTCAACA AGAT CCATACCACAGGCT T CAAGGAT CAGAAT GAGATGCT CA GCTCCCTG CAGAAACTGGCTGTGCTGAAGGCTGACCTGGAAAAGAAAAAG CAGTCCAT GGGCAAGCTCTACAGCCTGAAGCAGGACCTGCTGTCTACCCT GAAGAACA AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTTTG CTAGATGC TGGGACAACCTGGTGCAGAAGCTGGAAAAGTCTACAGCCCAG ATCAGCCA GCAACCTGATCTTGCCCCTGGCCTGACCACAATTGGAGCCTC TCCAACAC AGACTGTGACCCTGGTTACCCAGCCAGTGGTCACCAAAGAGA CAGCCATC Structure SEQID Nucleic Acid Sequence AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCCCC ACACTGGA AAGGCTGCAAGAACTTCAAGAGGCCACAGATGAGCTGGACCT GAAGCTGA GACAGGCTGAAGTGATCAAAGGCAGCTGGCAGCCAGTTGGGG ACCTGCTC ATTGATAGCCTGCAGGACCATCTGGAAAAAGTGAAAGCCCTG AGGGGAGA GATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGACCT GGCCAGAC AGCTGACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGA GCACCCTT GAGGACCTGAACACCAGGTGGAAGCTCCTCCAGGTGGCAGTG GAAGATAG AGTCAGGCAGCTGCATGAGGCCCACAGAGATTTTGGACCAGC CAGCCAGC ACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGCTA TCTCTCCT AACAAGGTGCCCTACTACATCAACCATGAGACACAGACCACC TGTTGGGA TCACCCCAAGATGACAGAGCTGTACCAGAGTCTGGCAGACCT CAACAATG TCAGATTCAGTGCCTACAGGACTGCCATGAAGCTCAGAAGGC TCCAGAAA GCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTGAT GCCCTGGA CCAGCACAATCTGAAGCAGAATGACCAGCCTATGGACATCCT CCAGATCA TCAACTGCCTCACCACCATCTATGATAGGCTGGAACAAGAGC ACAACAAT CTGGTCAATGTGCCCCTGTGTGTGGACATGTGCCTGAATTGG CTGCTGAA TGTGTATGACACAGGCAGAACAGGCAGGATCAGAGTCCTGTC CTTCAAGA CAGGCATCATCTCCCTGTGCAAAGCCCACTTGGAGGACAAGT ACAGATAC CTGTTCAAGCAAGTGGCCTCCAGCACAGGCTTTTGTGACCAG AGAAGGCT GGGCCTGCTCCTGCATGACAGCATTCAGATCCCTAGACAGCT GGGAGAAG TGGCTTCCTTTGGAGGCAGCAATATTGAGCCATCAGTCAGGT CCTGTTTT CAGTTTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTC CTGGACTG GATGAGACTTGAGCCTCAGAGCATGGTCTGGCTGCCTGTGCT TCATAGAG TGGCTGCTGCTGAGACTGCCAAGCACCAGGCCAAGTGCAACA TCTGCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGCAC TTCAACTA Structure SEQID Nucleic Acid Sequence TGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGGGTTGCCAA GGGCCACA AAAT GCAC TAC C C CAT GGT GGAATAC T GCAC C C CAACAAC C T CTGGGGAA GATGTTAGAGACTTTGCCAAGGTGCTGAAAAACAAGTTCAGG ACCAAGAG ATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTCCA GACAGTGC TTGAGGGTGACAACATGGAAACCCCTGTGACACTGATCAATT TCTGGCCA GTGGACTCTGCCCCTGCCTCAAGTCCACAGCTGTCCCATGAT GACACCCA CAGCAGAATTGAGCACTATGCCTCCAGACTGGCAGAGATGGA AAACAGCA ATGGCAGCTACCTGAATGATAGCATCAGCCCCAATGAGAGCA TTGATGAT GAGCATCTGCTGATCCAGCACTACTGTCAGTCCCTGAACCAG GACTCTCC ACTGAGCCAGCCTAGAAGCCCTGCTCAGATCCTGATCAGCCT TGAGTCTG AGGAAAGGGGAGAGCTGGAAAGAATCCTGGCAGATCTTGAGG AAGAGAAC AGAAACCTGCAGGCAGAGTATGACAGGCTCAAACAGCAGCAT GAGCACAA GGGACTGAGCCCTCTGCCTTCTCCTCCTGAAATGATGCCCAC CTCTCCAC AGTCTCCAAGGTGATGA DYS3 21 ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAA GATGTGCA GAAGAAAACCTTCACCAAATGGGTCAATGCCCAGTTCAGCAA GTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGGCA GAAGGCTG CTGGATCTGCTGGAAGGCCTGACAGGCCAGAAGCTGCCTAAA GAGAAGGG CAGCACAAGAGTGCATGCCCTGAACAATGTGAACAAGGCCCT GAGAGTGC T G CAGAACAACAAT GT GGAC C T GGT CAATAT T GGCAGCACAG ACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACATC ATCCTGCA CTGGCAAGTGAAGAATGTGATGAAGAACATCATGGCTGGCCT GCAGCAGA CCAACTCTGAGAAGATCCTGCTGAGCTGGGTCAGACAGAGCA CCAGAAAC TACCCTCAAGTGAATGTGATCAACTTCACCACCTCTTGGAGT GATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTT TGACTGGA Structure SEQID Nucleic Acid Sequence ACTCTGTTGTGTGCCAGCAGTCTGCCACACAGAGACTGGAAC ATGCCTTC AACATTGCCAGATACCAGCTGGGAATTGAGAAACTGCTGGAC CCTGAGGA TGTGGACACCACCTATCCTGACAAGAAATCCATCCTCATGTA CATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGCCA TTCAAGAG GTTGAGATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAA CACTTCCA GCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTGTC TCTGGCCC AGGGATATGAGAGAACAAGCAGCCCCAAGCCTAGGTTCAAGA GCTATGCC TACACACAGGC T GC C TAT GT GAG GAGAT C T GAG C C CACAAGA AGCCCATT TCCAAGCCAGCATCTGGAAGCCCCTGAGGACAAGAGCTTTGG CAGCAGCC TGATGGAATCTGAAGTGAACCTGGATAGATACCAGACAGCCC TGGAAGAA GTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCT CAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCACAC CCATGAGG GCTACATGATGGACCTGACAGCCCACCAGGGCAGAGTGGGAA ATATCCTG CAGCTGGGCTCCAAGCTGATTGGCACAGGCAAGCTGTCTGAG GATGAAGA GACAGAGGTGCAAGAGCAGATGAACCTGCTGAACAGCAGATG GGAGTGTC TGAGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAGAG TGCTCATG GACCTGCAGAATCAGAAACTGAAAGAACTGAATGACTGGCTG ACCAAGAC AGAAGAAAGGAC TAGGAAGAT GGAAGAGGAAC CTCTGGGACC AGACCTGG AAGATCTGAAAAGACAGGTGCAGCAGCATAAGGTGCTGCAAG AGGACCTT GAGCAAGAGCAAGT CAGAGT GAACAGCCT GACACACAT GGT G GTGGTTGT GGATGAGTCCTCTGGGGATCATGCCACAGCTGCTCTGGAAGA ACAGCTGA AGGTGCTGGGAGACAGATGGGCCAACATCTGTAGGTGGACAG AGGATAGA TGGGTGCTGCTCCAGGACATTCTGCTGAAGTGGCAGAGACTG ACAGAGGA ACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGATGC TGTCAACA AGAT CCATACCACAGGCT T CAAGGAT CAGAAT GAGATGCT CA GCTCCCTG Structure SEQID Nucleic Acid Sequence CAGAAACTGGCTGTGCTGAAGGCTGACCTGGAAAAGAAAAAG CAGTCCAT GGGCAAGCTCTACAGCCTGAAGCAGGACCTGCTGTCTACCCT GAAGAACA AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTTTG CTAGATGC TGGGACAACCTGGTGCAGAAGCTGGAAAAGTCTACAGCCCAG ATCAGCCA GCAACCTGATCTTGCCCCTGGCCTGACCACAATTGGAGCCTC TCCAACAC AGACTGTGACCCTGGTTACCCAGCCAGTGGTCACCAAAGAGA CAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCCCC ACACTGGA AAGGCTGCAAGAACTTCAAGAGGCCACAGATGAGCTGGACCT GAAGCTGA GACAGGCTGAAGTGATCAAAGGCAGCTGGCAGCCAGTTGGGG ACCTGCTC ATTGATAGCCTGCAGGACCATCTGGAAAAAGTGAAAGCCCTG AGGGGAGA GATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGACCT GGCCAGAC AGCTGACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGA GCACCCTT GAGGACCTGAACACCAGGTGGAAGCTCCTCCAGGTGGCAGTG GAAGATAG AGTCAGGCAGCTGCATGAGGCCCACAGAGATTTTGGACCAGC CAGCCAGC ACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGCTA TCTCTCCT AACAAGGTGCCCTACTACATCAACCATGAGACACAGACCACC TGTTGGGA TCACCCCAAGATGACAGAGCTGTACCAGAGTCTGGCAGACCT CAACAATG TCAGATTCAGTGCCTACAGGACTGCCATGAAGCTCAGAAGGC TCCAGAAA GCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTGAT GCCCTGGA CCAGCACAATCTGAAGCAGAATGACCAGCCTATGGACATCCT CCAGATCA TCAACTGCCTCACCACCATCTATGATAGGCTGGAACAAGAGC ACAACAAT CTGGTCAATGTGCCCCTGTGTGTGGACATGTGCCTGAATTGG CTGCTGAA TGTGTATGACACAGGCAGAACAGGCAGGATCAGAGTCCTGTC CTTCAAGA CAGGCATCATCTCCCTGTGCAAAGCCCACTTGGAGGACAAGT ACAGATAC CTGTTCAAGCAAGTGGCCTCCAGCACAGGCTTTTGTGACCAG AGAAGGCT Structure SEQID Nucleic Acid Sequence GGGCCTGCTCCTGCATGACAGCATTCAGATCCCTAGACAGCT GGGAGAAG TGGCTTCCTTTGGAGGCAGCAATATTGAGCCATCAGTCAGGT CCTGTTTT CAGTTTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTC CTGGACTG GATGAGACTTGAGCCTCAGAGCATGGTCTGGCTGCCTGTGCT TCATAGAG TGGCTGCTGCTGAGACTGCCAAGCACCAGGCCAAGTGCAACA TCTGCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGCAC TTCAACTA TGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGGGTTGCCAA GGGCCACA AAAT GCAC TAC C C CAT GGT GGAATAC T GCAC C C CAACAAC C T CTGGGGAA GATGTTAGAGACTTTGCCAAGGTGCTGAAAAACAAGTTCAGG ACCAAGAG ATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTCCA GACAGTGC TTGAGGGTGACAACATGGAAACC DYS5 81 ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAA GATGTGCAGAAGAAAACCTTCACCAAATGGGTCAATGCCCAG TTCAGCAAGTTTGGCAAGCAGCACATTGAGAACCTGTTCAGT GACCTGCAGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGC CTGACAGGCCAGAAGCTGCCTAAAGAGAAGGGCAGCACAAGA GTGCATGCCCTGAACAATGTGAACAAGGCCCTGAGAGTGCTG CAGAACAACAAT GT GGACCT GGT CAATAT TGGCAGCACAGAC ATTGTGGATGGCAACCACAAGCTGACCCTGGGCCTGATCTGG AACAT CAT CCT GCACT GGCAAGT GAAGAATGT GAT GAAGAAC ATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGTCAGACAGAGCACCAGAAACTACCCTCAAGTG AATGTGATCAACTTCACCACCTCTTGGAGTGATGGACTGGCC CTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGAC TGGAACTCTGTTGTGTGCCAGCAGTCTGCCACACAGAGACTG GAACATGCCTTCAACATTGCCAGATACCAGCTGGGAATTGAG AAAC TGCTGGACCCT GAGGAT GT GGACAC CACCTATCCTGAC AAGAAATCCATCCTCATGTACATCACCAGCCTGTTCCAGGTG CTGCCCCAGCAAGTGTCCATTGAGGCCATTCAAGAGGTTGAG ATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAACACTTC CAGCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTG TCTCTGGCCCAGGGATATGAGAGAACAAGCAGCCCCAAGCCT AGGTTCAAGAGCTATGCCTACACACAGGCTGCCTATGTGACC ACAT C T GACC C CACAAGAAGC C CAT T T C CAAGC CAGCAT C T G GAAGCCCCTGAGGACAAGAGCTTTGGCAGCAGCCTGATGGAA TCTGAAGTGAACCTGGATAGATACCAGACAGCCCTGGAAGAA GTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCT CAGGGT GAAAT CAGCAAT GAT GT GGAAGT GGT CAAGGACCAG TTTCACACCCATGAGGGCTACATGATGGACCTGACAGCCCAC CAGGGCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTG Structure SEQID Nucleic Acid Sequence ATTGGCACAGGCAAGCTGTCTGAGGATGAAGAGACAGAGGTG CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTCTG AGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAGAGTG CT CAT GGACCT GCAGAAT CAGAAACT GAAAGAACT GAAT GAC T G GC T GAC CAAGACAGAAGAAAGGAC TAGGAAGAT GGAAGAG GAACCTCTGGGACCAGACCTGGAAGATCTGAAAAGACAGGTG CAGCAGCATAAGGTGCTGCAAGAGGACCTTGAGCAAGAGCAA GTCAGAGTGAACAGCCTGACACACATGGTGGTGGTTGTGGAT GAGTCCTCTGGGGATCATGCCACAGCTGCTCTGGAAGAACAG CTGAAGGTGCTGGGAGACAGATGGGCCAACATCTGTAGGTGG ACAGAGGATAGATGGGTGCTGCTCCAGGACATTCTGCTGAAG T G GCAGAGAC T GACAGAGGAACAGT GC C TGTTTTCTGCCTGG CT CT CT GAGAAAGAGGAT GCT GT CAACAAGAT CCATACCACA GG CT T CAAGGAT CAGAAT GAGATGCTCAGCTCCC T GCAGAAA CTGGCTGTGCTGAAGGCTGACCTGGAAAAGAAAAAGCAGTCC ATGGGCAAGCTCTACAGCCTGAAGCAGGACCTGCTGTCTACC CTGAAGAACAAGTCTGTGACCCAGAAAACTGAGGCCTGGCTG GACAACTTTGCTAGATGCTGGGACAACCTGGTGCAGAAGCTG GAAAAGTCTACAGCCCAGATCAGCCAGCAACCTGATCTTGCC CCTGGCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTG ACCCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCCCC ACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGATGAG CTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAAGGCAGC TGGCAGCCAGTTGGGGACCTGCTCATTGATAGCCTGCAGGAC CATCTGGAAAAAGTGAAAGCCCTGAGGGGAGAGATTGCCCCT CTGAAAGAAAATGTGTCCCATGTGAATGACCTGGCCAGACAG CTGACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGAGC ACCCTTGAGGACCTGAACACCAGGTGGAAGCTCCTCCAGGTG GCAGTGGAAGATAGAGTCAGGCAGCTGCATGAGGCCCACAGA GATTTTGGACCAGCCAGCCAGCACTTTCTGTCTACCTCTGTG CAAGGCCCCTGGGAGAGAGCTATCTCTCCTAACAAGGTGCCC TACTACAT CAACCAT GAGACACAGACCACCT GT T GGGAT CAC CCCAAGATGACAGAGCTGTACCAGAGTCTGGCAGACCTCAAC AATGTCAGATTCAGTGCCTACAGGACTGCCATGAAGCTCAGA AGGCTCCAGAAAGCTCTGTGCCTGGACCTGCTTTCCCTGAGT GCAGCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAAT GACCAGCCTATGGACATCCTCCAGATCATCAACTGCCTCACC AC CAT CTATGATAGGCTGGAACAAGAGCACAACAAT CT GGT C AATGTGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTG AATGTGTATGACACAGGCAGAACAGGCAGGATCAGAGTCCTG TCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACTTG GAGGACAAGTACAGATACCTGTTCAAGCAAGTGGCCTCCAGC ACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTCCTGCAT GACAGCATTCAGATCCCTAGACAGCTGGGAGAAGTGGCTTCC TTTGGAGGCAGCAATATTGAGCCATCAGTCAGGTCCTGTTTT CAGTTTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTC CTGGACTGGATGAGACTTGAGCCTCAGAGCATGGTCTGGCTG CCTGTGCTTCATAGAGTGGCTGCTGCTGAGACTGCCAAGCAC CAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATTGGC Structure SEQID Nucleic Acid Sequence TTCAGATACAGATCCCTGAAGCACTTCAACTATGATATCTGC CAGAGCTGCTTCTTTAGTGGCAGGGTTGCCAAGGGCCACAAA ATGCACTACCCCATGGTGGAATACTGCACCCCAACAACCTCT GGGGAAGATGTTAGAGACTTTGCCAAGGTGCTGAAAAACAAG TTCAGGACCAAGAGATACTTTGCTAAGCACCCCAGAATGGGC TACCTGCCTGTCCAGACAGTGCTTGAGGGTGACAACATGGAA ACCCCTGTGACACTGATCAATTTCTGGCCAGTGGACTCTGCC CCTGCCTCAAGTCCACAGCTGTCCCATGATGACACCCACAGC AGAAT T GAGCACTAT GCCT CCAGACT GGCAGAGAT GGAAAAC AGCAATGGCAGCTACCTGAATGATAGCATCAGCCCCAATGAG AGCATTGATGATGAGCATCTGCTGATCCAGCACTACTGTCAG TCCCTGAACCAGGACTCTCCACTGAGCCAGCCTAGAAGCCCT GCTCAGATCCTGATCAGCCTTGAGTCTT GAT GA RGX-DYS6 (coding sequence 3867 bp) 101 ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAA GATGTGCAGAAGAAAACCTTCACCAAATGGGTCAATGCCCAG TTCAGCAAGTTTGGCAAGCAGCACATTGAGAACCTGTTCAGT GACCTGCAGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGC CTGACAGGCCAGAAGCTGCCTAAAGAGAAGGGCAGCACAAGA GTGCATGCCCTGAACAATGTGAACAAGGCCCTGAGAGTGCTG CAGAACAACAAT GT GGACCT GGT CAATAT TGGCAGCACAGAC ATTGTGGATGGCAACCACAAGCTGACCCTGGGCCTGATCTGG AACAT CAT GGT GCACT GGCAAGT GAAGAATGT GAT GAAGAAC ATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGTCAGACAGAGCACCAGAAACTACCCTCAAGTG AATGTGATCAACTTCACCACCTCTTGGAGTGATGGACTGGCC CTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGAC TGGAACTCTGTTGTGTGCCAGCAGTCTGCCACACAGAGACTG GAACATGCCTTCAACATTGCCAGATACCAGCTGGGAATTGAG AAAC TGCTGGACCCT GAGGAT GT GGACAC CACCTATCCTGAC AAGAAATCCATCCTCATGTACATCACCAGCCTGTTCCAGGTG CTGCCCCAGCAAGTGTCCATTGAGGCCATTCAAGAGGTTGAG ATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAACACTTC CAGCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTG TCTCTGGCCCAGGGATATGAGAGAACAAGCAGCCCCAAGCCT AGGTTCAAGAGCTATGCCTACACACAGGCTGCCTATGTGACC ACAT C T GACC C CACAAGAAGC C CAT T T C CAAGC CAGCAT C T G GAAGCCCCTGAGGACAAGAGCTTTGGCAGCAGCCTGATGGAA TCTGAAGTGAACCTGGATAGATACCAGACAGCCCTGGAAGAA GTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCT CAGGGT GAAAT CAGCAAT GAT GT GGAAGT GGT CAAGGACCAG TTTCACACCCATGAGGGCTACATGATGGACCTGACAGCCCAC CAGGGCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTG ATTGGCACAGGCAAGCTGTCTGAGGATGAAGAGACAGAGGTG CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTCTG AGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAGAGTG CT CAT GGACCT GCAGAAT CAGAAACT GAAAGAACT GAAT GAC T G GC T GAC CAAGACAGAAGAAAGGAC TAGGAAGAT GGAAGAG GAACCTCTGGGACCAGACCTGGAAGATCTGAAAAGACAGGTG CAGCAGCATAAGGTGCTGCAAGAGGACCTTGAGCAAGAGCAA GTCAGAGTGAACAGCCTGACACACATGGTGGTGGTTGTGGAT Structure SEQID Nucleic Acid Sequence GAGTCCTCTGGGGATCATGCCACAGCTGCTCTGGAAGAACAG CTGAAGGTGCTGGGAGACAGATGGGCCAACATCTGTAGGTGG ACAGAGGATAGATGGGTGCTGCTCCAGGACATTCTGCTGAAG T G GCAGAGAC T GAGAGAGGAACAGT GC C TGTTTTCTGCCTGG CT CT CT GAGAAAGAGGAT GCT GT CAACAAGAT CCATACCACA GG CT T CAAGGAT CAGAAT GAGATGCTCAGCTCCC T GCAGAAA CTGGCTGTGCTGAAGGCTGACCTGGAAAAGAAAAAGCAGTCC ATGGGCAAGCTCTACAGCCTGAAGCAGGACCTGCTGTCTACC CTGAAGAACAAGTCTGTGACCCAGAAAACTGAGGCCTGGCTG GACAACTTTGCTAGATGCTGGGACAACCTGGTGCAGAAGCTG GAAAAGTCTACAGCCCAGATCAGCCAGCAACCTGATCTTGCC CCTGGCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTG ACCCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCCCC ACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGATGAG CTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAAGGCAGC TGGCAGCCAGTTGGGGACCTGCTCATTGATAGCCTGCAGGAC CATCTGGAAAAAGTGAAAGCCCTGAGGGGAGAGATTGCCCCT CTGAAAGAAAATGTGTCCCATGTGAATGACCTGGCCAGACAG CTGACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGAGC ACCCTTGAGGACCTGAACACCAGGTGGAAGCTCCTCCAGGTG GCAGTGGAAGATAGAGTCAGGCAGCTGCATGAGGCCCACAGA GATTTTGGACCAGCCAGCCAGCACTTTCTGTCTACCTCTGTG CAAGGCCCCTGGGAGAGAGCTATCTCTCCTAACAAGGTGCCC TACTACAT CAACCAT GAGACACAGACCACCT GT T GGGAT CAC CCCAAGATGACAGAGCTGTACCAGAGTCTGGCAGACCTCAAC AATGTCAGATTCAGTGCCTACAGGACTGCCATGAAGCTCAGA AGGCTCCAGAAAGCTCTGTGCCTGGACCTGCTTTCCCTGAGT GCAGCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAAT GACCAGCCTATGGACATCCTCCAGATCATCAACTGCCTCACC AC CAT CTATGATAGGCTGGAACAAGAGCACAACAAT CT GGT C AATGTGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTG AATGTGTATGACACAGGCAGAACAGGCAGGATCAGAGTCCTG TCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACTTG GAGGACAAGTACAGATACCTGTTCAAGCAAGTGGCCTCCAGC ACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTCCTGCAT GACAGCATTCAGATCCCTAGACAGCTGGGAGAAGTGGCTTCC TTTGGAGGCGCCAAGCACCAGGCCAAGTGCAACATCTGCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGCAC TTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGG GTTGCCAAGGGCCACAAAATGCACTACCCCATGGTGGAATAC TGCACCCCAACAACCTCTGGGGAAGATGTTAGAGACTTTGCC AAGGTGCTGAAAAACAAGTTCAGGACCAAGAGATACTTTGCT AAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTT GAGGGTGACAACATGGAAACCCCTGTGACACTGATCAATTTC TGGCCAGTGGACTCTGCCCCTGCCTCAAGTCCACAGCTGTCC CAT GAT GACAC C CACAGCAGAAT TGAGCACTATGCCTCCAGA CTGGCAGAGATGGAAAACAGCAATGGCAGCTACCTGAATGAT AGCATCAGCCCCAATGAGAGCATTGATGATGAGCATCTGCTG ATCCAGCACTACTGTCAGTCCCTGAACCAGGACTCTCCACTG Structure SEQID Nucleic Acid Sequence AGCCAGCCTAGAAGCCCTGCTCAGATCCTGATCAGCCTTGAG TCTGAGGAAAGGGGAGAGCTGGAAAGAATCCTGGCAGATCTT GAGGAAGAGAACAGAAACCTGCAGGCAGAGTATGACAGGCTC AAACAGCAGCATGAGCACAAGGGACTGAGCCCTCTGCCTTCT CCTCCTGAAATGATGCCCACCTCTCCACAGTCTCCAAGGTGA TGA Stop codons underlined RGX-DYS7 (coding sequence 4041 bp) 102 ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAA GATGTGCAGAAGAAAACCTTCACCAAATGGGTCAATGCCCAG TTCAGCAAGTTTGGCAAGCAGCACATTGAGAACCTGTTCAGT GACCTGCAGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGC CTGACAGGCCAGAAGCTGCCTAAAGAGAAGGGCAGCACAAGA GTGCATGCCCTGAACAATGTGAACAAGGCCCTGAGAGTGCTG CAGAACAACAAT GT GGACCT GGT CAATAT TGGCAGCACAGAC ATTGTGGATGGCAACCACAAGCTGACCCTGGGCCTGATCTGG AACAT CAT GGT GCACT GGCAAGT GAAGAATGT GAT GAAGAAC ATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGTCAGACAGAGCACCAGAAACTACCCTCAAGTG AATGTGATCAACTTCACCACCTCTTGGAGTGATGGACTGGCC CTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGAC TGGAACTCTGTTGTGTGCCAGCAGTCTGCCACACAGAGACTG GAACATGCCTTCAACATTGCCAGATACCAGCTGGGAATTGAG AAAC TGCTGGACCCT GAGGAT GT GGACAC CACCTATCCTGAC AAGAAATCCATCCTCATGTACATCACCAGCCTGTTCCAGGTG CTGCCCCAGCAAGTGTCCATTGAGGCCATTCAAGAGGTTGAG ATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAACACTTC CAGCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTG TCTCTGGCCCAGGGATATGAGAGAACAAGCAGCCCCAAGCCT AGGTTCAAGAGCTATGCCTACACACAGGCTGCCTATGTGACC ACAT C T GACC C CACAAGAAGC C CAT T T C CAAGC CAGCAT C T G GAAGCCCCTGAGGACAAGAGCTTTGGCAGCAGCCTGATGGAA TCTGAAGTGAACCTGGATAGATACCAGACAGCCCTGGAAGAA GTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCT CAGGGT GAAAT CAGCAAT GAT GT GGAAGT GGT CAAGGACCAG TTTCACACCCATGAGGGCTACATGATGGACCTGACAGCCCAC CAGGGCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTG ATTGGCACAGGCAAGCTGTCTGAGGATGAAGAGACAGAGGTG CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTCTG AGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAGAGTG CT CAT GGACCT GCAGAAT CAGAAACT GAAAGAACT GAAT GAC T G GC T GAC CAAGACAGAAGAAAGGAC TAGGAAGAT GGAAGAG GAACCTCTGGGACCAGACCTGGAAGATCTGAAAAGACAGGTG CAGCAGCATAAGGTGCTGCAAGAGGACCTTGAGCAAGAGCAA GTCAGAGTGAACAGCCTGACACACATGGTGGTGGTTGTGGAT GAGTCCTCTGGGGATCATGCCACAGCTGCTCTGGAAGAACAG CTGAAGGTGCTGGGAGACAGATGGGCCAACATCTGTAGGTGG ACAGAGGATAGATGGGTGCTGCTCCAGGACATTCTGGAGATC AGCTATGTGCCCAGCACCTACCTGACAGAGATCACCCATGTG TCTCAGGCCCTGCTGGAAGTGGAACAGCTGCTGAATGCCCCT Structure SEQID Nucleic Acid Sequence GACCTGTGTGCCAAGGACTTTGAGGACCTGTTCAAGCAAGAG GAAAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGTCCTCT GG CAGAAT T GAGAT CAT C CACAGCAAGAAAACAGC T GC C C T G CAGTCTGCCACACCTGTGGAAAGAGTGAAGCTGCAAGAGGCC CTGAGCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAGATG TACAAGGACAGGCAGGGCAGATTTGATAGAAGTGTGGAAAAG TGGAGAAGGTTCCACTATGACATCAAGATCTTCAACCAGTGG CTGACAGAGGCTGAGCAGTTCCTGAGAAAGACACAGATCCCT GAGAACTGGGAGCATGCCAAGTACAAGTGGTATCTGAAAGAA CT GCAGGATGGCAT T GGCCAGAGACAGACAGT T GT CAGAACC CT GAAT GCCACAGGGGAAGAGAT CAT CCAGCAGAGCAGCAAG ACAGATGCCAGCATCCTGCAAGAGAAGCTGGGCAGCCTGAAC CT GAGAT GGCAAGAAGTGT GCAAGCAGCT GT CT GACAGAAAG AAGAGGCTGGAAGAACAGACACTGGAAAGGCTGCAAGAACTT CAAGAGGCCACAGATGAGCTGGACCTGAAGCTGAGACAGGCT GAAGTGATCAAAGGCAGCTGGCAGCCAGTTGGGGACCTGCTC ATTGATAGCCTGCAGGACCATCTGGAAAAAGTGAAAGCCCTG AGGGGAGAGATTGCCCCTCTGAAAGAAAATGTGTCCCATGTG AATGACCTGGCCAGACAGCTGACCACACTGGGAATCCAGCTG AGCCCCTACAACCTGAGCACCCTTGAGGACCTGAACACCAGG TGGAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAG CTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGCAC TTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGCTATC TCTCCTAACAAGGTGCCCTACTACATCAACCATGAGACACAG ACCACCTGTTGGGATCACCCCAAGATGACAGAGCTGTACCAG AGTCTGGCAGACCTCAACAATGTCAGATTCAGTGCCTACAGG ACTGCCATGAAGCTCAGAAGGCTCCAGAAAGCTCTGTGCCTG GACCTGCTTTCCCTGAGTGCAGCTTGTGATGCCCTGGACCAG CACAATCTGAAGCAGAATGACCAGCCTATGGACATCCTCCAG AT CAT CAACT GCCT CACCACCAT CTAT GATAGGCT GGAACAA GAGCACAACAATCTGGTCAATGTGCCCCTGTGTGTGGACATG TGCCTGAATTGGCTGCTGAATGTGTATGACACAGGCAGAACA GGCAGGATCAGAGTCCTGTCCTTCAAGACAGGCATCATCTCC CTGTGCAAAGCCCACTTGGAGGACAAGTACAGATACCTGTTC AAGCAAGTGGCCTCCAGCACAGGCTTTTGTGACCAGAGAAGG CTGGGCCTGCTCCTGCATGACAGCATTCAGATCCCTAGACAG CTGGGAGAAGTGGCTTCCTTTGGAGGCAGCAATATTGAGCCA TCAGTCAGGTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAG ATTGAGGCTGCCCTGTTCCTGGACTGGATGAGACTTGAGCCT CAGAGCATGGTCTGGCTGCCTGTGCTTCATAGAGTGGCTGCT GCTGAGACTGCCAAGCACCAGGCCAAGTGCAACATCTGCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGCAC TTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGG GTTGCCAAGGGCCACAAAATGCACTACCCCATGGTGGAATAC TGCACCCCAACAACCTCTGGGGAAGATGTTAGAGACTTTGCC AAGGTGCTGAAAAACAAGTTCAGGACCAAGAGATACTTTGCT AAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTT GAGGGTGACAACATGGAAACCCCTGTGACACTGATCAATTTC TGGCCAGTGGACTCTGCCCCTGCCTCAAGTCCACAGCTGTCC CAT GAT GACAC C CACAGCAGAAT TGAGCACTATGCCTCCAGA Structure SEQID Nucleic Acid Sequence CTGGCAGAGATGGAAAACAGCAATGGCAGCTACCTGAATGAT AGCATCAGCCCCAATGAGAGCATTGATGATGAGCATCTGCTG ATCCAGCACTACTGTCAGTCCCTGAACCAGGACTCTCCACTG AGCCAGCCTAGAAGCCCTGCTCAGATCCTGATCAGCCTTGAG TCTTGATGA Stop codons underlined RGX-DYS8 (coding sequence 3765 bp) 103 ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAA GATGTGCAGAAGAAAACCTTCACCAAATGGGTCAATGCCCAG TTCAGCAAGTTTGGCAAGCAGCACATTGAGAACCTGTTCAGT GACCTGCAGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGC CTGACAGGCCAGAAGCTGCCTAAAGAGAAGGGCAGCACAAGA GTGCATGCCCTGAACAATGTGAACAAGGCCCTGAGAGTGCTG CAGAACAACAAT GT GGACCT GGT CAATAT TGGCAGCACAGAC ATTGTGGATGGCAACCACAAGCTGACCCTGGGCCTGATCTGG AACAT CAT GGT GCACT GGCAAGT GAAGAATGT GAT GAAGAAC ATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGTCAGACAGAGCACCAGAAACTACCCTCAAGTG AATGTGATCAACTTCACCACCTCTTGGAGTGATGGACTGGCC CTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGAC TGGAACTCTGTTGTGTGCCAGCAGTCTGCCACACAGAGACTG GAACATGCCTTCAACATTGCCAGATACCAGCTGGGAATTGAG AAAC TGCTGGACCCT GAGGAT GT GGACAC CACCTATCCTGAC AAGAAATCCATCCTCATGTACATCACCAGCCTGTTCCAGGTG CTGCCCCAGCAAGTGTCCATTGAGGCCATTCAAGAGGTTGAG ATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAACACTTC CAGCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTG TCTCTGGCCCAGGGATATGAGAGAACAAGCAGCCCCAAGCCT AGGTTCAAGAGCTATGCCTACACACAGGCTGCCTATGTGACC ACAT C T GACC C CACAAGAAGC C CAT T T C CAAGC CAGCAT C T G GAAGCCCCTGAGGACAAGAGCTTTGGCAGCAGCCTGATGGAA TCTGAAGTGAACCTGGATAGATACCAGACAGCCCTGGAAGAA GTGCTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCT CAGGGT GAAAT CAGCAAT GAT GT GGAAGT GGT CAAGGACCAG TTTCACACCCATGAGGGCTACATGATGGACCTGACAGCCCAC CAGGGCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTG ATTGGCACAGGCAAGCTGTCTGAGGATGAAGAGACAGAGGTG CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTCTG AGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACAGAGTG CT CAT GGACCT GCAGAAT CAGAAACT GAAAGAACT GAAT GAC T G GC T GAC CAAGACAGAAGAAAGGAC TAGGAAGAT GGAAGAG GAACCTCTGGGACCAGACCTGGAAGATCTGAAAAGACAGGTG CAGCAGCATAAGGTGCTGCAAGAGGACCTTGAGCAAGAGCAA GTCAGAGTGAACAGCCTGACACACATGGTGGTGGTTGTGGAT GAGTCCTCTGGGGATCATGCCACAGCTGCTCTGGAAGAACAG CTGAAGGTGCTGGGAGACAGATGGGCCAACATCTGTAGGTGG ACAGAGGATAGATGGGTGCTGCTCCAGGACATTCTGGAGATC AGCTATGTGCCCAGCACCTACCTGACAGAGATCACCCATGTG TCTCAGGCCCTGCTGGAAGTGGAACAGCTGCTGAATGCCCCT GACCTGTGTGCCAAGGACTTTGAGGACCTGTTCAAGCAAGAG Structure SEQID Nucleic Acid Sequence GAAAGCCTGAAGAACATCAAGGACAGCCTGCAGCAGTCCTCT GG CAGAAT T GAGAT CAT C CACAGCAAGAAAACAGC T GC C C T G CAGTCTGCCACACCTGTGGAAAGAGTGAAGCTGCAAGAGGCC CTGAGCCAGCTGGACTTCCAGTGGGAGAAAGTGAACAAGATG TACAAGGACAGGCAGGGCAGATTTGATAGAAGTGTGGAAAAG TGGAGAAGGTTCCACTATGACATCAAGATCTTCAACCAGTGG CTGACAGAGGCTGAGCAGTTCCTGAGAAAGACACAGATCCCT GAGAACTGGGAGCATGCCAAGTACAAGTGGTATCTGAAAGAA CT GCAGGATGGCAT T GGCCAGAGACAGACAGT T GT CAGAACC CT GAAT GCCACAGGGGAAGAGAT CAT CCAGCAGAGCAGCAAG ACAGATGCCAGCATCCTGCAAGAGAAGCTGGGCAGCCTGAAC CT GAGAT GGCAAGAAGTGT GCAAGCAGCT GT CT GACAGAAAG AAGAGGCTGGAAGAACAGACACTGGAAAGGCTGCAAGAACTT CAAGAGGCCACAGATGAGCTGGACCTGAAGCTGAGACAGGCT GAAGTGATCAAAGGCAGCTGGCAGCCAGTTGGGGACCTGCTC ATTGATAGCCTGCAGGACCATCTGGAAAAAGTGAAAGCCCTG AGGGGAGAGATTGCCCCTCTGAAAGAAAATGTGTCCCATGTG AATGACCTGGCCAGACAGCTGACCACACTGGGAATCCAGCTG AGCCCCTACAACCTGAGCACCCTTGAGGACCTGAACACCAGG TGGAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAG CTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGCAC TTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGCTATC TCTCCTAACAAGGTGCCCTACTACATCAACCATGAGACACAG ACCACCTGTTGGGATCACCCCAAGATGACAGAGCTGTACCAG AGTCTGGCAGACCTCAACAATGTCAGATTCAGTGCCTACAGG ACTGCCATGAAGCTCAGAAGGCTCCAGAAAGCTCTGTGCCTG GACCTGCTTTCCCTGAGTGCAGCTTGTGATGCCCTGGACCAG CACAATCTGAAGCAGAATGACCAGCCTATGGACATCCTCCAG AT CAT CAACT GCCT CACCACCAT CTAT GATAGGCT GGAACAA GAGCACAACAATCTGGTCAATGTGCCCCTGTGTGTGGACATG TGCCTGAATTGGCTGCTGAATGTGTATGACACAGGCAGAACA GGCAGGATCAGAGTCCTGTCCTTCAAGACAGGCATCATCTCC CTGTGCAAAGCCCACTTGGAGGACAAGTACAGATACCTGTTC AAGCAAGTGGCCTCCAGCACAGGCTTTTGTGACCAGAGAAGG CTGGGCCTGCTCCTGCATGACAGCATTCAGATCCCTAGACAG CTGGGAGAAGTGGCTTCCTTTGGAGGCAGCAATATTGAGCCA TCAGTCAGGTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAG ATTGAGGCTGCCCTGTTCCTGGACTGGATGAGACTTGAGCCT CAGAGCATGGTCTGGCTGCCTGTGCTTCATAGAGTGGCTGCT GCTGAGACTGCCAAGCACCAGGCCAAGTGCAACATCTGCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGCAC TTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGG GTTGCCAAGGGCCACAAAATGCACTACCCCATGGTGGAATAC TGCACCCCAACAACCTCTGGGGAAGATGTTAGAGACTTTGCC AAGGTGCTGAAAAACAAGTTCAGGACCAAGAGATACTTTGCT AAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTT GAGGGTGACAACATGGAAACCTGATGA Stop codons underlined 5.2.2.1 Codon Optimization and CpG Depletion

[0099] In one aspect the nucleotide sequence encoding the microdystrophin cassette is modified by codon optimization and CpG dinucleotide and CpG island depletion. Immune response against microdystrophin transgene is a concern for human clinical application, as evidenced in the first Duchenne Muscular Dystrophy (DMD) gene therapy clinical trials and in several adeno-associated vial (AAV)-minidystrophin gene therapy in canine models [Mendell, J.R., et al., Dystrophin immunity in Duchenne's muscular dystrophy. N Engl J Med, 2010. 363(15): p. 1429-37; and Kornegay, J.N., et al., Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophindeficient dogs. Mol Ther, 2010. 18(8): p. 1501-8],

[00100] AAV-directed immune responses can be inhibited by reducing the number of CpG di-nucleotides in the AAV genome [Faust, S.M., et al., CpG-depleted adeno-associated virus vectors evade immune detection. J Clin Invest, 2013. 123(7): p. 2994-3001], Depleting the transgene sequence of CpG motifs may diminish the role of TLR9 in activation of innate immunity upon recognition of the transgene as non-self, and thus provide stable and prolonged transgene expression. [See also Wang, D., P.W.L. Tai, and G. Gao, Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov, 2019. 18(5): p. 358-378.; and Rabinowitz, J., Y.K. Chan, and R.J. Samulski, Adeno-associated Virus (AAV) versus Immune Response. Viruses, 2019. 11(2)]. In embodiments, the microdystrophin cassette is human codon-optimized with CpG depletion. Codon-optimized and CpG depleted nucleotide sequences may be designed by any method known in the art, including for example, by Thermo Fisher Scientific GeneArt Gene Synthesis tools utilizing GeneOptimizer (Waltham, MA USA)). Nucleotide sequences SEQ ID NOs: 20, 21, 57-72, 80, 81, and 101-103 described herein represent codon-optimized and CpG depleted sequences.

[00101] Provided are microdystrophin transgenes that have reduced numbers of CpG dinucleotide sequences and, as a result, have reduced number of CpG islands. In certain embodiments, the microdystrophin nucleotide sequence has fewer than two (2) CpG islands, or one (1) CpG island orzero (0) CpG islands. In embodiments, provided are microdystrophin transgenes having fewer than 2, or 1 CpG islands, or 0 CpG islands that have reduced immunogenicity, as measured by anti-drug antibody titer compared to a microdystrophin transgene having more than 2 CpG islands. In certain embodiments, the microdystrophin nucleotide sequence consisting essentially of SEQ ID NO: 20, 21, 81, 101, 102 or 103 has zero (0) CpG islands. In other embodiments, the microdystrophin transgene nucleotide sequence consisting essentially of a microdystrophin gene operably linked to a promoter, wherein the microdystrophin consists of SEQ ID NO: 20, 21, 81, 101, 102 or 103, has less than two (2) CpG islands. In still other embodiments, the microdystrophin transgene nucleotide sequence consisting essentially of a microdystrophin gene operably linked to a promoter, wherein the microdystrophin consists of SEQ ID NO: 20, 21, 81, 101, 102 or 103, has one (1) CpG island. 5.3. Gene Cassettes and Regulatory Elements

[00102] Another aspect of the present invention relates to nucleic acid expression cassettes comprising regulatory elements designed to confer or enhance expression of the microdystrophins. The invention further involves engineering regulatory elements, including promoter elements, and optionally enhancer elements and / or introns, to enhance or facilitate expression of the transgene. In some embodiments, the rAAV vector also includes such regulatory control elements known to one skilled in the art to influence the expression of the RNA and / or protein products encoded by nucleic acids (transgenes) within target cells of the subject. Regulatory control elements and may be tissue-specific, that is, active (or substantially more active or significantly more active) only in the target cell / tissue. 5.3.1 Promoters 5.3.1.1 Tissue-specific promoters

[00103] In specific embodiments, the expression cassette of an AAV vector comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues. The promoter may be a constitutive promoter, for example, the CB7 promoter. Additional promoters include: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter (SEQ ID NO: 118), UB6 promoter, chicken beta-actin promoter, CAG promoter (SEQ ID NO: 116), RPE65 promoter, opsin promoter, the TBG (Thyroxine-binding Globulin) promoter, the APOA2 promoter, SERPINA1 (hAAT) promoter, or MIR122 promoter. In some embodiments, particularly where it may be desirable to turn off transgene expression, an inducible promoter is used, e.g., hypoxia-inducible or rapamycin-inducible promoter.

[00104] In certain embodiments, the promoter is a muscle-specific promoter. The phrase “muscle-specific”, “muscle-selective” or “muscle-directed” refers to nucleic acid elements that have adapted their activity in muscle cells or tissue due to the interaction of such elements with the intracellular environment of the muscle cells. Such muscle cells may include myocytes, myotubes, cardiomyocytes, and the like. Specialized forms of myocytes with distinct properties such as cardiac, skeletal, and smooth muscle cells are included. Various therapeutics may benefit from musclespecific expression of a transgene. In particular, gene therapies that treat various forms of muscular dystrophy delivered to and enabling high transduction efficiency in muscle cells have the added benefit of directing expression of the transgene in the cells where the transgene is most needed. Cardiac tissue will also benefit from muscle-directed expression of the transgene. Muscle-specific promoters may be operably linked to the transgenes of the invention. In some embodiments, the muscle-specific promoter is selected from an SPc5-12 promoter, a muscle creatine kinase myosin light chain (MLC) promoter, a myosin heavy chain (MHC) promoter, a desmin promoter (SEQ ID NO: 119), a MHCK7 promoter (SEQ ID NO: 120), a CK6 promoter, a CK8 promoter (SEQ ID NO: 115), a MCK promoter (or a truncated form thereof) (SEQ ID NO: 121), an alpha actin promoter, an beta actin promoter, an gamma actin promoter, an E-syn promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, or a muscle-selective promoter residing within intron 1 of the ocular form of Pitx3.

[00105] Synthetic promoter c5-12 (Li, X. et al. Nature Biotechnology Vol. 17, pp. 241-245, MARCH 1999), known as the SPc5-12 promoter, has been shown to have cell type restricted expression, specifically muscle-cell specific expression. At less than 350 bp in length, the SPc5-12 promoter is smaller in length than most endogenous promoters, which can be advantageous when the length of the nucleic acid encoding the therapeutic protein is relatively long. In embodiments, provided are gene therapy cassettes with an SPc5-12 promoter (SEQ ID NO: 39).

[00106] In order to further reduce the length of a vector, regulatory elements can be a reduced or shortened version (referred to herein as a “minimal promoter”) of any one of the promoters described herein. A minimal promoter comprises at least the transcriptionally active domain of the full-length version and is therefore still capable of driving expression. For example, in some embodiments, an AAV vector can comprise the transcriptionally active domain of a muscle-specific promoter, e.g., a minimal SPc5-12 promoter (e.g., SEQ ID NO: 40), operably linked to a therapeutic protein transgene. In embodiments, the therapeutic protein is microdystrophin as described herein. A minimal promoter of the present disclosure may or may not contain the portion of the promoter sequence that contributes to regulating expression in a tissue-specific manner.

[00107] Accordingly, in embodiments, provided are gene therapy cassettes with an SPc5-12 promoter (SEQ ID NO: 39). In embodiments, provided are gene therapy cassettes with minimal promoters that direct expression of the microdystrophin in muscle cells. One such promoter is a minimal SPc5-12 promoter of SEQ ID NO: 40. Sequences of these promoters are provided in Table 6. Table 6: Promoter sequences Promoter SEQ ID Nucleic Acid Sequence SPc5-12 39 GGCCGTCCGCCCTCGGCACCATCCTCACGACACCC AAATATGGCGACGGGTGAGGAATGGTGGGGAGTTA TTTTTAGAGCGGTGAGGAAGGTGGGCAGGCAGCAG GTGTTGGCGCTCTAAAAATAACTCCCGGGAGTTAT TTTTAGAGCGGAGGAATGGTGGACACCCAAATATG GCGACGGTTCCTCACCCGTCGCCATATTTGGGTGT CCGCCCTCGGCCGGGGCCGCATTCCTGGGGGCCGG GCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGG GGCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCG GGAGGCGCCAAGC minSPc5-12 40 GAATGGTGGACACCCAAATATGGCGACGGTTCCTC ACCCGTCGCCATATTTGGGTGTCCGCCCTCGGCCG GGGCCGCATTCCTGGGGGCCGGGCGGTGCTCCCGC Promoter SEQ ID Nucleic Acid Sequence CCGCCTCGATAAAAGGCTCCGGGGCCGGCGGCGGC CCACGAGCTACCCGGAGGAGCGGGAGGCGCCAAG CK8 115 ccactacgggtttaggctgcccatgtaaggaggcaagg cctggggacacccgagatgcctggttataattaaccca gacatgtggctgccccccccccccccaacacctgctgc ctctaaaaataaccctgtccctggtggatcccactacg ggtttaggctgcccatgtaaggaggcaaggcctgggga cacccgagatgcctggttataattaacccagacatgtg gctgccccccccccccccaacacctgctgcctctaaaa ataaccctgtccctggtggatcccactacgggtttagg ctgcccatgtaaggaggcaaggcctggggacacccgag atgcctggttataattaacccagacatgtggctgcccc ccccccccccaacacctgctgcctctaaaaataaccct gtccctggtggatcccctgcatgcgaagatcttcgaac aaggctgtgggggactgagggcaggctgtaacaggctt gggggccagggcttatacgtgcctgggactcccaaagt attactgttccatgttcccggcgaagggccagctgtcc cccgccagctagactcagcacttagtttaggaaccagt gagcaagtcagcccttggggcagcccatacaaggccat ggggctgggcaagctgcacgcctgggtccggggtgggc acggtgcccgggcaacgagctgaaagctcatctgctct caggggcccctccctggggacagcccctcctggctagt cacaccctgtaggctcctctatataacccaggggcaca ggggctgccctcattctaccaccacctccacagcacag acagacactcaggagccagccagcgtcga CAG 116 gacattgattattgactagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccg cgttacataacttacggtaaatggcccgcctggctgac cgcccaacgacccccgcccattgacgtcaataatgacg tatgttcccatagtaacgccaatagggactttccattg acgtcaatgggtggagtatttacggtaaactgcccact tggcagtacatcaagtgtatcatatgccaagtacgccc cctattgacgtcaatgacggtaaatggcccgcctggca ttatgcccagtacatgaccttatgggactttcctactt ggcagtacatctacgtattagtcatcgctattaccatg gtcgaggtgagccccacgttctgcttcactctccccat ctcccccccctccccacccccaattttgtatttattta ttttttaattattttgtgcagcgatgggggcggggggg gggggggggcgcgcgccaggcggggcggggcggggcga ggggcggggcggggcgaggcggagaggtgcggcggcag ccaatcagagcggcgcgctccgaaagtttccttttatg gcgaggcggcggcggcggcggccctataaaaagcgaag cgcgcggcgggcgggagtcgctgcgcgctgccttcgcc ccgtgccccgctccgccgccgcctcgcgccgcccgccc cggctctgactgaccgcgttactcccacaggtgagcgg gcgggacggcccttctcctccgggctgtaattagcgct tggtttaatgacggcttgtttcttttctgtggctgcgt gaaagccttgaggggctccgggagggccctttgtgcgg ggggagcggctcggggggtgcgtgcgtgtgtgtgtgcg tggggagcgccgcgtgcggctccgcgctgcccggcggc tgtgagcgctgcgggcgcggcgcggggctttgtgcgct ccgcagtgtgcgcgaggggagcgcggccgggggcggtg ccccgcggtgcggggggggctgcgaggggaacaaaggc Promoter SEQ ID Nucleic Acid Sequence tgcgtgcggggtgtgtgcgtgggggggtgagcaggggg tgtgggcgcgtcggtcgggctgcaaccccccctgcacc cccctccccgagttgctgagcacggcccggcttcgggt gcggggctccgtacggggcgtggcgcggggctcgccgt gccgggcggggggtggcggcaggtgggggtgccgggcg gggcggggccgcctcgggccggggagggctcgggggag gggcgcggcggcccccggagcgccggcggctgtcgagg cgcggcgagccgcagccattgccttttatggtaatcgt gcgagagggcgcagggacttcctttgtcccaaatctgt gcggagccgaaatctgggaggcgccgccgcaccccctc tagcgggcgcggggcgaagcggtgcggcgccggcagga aggaaatgggcggggagggccttcgtgcgtcgccgcgc cgccgtccccttctccctctccagcctcggggctgtcc gcggggggacggctgccttcgggggggacggggcaggg cggggttcggcttctggcgtgtgaccggcggctctaga gcctctgctaaccatgttcatgccttcttctttttcct acagctcctgggcaacgtgctggttattgtgctgtctc atcattttggcaaag mUla 117 atggaggcggtactatgtagatgagaattcaggagcaa actgggaaaagcaactgcttccaaatatttgtgatttt tacagtgtagttttggaaaaactcttagcctaccaatt cttctaagtgttttaaaatgtgggagccagtacacatg aagttatagagtgttttaatgaggcttaaatatttacc gtaactatgaaatgctacgcatatcatgctgttcaggc tccgtggccacgcaactcatact EF-1D 118 gggcagagcgcacatcgcccacagtccccgagaagttg gggggaggggtcggcaattgaacgggtgcctagagaag gtggcgcggggtaaactgggaaagtgatgtcgtgtact ggctccgcctttttcccgagggtgggggagaaccgtat ataagtgcagtagtcgccgtgaacgttctttttcgcaa cgggtttgccgccagaacacag Human desmin 119 ctgcagacatgcttgctgcctgccctggcgtgccctggcg aggcttgccgtcacaggacccccgctggctgactcagggg cgcaggctcttgcgggggagctggcctcccgcccccacgg ccacgggccctttcctggcaggacagcgggatcttgcagc tgtcaggggaggggatgacgggggactgatgtcaggaggg gatacaaatagtgccgaacaaggaccggattagatctacc MHCK7 120 aagcttgcat gtctaagcta gacccttcag attaaaaata actgaggtaa gggcctgggt aggggaggtg gtgtgagacg ctcctgtctc tcctctatct gcccatcggc cctttgggga ggaggaatgt gcccaaggac taaaaaaagg ccatggagcc agaggggcga gggcaacaga cctttcatgg gcaaaccttg gggccctgct gtctagcatg ccccactacg ggtctaggct gcccatgtaa ggaggcaagg cctggggaca cccgagatgc ctggttataa ttaacccaga catgtggctg cccccccccc cccaacacct gctgcctcta aaaataaccc tgtccctggt ggatcccctg catgcgaaga tcttcgaaca aggctgtggg ggactgaggg caggctgtaa caggcttggg ggccagggct tatacgtgcc tgggactccc aaagtattac tgttccatgt tcccggcgaa gggccagctg tcccccgcca Promoter SEQ ID Nucleic Acid Sequence gctagactca gcacttagtt taggaaccag tgagcaagtc agcccttggg gcagcccata caaggccatg gggctgggca agctgcacgc ctgggtccgg ggtgggcacg gtgcccgggc aacgagctga aagctcatct gctctcaggg gcccctccct ggggacagcc cctcctggct agtcacaccc tgtaggctcc tctatataac ccaggggcac aggggctgcc ctcattctac caccacctcc acagcacaga cagacactca ggagcagcca gc Truncated MCK 121 ccactacggg tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct ggttataatt aaccccaaca cctgctgccc cccccccccc aacacctgct gcctgagcct gagcggttac cccaccccgg tgcctgggtc ttaggctctg tacaccatgg aggagaagct cgctctaaaa ataaccctgt ccctggtgga tccactacgg gtctatgctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc tggttataat taaccccaac acctgctgcc cccccccccc caacacctgc tgcctgagcc tgagcggtta ccccaccccg gtgcctgggt cttaggctct gtacaccatg gaggagaagc tcgctctaaa aataaccctg tccctggtgg accactacgg gtctaggctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc tggttataat taaccccaac acctgctgcc cccccccccc aacacctgct gcctgagcct gagcggttac cccaccccgg tgcctgggtc ttaggctctg tacaccatgg aggagaagct cgctctaaaa ataaccctgt ccctggtcct ccctggggac agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg gcacaggggc tgcccccggg tcac

[00108] In certain embodiments, the promoter is a CNS-specific promoter. For example, an expression cassette can comprise a promoter selected from a promoter isolated from the genes of neuron specific enolase (NSE), any neuronal promoter such as the promoter of Dopamine-1 receptor or Dopamine-2 receptor, the synapsin promoter, CB7 promoter (a chicken [3-actin promoter and CMV enhancer), RSV promoter, GFAP promoter (glial fibrillary acidic protein), MBP promoter (myelin basic protein), MMT promoter, EF-la, U86 promoter, RPE65 promoter or opsin promoter, an inducible promoter, for example, a hypoxia-inducible promoter, and a drug inducible promoter, such as a promoter induced by rapamycin and related agents.

[00109] In still other embodiments, expression cassettes can comprise multiple promoters which may be placed in tandem in the expression cassette comprising a microdystrophin transgene. As such, tandem or hybrid promoters may be employed in order to enhance expression and / or direct expression to multiple tissue types, (see, e.g. PCT International Publication No. WO2019154939A1, published Aug. 15, 2019, incorporated herein by reference) and, in particular, LMTP6, LMTP13, LMTP14, LMTP15, LMTP18, LMTP19, or LMTP20 as disclosed in PCT International Application No. PCT / US2020 / 043578, filed July 24, 2020, hereby incorporated by reference). 5.3.2 Introns

[00110] Another aspect of the present disclosure relates to an AAV vector comprising an intron within the regulatory cassette. Example 2 demonstrates that the VH4 intron 5’ of the microdystrophin coding sequence enhances proper splicing and, thus, microdystrophin expression. Accordingly, in some embodiments, an intron is coupled to the 5’ end of a sequence encoding a microdystrophin protein, eg., ABD-H1-R1-R2-R3-H3-R24-H4-CR, ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT. In particular, the intron can be linked to the actin-binding domain. In other embodiments, the intron is less than 100 nucleotides in length.

[00111] In embodiments, the intron is a VH4 intron. The VH4 intron nucleic acid can comprise SEQ ID NO: 41 as shown in Table 7 below. Table 7: Nucleotide sequences for different introns Structure SEQ ID Sequence VH4 intron 41 GTGAGTATCTCAGGGATCCAGACATGGGGATATGGGAGGTGC CTCTGATC CCAGGGCTCACTGTGGGTCTCTCTGTTCACAG Chimeric intron 75 GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAG AAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGA TAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTC TCCACAG SV40 intron 76 GTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCC GGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTG CCTTTACTTCTAG

[00112] In other embodiments, the intron is a chimeric intron derived from human P-globin and Ig heavy chain (also known as P-globin splice donor / immunoglobulin heavy chain splice acceptor intron, or P-globin / IgG chimeric intron) (Table 7, SEQ ID NO: 75). Other introns well known to the skilled person may be employed, such as the chicken P-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), P-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin splice acceptor intron, SV40 late splice donor / splice acceptor (19S / 16S) intron (Table 7, SEQ ID NO: 76). 5.3.3 Other regulatory elements 5.3.3.1 polyA

[00113] Another aspect of the present disclosure relates to expression cassettes comprising a polyadenylation (polyA) site downstream of the coding region of the microdystrophin transgene. Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure. Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit P-globin gene, the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, and the synthetic polyA (SPA) site. In one embodiment, the polyA signal comprises SEQ ID NO: 42 as shown in Table 8. Table 8: Nucleotide sequence of the polyA signal Structure SEQ ID Sequence polyA 42 AGGCCTAATAAAGAGCTCAGATGCATCGATCAGAGTGTGT TGGTTTTTTG 5.3.4 Viral vectors

[00114] The microdystrophin transgene in accordance with the present disclosure can be included in an AAV vector for gene therapy administration to a human subject. In some embodiments, recombinant AAV (rAAV) vectors can comprise an AAV viral capsid and a viral or artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs) wherein the expression cassette comprises a microdystrophin transgene, operably linked to one or more regulatory sequences that control expression of the transgene in human muscle or CNS cells to express and deliver the microdystrophin. The provided methods are suitable for use in the production of any isolated recombinant AAV particles for delivery of a microdystrophins described herein, in the production of a composition comprising any isolated recombinant AAV particles encoding a microdystrophin, or in the method for treating a disease or disorder amenable for treatment with a microdystrophin in a subject in need thereof comprising the administration of any isolated recombinant AAV particles encoding a microdystrophin described herein. As such, the rAAV can be of any serotype, variant, modification, hybrid, or derivative thereof, known in the art, or any combination thereof (collectively referred to as “serotype”). In particular embodiments, the AAV serotype has a tropism for muscle tissue. In other embodiments, the AAV serotype has a tropism for the CNS. In other embodiments, the AAV serotype has a tropism for both muscle tissue and the CNS. And, in other embodiments, the AAV serotype has a tropism for the liver, in which case the liver cells transduced with the AAV form a depot of microdystrophin secreting cells, secretin the microdystrophin into the circulation.

[00115] In some embodiments, rAAV particles have a capsid protein from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g, VP1, VP2 and / or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof.

[00116] For example, a population of rAAV particles can comprise two or more serotypes, e.g., comprising two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof.)

[00117] In some embodiments, rAAV particles comprise the capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the rAAV particles comprise the capsid with one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in United States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV.7m8, as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016 / 0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,585,971, such as AAVPHP.B. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015 / 013313, such as AAV.Rh74andRHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014 / 172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2 / 5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017 / 070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016 / 049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety.

[00118] In some embodiments, rAAV particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[00119] In some embodiments, rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003 / 052051 (see, e.g, SEQ ID NO: 2 of '051), WO 2005 / 033321 (see, e.g, SEQ ID NOs: 123 and 88 of '321), WO 03 / 042397 (see, e.g, SEQ ID NOs: 2, 81, 85, and 97 of '397), WO 2006 / 068888 (see, e.g, SEQ ID NOs: 1 and 3-6 of '888), WO 2006 / 110689, (see, e.g, SEQ ID NOs: 5-38 of '689) WO2009 / 104964 (see, e.g, SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964), WO 2010 / 127097 (see, e.g, SEQ ID NOs: 5-38 of '097), and WO 2015 / 191508 (see, e.g, SEQ ID NOs: 80-294 of '508), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of '924), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003 / 052051 (see, e.g, SEQ ID NO: 2 of '051), WO 2005 / 033321 (see, e.g, SEQ ID NOs: 123 and 88 of '321), WO 03 / 042397 (see, e.g, SEQ ID NOs: 2, 81, 85, and 97 of '397), WO 2006 / 068888 (see, e.g, SEQ ID NOs: 1 and 3-6 of '888), WO 2006 / 110689 (see, e.g, SEQ ID NOs: 5-38 of '689) WO2009 / 104964 (see, e.g, SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of 964), W0 2010 / 127097 (see, e.g, SEQ ID NOs: 5-38 of '097), and WO 2015 / 191508 (see, e.g, SEQ ID NOs: 80-294 of '508), and U.S. Appl. Publ. No. 20150023924 (see, e.g, SEQ ID NOs: 1, 5-10 of '924).

[00120] Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335; WO 2003 / 052051, WO 2005 / 033321, WO 03 / 042397, WO 2006 / 068888, WO 2006 / 110689, WO2009 / 104964, W0 2010 / 127097, and WO 2015 / 191508, and U. S. Appl. Publ. No. 20150023924.

[00121] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

[00122] In certain embodiments, a single-stranded AAV (ssAAV) can be used. In certain embodiments, a self-complementary vector, e.g., scAAV, can be used (see, e.g, Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[00123] In some embodiments, rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from the group consisting of AAV7, AAV8, AAV9, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, and AAV.7m8. In some embodiments, the rAAV particles comprise a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, and AAV.hu37. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV1 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV4 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV5 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9 or a derivative, modification, or pseudotype thereof.

[00124] In some embodiments, rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV8 or AAV9 capsid protein. In some embodiments, rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more identical, e.g, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and / or VP3 sequence of AAV8 capsid protein. In some embodiments, rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV9 capsid protein. In some embodiments, rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more identical, e.g, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. upto 100%identical, tothe VP1, VP2 and / or VP3 sequence of AAV9 capsid protein.

[00125] In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identity, to the VP1, VP2 and / or VP3 sequence of AAV7, AAV8, AAV9, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, AAV.hu37, AAV.PHP.B, AAV.PHP.eB, or AAV.7m8 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identity, to the VP1, VP2 and / or VP3 sequence of an AAV capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, and AAV.hu37.

[00126] In additional embodiments, rAAV particles comprise a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[00127] In some embodiments, rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAVrh.8, and AAVrh.10.

[00128] In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle. In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from AAVx (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16). In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle comprised of a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu31, AAV.hu32, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle containing AAV8 capsid protein. In additional embodiments, rAAV particles comprise a pseudotyped rAAV particle is comprised of AAV9 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2 / 8 or rAAV2 / 9 pseudotyped particles. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g, Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

[00129] In additional embodiments, rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In further embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In further embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[00130] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh.8, and AAVrh.10.

[00131] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16.

[00132] In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10.

[00133] In some embodiments the rAAV particles comprises a Clade A, B, E, or F AAV capsid protein. . In some embodiments, the rAAV particles comprises a Clade F AAV capsid protein. In some embodiments the rAAV particles comprises a Clade E AAV capsid protein.

[00134] Table 9 below provides examples of amino acid sequences for an AAV8, AAV9, AAV.rh74, AAV.hu31, AAV.hu32, and AAV.hu37 capsid proteins and the nucleic acid sequence of AAV2 5’- and 3’ ITRs. Table 9: Structure SEQ ID Sequence 5-ITR 73 cgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcga cctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggcca actccatcactaggggttcct Rep protein binding site (rps) is underlined. 3-ITR 74 aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctca ctgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcct cagtgagcgagcgagcgcgcag Structure SEQ ID Sequence Rep protein binding site (rps) is underlined. AAV8 Capsid 77 MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLQAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPARK RLNFGQTGDS ESVPDPQPLG EPPAAPSGVG PNTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL PTYNNHLYKQ ISNGTSGGAT NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFQFTYTFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQTTGGTANT QTLGFSQGGP NTMANQAKNW LPGPCYRQQR VSTTTGQNNN SNFAWTAGTK YHLNGRNSLA NPGIAMATHK DDEERFFPSN GILIFGKQNA ARDNADYSDV MLTSEEEIKT TNPVATEEYG IVADNLQQQN TAPQIGTVNS QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP PTTFNQSKLN SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TSVDFAVNTE GVYSEPRPIG TRYLTRNL AAV9 Capsid 78 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV YSEPRPIGTR YLTRNL hu.37 Capsid 112 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEAAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPAKK RLNFGQTGDS ESVPDPQPIG EPPAGPSGLG SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFEFSYTFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQSTGGTQGT QQLLFSQAGP ANMSAQAKNW LPGPCYRQQR VSTTLSQNNN SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS GVLMFGKQGA GRDNVDYSSV MLTSEEEIKT TNPVATEQYG WADNLQQTN TGPIVGNVNS QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TNVDFAVNTE GTYSEPRPIG TRYLTRNL Structure SEQ ID Sequence hu.31 Capsid 113 MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGSQPAKKK LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGGQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVSTEGV YSEPRPIGTR YLTRNL hu.32 Capsid 114 MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGSQPAKKK LNFGQTGDTE SVPDPGQPIG EPPAAPSGVG SLTMASGGGA PVADNNEGAD GVGSSSGNWH CDSQWLGDRV ITTSTRTWAL PTYNNHLYKQ ISNSTSGGSS NDNAYFGYST PWGYFDFNRF HCHFSPRDWQ RLINNNWGFR PKRLNFKLFN IQVKEVTDNN GVKTIANNLT STVQVFTDSD YQLPYVLGSA HEGCLPPFPA DVFMIPQYGY LTLNDGSQAV GRSSFYCLEY FPSQMLRTGN NFQFSYEFEN VPFHSSYAHS QSLDRLMNPL IDQYLYYLSK TINGSGQNQQ TLKFSVAGPS NMAVQGRNYI PGPSYRQQRV STTVTQNNNS EFAWPGASSW ALNGRNSLMN PGPAMASHKE GEDRFFPLSG SLIFGKQGTG RDNVDADKVM ITNEEEIKTT NPVATESYGQ VATNHQSAQA QAQTGWVQNQ GILPGMVWQD RDVYLQGPIW AKIPHTDGNF HPSPLMGGFG MKHPPPQILI KNTPVPADPP TAFNKDKLNS FITQYSTGQV SVEIEWELQK ENSKRWNPEI QYTSNYYKSN NVEFAVNTEG VYSEPRPIGT RYLTRNL Rh.74 version 1 127 MAADGYLPD WLEDNLSEG IREWWDLKP GAPKPKANQ QKQDNGRGL VLPGYKYLG PFNGLDKGE PVNAADAAA LEHDKAYDQ QLQAGDNPY LRYNHADAE FQERLQEDT SFGGNLGRA VFQAKKRVL EPLGLVESP VKTAPGKKR PVEPSPQRS PDSSTGIGK KGQQPAKKR LNFGQTGDS ESVPDPQPI GEPPAGPSG LGSGTMAAG GGAPMADNN EGADGVGSS SGNWHCDST WLGDRVITT STRTWALPT YNNHLYKQI SNGTSGGST NDNTYFGYS TPWGYFDFN RFHCHFSPR DWQRLINNN WGFRPKRLN FKLFNIQVK EVTQNEGTK TIANNLTST IQVFTDSEY QLPYVLGSA HQGCLPPFP ADVFMIPQY GYLTLNNGS QAVGRSSFY CLEYFPSQM LRTGNNFEF SYNFEDVPF HSSYAHSQS LDRLMNPLI DQYLYYLSR TQSTGGTAG TQQLLFSQA GPNNMSAQA KNWLPGPCY RQQRVSTTL SQNNNSNFA WTGATKYHL NGRDSLVNP GVAMATHKD DEERFFPSS GVLMFGKQG AGKDNVDYS SVMLTSEEE IKTTNPVAT EQYGWADN LQQQNAAPI VGAVNSQGA LPGMVWQNR DVYLQGPIW AKIPHTDGN FHPSPLMGG FGLKHPPPQ ILIKNTPVP ADPPTTFNQ AKLASFITQ Structure SEQ ID Sequence YSTGQVSVE IEWELQKEN SKRWNPEIQ YTSNYYKST NVDFAVNTE GTYSEPRPI GTRYLTRNL Rh.74 version 2 85 MAADGYLPD WLEDNLSEG IREWWDLKP GAPKPKANQ QKQDNGRGL VLPGYKYLG PFNGLDKGE PVNAADAAA LEHDKAYDQ QLQAGDNPY LRYNHADAE FQERLQEDT SFGGNLGRA VFQAKKRVL EPLGLVESP VKTAPGKKR PVEPSPQRS PDSSTGIGK KGQQPAKKR LNFGQTGDS ESVPDPQPI GEPPAAPSG VGPNTMAAG GGAPMADNN EGADGVGSS SGNWHCDST WLGDRVITT STRTWALPT YNNHLYKQI SNGTSGGST NDNTYFGYS TPWGYFDFN RFHCHFSPR DWQRLINNN WGFRPKRLN FKLFNIQVK EVTQNEGTK TIANNLTST IQVFTDSEY QLPYVLGSA HQGCLPPFP ADVFMIPQY GYLTLNNGS QAVGRSSFY CLEYFPSQM LRTGNNFEF SYNFEDVPF HSSYAHSQS LDRLMNPLI DQYLYYLSR TQSTGGTAG TQQLLFSQA GPNNMSAQA KNWLPGPCY RQQRVSTTL SQNNNSNFA WTGATKYHL NGRDSLVNP GVAMATHKD DEERFFPSS GVLMFGKQG AGKDNVDYS SVMLTSEEE IKTTNPVAT EQYGWADN LQQQNAAPI VGAVNSQGA LPGMVWQNR DVYLQGPIW AKIPHTDGN FHPSPLMGG FGLKHPPPQ ILIKNTPVP ADPPTTFNQ AKLASFITQ YSTGQVSVE IEWELQKEN SKRWNPEIQ YTSNYYKST NVDFAVNTE GTYSEPRPI GTRYLTRNL

[00135] The provided methods are suitable for use in the production of recombinant AAV encoding a transgene. In certain embodiments, the transgene is a microdystrophin as described herein. In some embodiments, the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter / enhancers, b) a poly A signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the described transgene. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 or AAV8 inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a muscle-specific SPc5.12 promoter and a small poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid encoding microdystrophin as described herein. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle-specific SPc5.12 promoter, b) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus, ABD 1 -H1 -RI -R2-R3-H3 -R24-H4-CR, ABD 1 -H1 -RI -R2-R3-H3 -R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) a CNS promoter, b) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus, ABD1-H1-R1-R2-R3-H3-R24-H4-CR, ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle-specific SPc5.12 promoter, b) an intron (e.g., VH4) and c) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus ABD 1 -H1 -RI -R2-R3-H3 -R24-H4-CR, ABD 1 -H1 -RI -R2-R3-H3 -R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT, ABDI being directly coupled to VH4. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) a CNS promoter, b) an intron (e.g., VH4) and c) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus ABD 1 -H1 -RI -R2-R3-H3 -R24-H4-CR, ABD 1 -H1 -RI -R2-R3-H3 -R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT, ABDI being directly coupled to VH4. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) a minimal SPc promoter for muscle-specific transgene expression, b) optionally, a human immunoglobin heavy chain variable region intron (e.g., VH4) and c) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus ABD1-H1-R1-R2-R3-H3-R24-H4-CR, ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) the musclespecific SPc5.12 promoter or a CNS promoter, b) an intron (e.g., VH4) and c) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus ABD1-H1-R1-R2-R3-H2-R24-H4-CR, ABD1-H1-R1-R2-R3-H2-R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT, ABDI being directly coupled to VH4. In some embodiments, constructs described herein comprising AAV ITRs flanking a microdystrophin expression cassette, which includes from the N-terminus to the C-terminus ABD1-H1-R1-R2-R3-H2-R24-H4-CR, ABD1-H1-R1-R2-R3-H2-R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT can be between 4000 nt and 5000 nt in length. In some embodiments, such constructs are less than 4900 nt, 4800 nt, 4700 nt, 4600 nt, 4500 nt, 4400 nt, or 4300 nt in length.

[00136] Some nucleic acid embodiments of the present disclosure comprise rAAV vectors encoding microdystrophin comprising or consisting of a nucleotide sequence of SEQ ID NO: 53, 54, 55, 56, or 82 provided in Table 10 below. In various embodiments, an rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 53, 54, 55, 56, 82 or the reverse complement thereof and encodes a rAAV vector suitable for expression of a therapeutically effective microdystrophin in muscle cells. Table 10: RGX-DYS cassette nucleotide sequences Structure SEQ ID Nucleic Acid Sequence RGX-DYS 1 (full cassette SPc5-12 to polyA including intervening seqs) 53 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGcagggtaatggggatcctCTAGAG GCCGTCCGCCCTCGGCACCATCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTAGAGCG GTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAA AATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTG Structure SEQ ID Nucleic Acid Sequence 4734bp ITRs shown in lower case GACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATA TTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGG GCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGC GCCAAGCGgAATTCGCCACCATGCTTTGGTGGGAAGAGGT GGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGG CAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTGCATGCCC TGAACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAA CAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACA TCATCCTGCAC T GGCAAGT GAAGAAT GT GAT GAAGAACAT CATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGT CAGACAGAGCAC CAGAAAC TAC C C T CAAG TGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTG TTTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAC AGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCT GGGAATTGAGAAACTGCTGGACCCTGAGGATGTGGACACC ACCTAT CCT GACAAGAAAT CCAT CCT CAT GTACAT CACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGC CATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTG ACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACT ACT CT CAGCAGAT CACAGT GT CT CT GGCCCAGGGATAT GA GAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCC TAGACACAG GCTGCCTATGTGACCACATCTGACCC CACAA GAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGA CAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAAC CTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCT GGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCAC ACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGG GCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGAT T GGCACAGGCAAGCT GTCT GAGGAT GAAGAGACAGAGGT G CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTC T GAGAGT GGCCAGCAT GGAAAAGCAGAGCAACCT GCACAG AGT GC T CAT GGAC C T GCAGAAT CAGAAAC T GAAAGAAC T G AAT GACTGGCTGAC CAAGACAGAAGAAAGGAC TAGGAAGA T GGAAGAGGAAC CTCTGGGACCAGACC T GGAAGAT C T GAA AAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTT GAGCAAGAGCAAGT GAGAGT GAACAGCCT GACACACAT GG TGGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGC TGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGG GCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGC TCCAGGACATTCTGCTGAAGTGGCAGAGACTGACAGAGGA ACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGAT GCT GT CAACAAGAT CCATACCACAGGCT T CAAGGAT CAGA Structure SEQ ID Nucleic Acid Sequence ATGAGATGCTCAGCTCCCTGCAGAAACTGGCTGTGCTGAA GGCTGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAGCTC TACAGCCTGAAGCAGGACCTGCTGTCTACCCTGAAGAACA AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTT TGCTAGATGCTGGGACAACCTGGTGCAGAAGCTGGAAAAG TCTACAGCCCAGATCAGCCAGCAACCTGATCTTGCCCCTG GCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTGAC CCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCC CCACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGA TGAGCTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAA GGCAGCTGGCAGCCAGTTGGGGACCTGCTCATTGATAGCC TGCAGGACCATCTGGAAAAAGTGAAAGCCCTGAGGGGAGA GATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGAC CTGGCCAGACAGCTGACCACACTGGGAATCCAGCTGAGCC CCTACAACCTGAGCACCCTTGAGGACCTGAACACCAGGTG GAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAG CTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGC ACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGC TATCTCTCCTAACAAGGTGCCCTACTACATCAACCATGAG ACACAGACCACCT GT T GGGAT CACCCCAAGAT GACAGAGC TGTACCAGAGTCTGGCAGACCTCAACAATGTCAGATTCAG TGCCTACAGGACTGCCATGAAGCTCAGAAGGCTCCAGAAA GCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTG AT GC C C T GGAC CAGCACAAT C T GAAGCAGAAT GAC CAGC C TAT GGACAT CC T C CAGAT CAT CAAC TGCCTCACCACCATC TAT GATAGGCT GGAACAAGAGCACAACAAT CT GGT CAAT G TGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAA T GT GTAT GACACAGGCAGAACAGGCAGGAT CAGAGT CCT G TCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACT TGGAGGACAAGTACAGATACCTGTTCAAGCAAGTGGCCTC CAGCACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTC CTGCATGACAGCATTCAGATCCCTAGACAGCTGGGAGAAG TGGCTTCCTTTGGAGGCAGCAATATTGAGCCATCAGTCAG GTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAGATTGAG GCTGCCCTGTTCCTGGACTGGATGAGACTTGAGCCTCAGA GCATGGTCTGGCTGCCTGTGCTTCATAGAGTGGCTGCTGC T GAGACT GC CAAGCACCAGGCCAAGT GCAACAT CT GCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGC ACTTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGG CAGGGTTGCCAAGGGCCACAAAATGCACTACCCCATGGTG GAATACTGCACCCCAACAACCTCTGGGGAAGATGTTAGAG ACT T T GCCAAGGT GCT GAAAAACAAGT T CAGGACCAAGAG ATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTC CAGACAGTGCTTGAGGGTGACAACATGGAAACCCCTGTGA CACTGATCAATTTCTGGCCAGTGGACTCTGCCCCTGCCTC AAGT C CACAGC T GT C C CAT GAT GACAC C CACAGCAGAAT T GAGCACTATGCCTCCAGACTGGCAGAGATGGAAAACAGCA ATGGCAGCTACCTGAATGATAGCATCAGCCCCAATGAGAG Structure SEQ ID Nucleic Acid Sequence CATTGATGATGAGCATCTGCTGATCCAGCACTACTGTCAG TCCCTGAACCAGGACTCTCCACTGAGCCAGCCTAGAAGCC CTGCTCAGATCCTGATCAGCCTTGAGTCTGAGGAAAGGGG AGAGCT GGAAAGAAT CCT GGCAGAT CT T GAGGAAGAGAAC AGAAACCTGCAGGCAGAGTATGACAGGCTCAAACAGCAGC ATGAGCACAAGGGACTGAGCCCTCTGCCTTCTCCTCCTGA AATGATGCCCACCTCTCCACAGTCTCCAAGGTGATGACTC GAGAGGCCTAATAAAGAGCT CAGAT GCAT CGAT CAGAGT G TGTTGGTTTTTTGTGTGCCAGGGTAATGGGCTAGCTGCGG CCGCaggaacccctagtgatggagttggccactccctctc tgcgcgctcgctcgctcactgaggccgggcgaccaaaggt cgcccgacgcccgggctttgcccgggcggcctcagtgagc gagcgagcgcgcag RGX-DYS2 (full cassette SPc5-12 to polyA including intervening seqs) 4814 bp ITRs shown in lower case 54 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGcagggtaatggggatcctCTAGAG GCCGTCCGCCCTCGGCACCATCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTAGAGCG GTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAA AATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTG GACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATA TTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGG GCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGC GCCAAGGTGAGTATCTCAGGGATCCAGACATGGGGATATG GGAGGTGCCTCTGATCCCAGGGCTCACTGTGGGTCTCTCT GTTCACAGGAATTCGCCACCATGCTTTGGTGGGAAGAGGT GGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGG CAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTGCATGCCC TGAACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAA CAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACA TCATCCTGCAC T GGCAAGT GAAGAAT GT GAT GAAGAACAT CATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGT CAGACAGAGCAC CAGAAAC TAC C C T CAAG TGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTG TTTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAC AGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCT GGGAATTGAGAAACTGCTGGACCCTGAGGATGTGGACACC ACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGC CATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTG ACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACT ACT CT CAGCAGAT CACAGT GT CT CT GGCCCAGGGATAT GA Structure SEQ ID Nucleic Acid Sequence GAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCC TAGACACAG GCTGCCTATGTGACCACATCTGACCC CACAA GAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGA CAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAAC CTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCT GGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCAC ACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGG GCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGAT T GGCACAGGCAAGCT GTCT GAGGAT GAAGAGACAGAGGT G CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTC T GAGAGT GGCCAGCAT GGAAAAGCAGAGCAACCT GCACAG AGT GC T CAT GGAC C T GCAGAAT CAGAAAC T GAAAGAAC T G AAT GACTGGCTGAC CAAGACAGAAGAAAGGAC TAGGAAGA T GGAAGAGGAAC CTCTGGGACCAGACC T GGAAGAT C T GAA AAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTT GAGCAAGAGCAAGT CAGAGT GAACAGCCT GACACACAT GG TGGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGC TGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGG GCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGC TCCAGGACATTCTGCTGAAGTGGCAGAGACTGACAGAGGA ACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGAT GCT GT CAACAAGAT CCATACCACAGGCT T CAAGGAT CAGA ATGAGATGCTCAGCTCCCTGCAGAAACTGGCTGTGCTGAA GGCTGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAGCTC TACAGCCTGAAGCAGGACCTGCTGTCTACCCTGAAGAACA AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTT TGCTAGATGCTGGGACAACCTGGTGCAGAAGCTGGAAAAG TCTACAGCCCAGATCAGCCAGCAACCTGATCTTGCCCCTG GCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTGAC CCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCC CCACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGA TGAGCTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAA GGCAGCTGGCAGCCAGTTGGGGACCTGCTCATTGATAGCC TGCAGGACCATCTGGAAAAAGTGAAAGCCCTGAGGGGAGA GATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGAC CTGGCCAGACAGCTGACCACACTGGGAATCCAGCTGAGCC CCTACAACCTGAGCACCCTTGAGGACCTGAACACCAGGTG GAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAG CTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGC ACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGC TATCTCTCCTAACAAGGTGCCCTACTACATCAACCATGAG ACACAGACCACCT GT T GGGAT CACCCCAAGAT GACAGAGC TGTACCAGAGTCTGGCAGACCTCAACAATGTCAGATTCAG TGCCTACAGGACTGCCATGAAGCTCAGAAGGCTCCAGAAA GCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTG AT GC C C T GGAC CAGCACAAT C T GAAGCAGAAT GAC CAGC C TAT GGACAT CC T C CAGAT CAT CAAC TGCCTCACCACCATC Structure SEQ ID Nucleic Acid Sequence TAT GATAGGCT GGAACAAGAGCACAACAAT CT GGT CAAT G TGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAA T GT GTAT GACACAGGCAGAACAGGCAGGAT CAGAGT GGT G TCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACT TGGAGGACAAGTACAGATACCTGTTCAAGCAAGTGGCCTC CAGCACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTC CTGCATGACAGCATTCAGATCCCTAGACAGCTGGGAGAAG TGGCTTCCTTTGGAGGCAGCAATATTGAGCCATCAGTCAG GTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAGATTGAG GCTGCCCTGTTCCTGGACTGGATGAGACTTGAGCCTCAGA GCATGGTCTGGCTGCCTGTGCTTCATAGAGTGGCTGCTGC T GAGACT GC CAAGCACCAGGCCAAGT GCAACAT CT GCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGC ACTTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGG CAGGGTTGCCAAGGGCCACAAAATGCACTACCCCATGGTG GAATACTGCACCCCAACAACCTCTGGGGAAGATGTTAGAG ACT T T GCCAAGGT GCT GAAAAACAAGT T CAGGACCAAGAG ATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTC CAGACAGTGCTTGAGGGTGACAACATGGAAACCCCTGTGA CACTGATCAATTTCTGGCCAGTGGACTCTGCCCCTGCCTC AAGT C CACAGC T GT C C CAT GAT GACAC C CACAGCAGAAT T GAGCACTATGCCTCCAGACTGGCAGAGATGGAAAACAGCA ATGGCAGCTACCTGAATGATAGCATCAGCCCCAATGAGAG CATTGATGATGAGCATCTGCTGATCCAGCACTACTGTCAG TCCCTGAACCAGGACTCTCCACTGAGCCAGCCTAGAAGCC CTGCTCAGATCCTGATCAGCCTTGAGTCTGAGGAAAGGGG AGAGCT GGAAAGAAT CCT GGCAGAT CT T GAGGAAGAGAAC AGAAACCTGCAGGCAGAGTATGACAGGCTCAAACAGCAGC ATGAGCACAAGGGACTGAGCCCTCTGCCTTCTCCTCCTGA AATGATGCCCACCTCTCCACAGTCTCCAAGGTGATGACTC GAGAGGCCTAATAAAGAGCT CAGAT GCAT CGAT CAGAGT G TGTTGGTTTTTTGTGTG CCAGGGTAATGGGCTAGCTGCGGCCGCaggaacccctagt gatggagttggccactccctctctgcgcgctcgctcgctc actgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgcag RGX-DYS3 (full cassette SPc5-12 to polyA including intervening seqs) 4364 bp) ITRs shown in lower case 55 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGcagggtaatggggatcctCTAGAG GCCGTCCGCCCTCGGCACCATCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTAGAGCG GTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAA AATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTG GACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATA TTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGG GCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGC GCCAAGGTGAGTATCTCAGGGATCCAGACATGGGGATATG Structure SEQ ID Nucleic Acid Sequence GGAGGTGCCTCTGATCCCAGGGCTCACTGTGGGTCTCTCT GTTCACAGGAATTCGCCACCATGCTTTGGTGGGAAGAGGT GGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGG CAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTGCATGCCC TGAACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAA CAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACA TCATCCTGCAC T GGCAAGT GAAGAAT GT GAT GAAGAACAT CATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGT CAGACAGAGCAC CAGAAAC TAC C C T CAAG TGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTG TTTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAC AGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCT GGGAATTGAGAAACTGCTGGACCCTGAGGATGTGGACACC ACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGC CATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTG ACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACT ACT CT CAGCAGAT CACAGT GT CT CT GGCCCAGGGATAT GA GAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCC TAGACACAG GCTGCCTATGTGACCACATCTGACCC CACAA GAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGA CAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAAC CTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCT GGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCAC ACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGG GCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGAT T GGCACAGGCAAGCT GTCT GAGGAT GAAGAGACAGAGGT G CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTC T GAGAGT GGCCAGCAT GGAAAAGCAGAGCAACCT GCACAG AGT GC T CAT GGAC C T GCAGAAT CAGAAAC T GAAAGAAC T G AAT GACTGGCTGAC CAAGACAGAAGAAAGGAC TAGGAAGA T GGAAGAGGAAC CTCTGGGACCAGACC T GGAAGAT C T GAA AAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTT GAGCAAGAGCAAGT GAGAGT GAACAGCCT GACACACAT GG TGGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGC TGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGG GCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGC TCCAGGACATTCTGCTGAAGTGGCAGAGACTGACAGAGGA ACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGAT GCT GT CAACAAGAT CCATACCACAGGCT T CAAGGAT CAGA ATGAGATGCTCAGCTCCCTGCAGAAACTGGCTGTGCTGAA GGCTGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAGCTC TACAGCCTGAAGCAGGACCTGCTGTCTACCCTGAAGAACA Structure SEQ ID Nucleic Acid Sequence AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTT TGCTAGATGCTGGGACAACCTGGTGCAGAAGCTGGAAAAG TCTACAGCCCAGATCAGCCAGCAACCTGATCTTGCCCCTG GCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTGAC CCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCC CCACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGA TGAGCTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAA GGCAGCTGGCAGCCAGTTGGGGACCTGCTCATTGATAGCC TGCAGGACCATCTGGAAAAAGTGAAAGCCCTGAGGGGAGA GATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGAC CTGGCCAGACAGCTGACCACACTGGGAATCCAGCTGAGCC CCTACAACCTGAGCACCCTTGAGGACCTGAACACCAGGTG GAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAG CTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGC ACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGC TATCTCTCCTAACAAGGTGCCCTACTACATCAACCATGAG ACACAGACCACCT GT T GGGAT CACCCCAAGAT GACAGAGC TGTACCAGAGTCTGGCAGACCTCAACAATGTCAGATTCAG TGCCTACAGGACTGCCATGAAGCTCAGAAGGCTCCAGAAA GCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTG AT GC C C T GGAC CAGCACAAT C T GAAGCAGAAT GAC CAGC C TAT GGACAT CC T C CAGAT CAT CAAC TGCCTCACCACCATC TAT GATAGGCT GGAACAAGAGCACAACAAT CT GGT CAAT G TGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAA T GT GTAT GACACAGGCAGAACAGGCAGGAT CAGAGT CCT G TCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACT TGGAGGACAAGTACAGATACCTGTTCAAGCAAGTGGCCTC CAGCACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTC CTGCATGACAGCATTCAGATCCCTAGACAGCTGGGAGAAG TGGCTTCCTTTGGAGGCAGCAATATTGAGCCATCAGTCAG GTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAGATTGAG GCTGCCCTGTTCCTGGACTGGATGAGACTTGAGCCTCAGA GCATGGTCTGGCTGCCTGTGCTTCATAGAGTGGCTGCTGC T GAGACT GC CAAGCACCAGGCCAAGT GCAACAT CT GCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGC ACTTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGG CAGGGTTGCCAAGGGCCACAAAATGCACTACCCCATGGTG GAATACTGCACCCCAACAACCTCTGGGGAAGATGTTAGAG ACT T T GCCAAGGT GCT GAAAAACAAGT T CAGGACCAAGAG ATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTC CAGACAGTGCTTGAGGGTGACAACATGGAAACCTGATGAG T CGACAGGC CTAATAAAGAGCT CAGAT GCATCGAT CAGAG TGTGTTGGTTTTTTGTGTG GCTAGCTGCGGCCGCaggaacccctagtgatggagttggc cactccctctctgcgcgctcgctcgctcactgaggccggg cgaccaaaggtcgcccgacgcccgggctttgcccgggcgg cctcagtgagcgagcgagcgcgcag Structure SEQ ID Nucleic Acid Sequence RGX-DYS4 (full cassette mini-SPc5-12 to polyA including intervening seqs) 4661 bp ITRs shown in lower case 56 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGcagggtaatggggatcctCTAGAG AATGGTGGACACCCAAATATGGCGACGGTTCCTCACCCGT CGCCATATTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTC CTGGGGGCCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGG CTCCGGGGCCGGCGGCGGCCCACGAGCTACCCGGAGGAGC GGGAGGCGCCAAGGTGAGTATCTCAGGGATCCAGACATGG GGATATGGGAGGTGCCTCTGATCCCAGGGCTCACTGTGGG TCTCTCTGTTCACAGGAATTCGCCACCATGCTTTGGTGGG AAGAGGTGGAAGATTGCTATGAGAGGGAAGATGTGCAGAA GAAAACCTTCACCAAATGGGTCAATGCCCAGTTCAGCAAG TTTGGCAAGCAGCACATTGAGAACCTGTTCAGTGACCTGC AGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGCCTGAC AGGCCAGAAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTG CATGCCCTGAACAATGTGAACAAGGCCCTGAGAGTGCTGC AGAACAACAAT GT GGACCT GGT CAATAT T GGCAGCACAGA CATTGTGGATGGCAACCACAAGCTGACCCTGGGCCTGATC T GGAACATCAT GGT GCACT GGCAAGT GAAGAAT GT GAT GA AGAACATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAA GATCCTGCTGAGCTGGGTCAGACAGAGCACCAGAAACTAC CCTCAAGTGAATGTGATCAACTTCACCACCTCTTGGAGTG ATGGACTGGCCCTGAATGCCCTGATCCACAGCCACAGACC TGACCTGTTTGACTGGAACTCTGTTGTGTGCCAGCAGTCT GCCACACAGAGACT GGAACAT GCCT T CAACAT T GCCAGAT ACCAGCTGGGAATTGAGAAACTGCTGGACCCTGAGGATGT GGACACCACCTATCCTGACAAGAAATCCATCCTCATGTAC ATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCA TTGAGGCCATTCAAGAGGTTGAGATGCTGCCCAGACCTCC TAAAGTGACCAAAGAGGAACACTTCCAGCTGCACCACCAG ATGCACTACTCTCAGCAGATCACAGTGTCTCTGGCCCAGG GATATGAGAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAG CTATGCCTACACACAGGCTGCCTATGTGACCACATCTGAC CCCACAAGAAGCCCATTTCCAAGCCAGCATCTGGAAGCCC CTGAGGACAAGAGCTTTGGCAGCAGCCTGATGGAATCTGA AGT GAAC CT GGATAGATAC CAGACAGC C C T GGAAGAAGT G CTGTCCTGGCTGCTGTCTGCTGAGGATACACTGCAGGCTC AGGGTGAAATCAGCAATGATGTGGAAGTGGTCAAGGACCA GTTTCACACCCATGAGGGCTACATGATGGACCTGACAGCC CACCAGGGCAGAGTGGGAAATATCCTGCAGCTGGGCTCCA AGCTGATTGGCACAGGCAAGCTGTCTGAGGATGAAGAGAC AGAGGTGCAAGAGCAGATGAACCTGCTGAACAGCAGATGG GAGTGTCTGAGAGTGGCCAGCATGGAAAAGCAGAGCAACC T GCACAGAG TGCTCATGGACC T GCAGAAT CAGAAAC T GAA AGAACTGAATGACTGGCTGACCAAGACAGAAGAAAGGACT AGGAAGATGGAAGAGGAACCTCTGGGACCAGACCTGGAAG ATCTGAAAAGACAGGTGCAGCAGCATAAGGTGCTGCAAGA GGACCTTGAGCAAGAGCAAGTCAGAGTGAACAGCCTGACA Structure SEQ ID Nucleic Acid Sequence CACATGGTGGTGGTTGTGGATGAGTCCTCTGGGGATCATG CCACAGCTGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGA CAGAT GGGC CAACAT CTGTAGGT GGACAGAGGATAGAT GG GTGCTGCTCCAGGACATTCTGCTGAAGTGGCAGAGACTGA CAGAGGAACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAA AGAGGAT GC T GT CAACAAGAT C CATAC CACAGGC T T CAAG GATCAGAATGAGATGCTCAGCTCCCTGCAGAAACTGGCTG TGCTGAAGGCTGACCTGGAAAAGAAAAAGCAGTCCATGGG CAAGCTCTACAGCCTGAAGCAGGACCTGCTGTCTACCCTG AAGAACAAGTCTGTGACCCAGAAAACTGAGGCCTGGCTGG ACAACTTTGCTAGATGCTGGGACAACCTGGTGCAGAAGCT GGAAAAGTCTAGAGCCCAGATCAGCCAGCAACCTGATCTT GCCCCTGGCCTGACCACAATTGGAGCCTCTCCAACACAGA CTGTGACCCTGGTTACCCAGCCAGTGGTCACCAAAGAGAC AGCCATCAGCAAACTGGAAATGCCCAGCTCTCTGATGCTG GAAGTCCCCACACTGGAAAGGCTGCAAGAACTTCAAGAGG CCACAGATGAGCTGGACCTGAAGCTGAGACAGGCTGAAGT GATCAAAGGCAGCTGGCAGCCAGTTGGGGACCTGCTCATT GATAGCCTGCAGGACCATCTGGAAAAAGTGAAAGCCCTGA GGGGAGAGATTGCCCCTCTGAAAGAAAATGTGTCCCATGT GAATGACCTGGCCAGACAGCTGACCACACTGGGAATCCAG CTGAGCCCCTACAACCTGAGCACCCTTGAGGACCTGAACA CCAGGTGGAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGT CAGGCAGCTGCATGAGGCCCACAGAGATTTTGGACCAGCC AGCCAGCACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGG AGAGAGCTATCTCTCCTAACAAGGTGCCCTACTACATCAA C CAT GAGACACAGAC CAC CTGTTGGGATCACCC CAAGAT G ACAGAGCTGTACCAGAGTCTGGCAGACCTCAACAATGTCA GATTCAGTGCCTACAGGACTGCCATGAAGCTCAGAAGGCT CCAGAAAGCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCA GCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAATG ACCAGCCTATGGACATCCTCCAGATCATCAACTGCCTCAC CACCATCTATGATAGGCTGGAACAAGAGCACAACAATCTG GTCAATGTGCCCCTGTGTGTGGACATGTGCCTGAATTGGC TGCTGAATGTGTATGACACAGGCAGAACAGGCAGGATCAG AGTCCTGTCCTTCAAGACAGGCATCATCTCCCTGTGCAAA GCCCACTTGGAGGACAAGTACAGATACCTGTTCAAGCAAG TGGCCTCCAGCACAGGCTTTTGTGACCAGAGAAGGCTGGG CCTGCTCCTGCATGACAGCATTCAGATCCCTAGACAGCTG GGAGAAGTGGCTTCCTTTGGAGGCAGCAATATTGAGCCAT CAGT CAGGT CCT GT T T TCAGT T T GCCAACAACAAGCCT GA GATTGAGGCTGCCCTGTTCCTGGACTGGATGAGACTTGAG CCTCAGAGCATGGTCTGGCTGCCTGTGCTTCATAGAGTGG CTGCTGCTGAGACTGCCAAGCACCAGGCCAAGTGCAACAT CTGCAAAGAGTGCCCCATCATTGGCTTCAGATACAGATCC CTGAAGCACTTCAACTATGATATCTGCCAGAGCTGCTTCT TTAGTGGCAGGGTTGCCAAGGGCCACAAAATGCACTACCC CATGGTGGAATACTGCACCCCAACAACCTCTGGGGAAGAT GTTAGAGACTTTGCCAAGGTGCTGAAAAACAAGTTCAGGA - Ill - Structure SEQ ID Nucleic Acid Sequence CCAAGAGATACTTTGCTAAGCACCCCAGAATGGGCTACCT GCCTGTCCAGACAGTGCTTGAGGGTGACAACATGGAAACC CCTGTGACACTGATCAATTTCTGGCCAGTGGACTCTGCCC C T GC C T CAAGT C CACAGC T GT C C CAT GAT GACAC C CACAG CAGAATTGAGCACTATGCCTCCAGACTGGCAGAGATGGAA AACAGCAATGGCAGCTACCTGAATGATAGCATCAGCCCCA ATGAGAGCATTGATGATGAGCATCTGCTGATCCAGCACTA CTGTCAGTCCCTGAACCAGGACTCTCCACTGAGCCAGCCT AGAAGCCCTGCTCAGATCCTGATCAGCCTTGAGTCTGAGG AAAGGGGAGAGCTGGAAAGAATCCTGGCAGATCTTGAGGA AGAGAACAGAAACCTGCAGGCAGAGTATGACAGGCTCAAA CAGCAGCATGAGCACAAGGGACTGAGCCCTCTGCCTTCTC CTCCTGAAATGATGCCCACCTCTCCACAGTCTCCAAGGTG ATGACTCGAGAGGCCTAATAAAGAGCTCAGATGCATCGAT CAGAGTGTGTTGGTTTTTTGTGTG CCAGGGTAATGGGCTAGCTGCGGCCGCaggaacccctagt gatggagttggccactccctctctgcgcgctcgctcgctc actgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgcag RGX-DYS5 (full cassette SPc5-12 to polyA including intervening seqs) 4560bp ITRs shown in lower case 82 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGcagggtaatggggatcctCTAGAG GCCGTCCGCCCTCGGCACCATCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTAGAGCG GTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAA AATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTG GACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATA TTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGG GCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGC GCCAAGCGGAATTCGCCACCATGCTTTGGTGGGAAGAGGT GGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGG CAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTGCATGCCC TGAACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAA CAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACA TCATCCTGCAC T GGCAAGT GAAGAAT GT GAT GAAGAACAT CATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGT CAGACAGAGCAC CAGAAAC TAC C C T CAAG TGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTG TTTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAC AGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCT GGGAATTGAGAAACTGCTGGACCCTGAGGATGTGGACACC ACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCA Structure SEQ ID Nucleic Acid Sequence GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGC CATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTG ACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACT ACT CT CAGCAGAT CACAGT GT CT CT GGCCCAGGGATAT GA GAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCC TAGACACAG GCTGCCTATGTGACCACATCTGACCC CACAA GAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGA CAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAAC CTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCT GGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCAC ACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGG GCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGAT T GGCACAGGCAAGCT GTCT GAGGAT GAAGAGACAGAGGT G CAAGAGCAGAT GAACCT GCT GAACAGCAGATGGGAGT GT C T GAGAGT GGCCAGCAT GGAAAAGCAGAGCAACCT GCACAG AGT GC T CAT GGAC C T GCAGAAT CAGAAAC T GAAAGAAC T G AAT GACTGGCTGAC CAAGACAGAAGAAAGGAC TAGGAAGA T GGAAGAGGAAC CTCTGGGACCAGACC T GGAAGAT C T GAA AAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTT GAGCAAGAGCAAGT CAGAGT GAACAGCCT GACACACAT GG TGGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGC TGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGG GCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGC TCCAGGACATTCTGCTGAAGTGGCAGAGACTGACAGAGGA ACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGAT GCT GT CAACAAGAT CCATACCACAGGCT T CAAGGAT CAGA ATGAGATGCTCAGCTCCCTGCAGAAACTGGCTGTGCTGAA GGCTGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAGCTC TACAGCCTGAAGCAGGACCTGCTGTCTACCCTGAAGAACA AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTT TGCTAGATGCTGGGACAACCTGGTGCAGAAGCTGGAAAAG TCTACAGCCCAGATCAGCCAGCAACCTGATCTTGCCCCTG GCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTGAC CCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCC CCACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGA TGAGCTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAA GGCAGCTGGCAGCCAGTTGGGGACCTGCTCATTGATAGCC TGCAGGACCATCTGGAAAAAGTGAAAGCCCTGAGGGGAGA GATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGAC CTGGCCAGACAGCTGACCACACTGGGAATCCAGCTGAGCC CCTACAACCTGAGCACCCTTGAGGACCTGAACACCAGGTG GAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAG CTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGC ACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGC TATCTCTCCTAACAAGGTGCCCTACTACATCAACCATGAG ACACAGACCACCT GT T GGGAT CACCCCAAGAT GACAGAGC TGTACCAGAGTCTGGCAGACCTCAACAATGTCAGATTCAG Structure SEQ ID Nucleic Acid Sequence TGCCTACAGGACTGCCATGAAGCTCAGAAGGCTCCAGAAA GCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTG AT GC C C T GGAC CAGCACAAT C T GAAGCAGAAT GAG CAGC C TAT GGACAT CC T C CAGAT CAT CAAC TGCCTCACCACCATC TAT GATAGGCT GGAACAAGAGCACAACAAT CT GGT CAAT G TGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAA T GT GTAT GACACAGGCAGAACAGGCAGGAT CAGAGT CCT G TCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACT TGGAGGACAAGTACAGATACCTGTTCAAGCAAGTGGCCTC CAGCACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTC CTGCATGACAGCATTCAGATCCCTAGACAGCTGGGAGAAG TGGCTTCCTTTGGAGGCAGCAATATTGAGCCATCAGTCAG GTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAGATTGAG GCTGCCCTGTTCCTGGACTGGATGAGACTTGAGCCTCAGA GCATGGTCTGGCTGCCTGTGCTTCATAGAGTGGCTGCTGC T GAGACT GC CAAGCACCAGGCCAAGT GCAACAT CT GCAAA GAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGC ACTTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGG CAGGGTTGCCAAGGGCCACAAAATGCACTACCCCATGGTG GAATACTGCACCCCAACAACCTCTGGGGAAGATGTTAGAG ACT T T GCCAAGGT GCT GAAAAACAAGT T CAGGACCAAGAG ATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTC CAGACAGTGCTTGAGGGTGACAACATGGAAACCCCTGTGA CACTGATCAATTTCTGGCCAGTGGACTCTGCCCCTGCCTC AAGT C CACAGC T GT C C CAT GAT GACAC C CACAGCAGAAT T GAGCACTATGCCTCCAGACTGGCAGAGATGGAAAACAGCA ATGGCAGCTACCTGAATGATAGCATCAGCCCCAATGAGAG CATTGATGATGAGCATCTGCTGATCCAGCACTACTGTCAG TCCCTGAACCAGGACTCTCCACTGAGCCAGCCTAGAAGCC CTGCTCAGATCCTGATCAGCCTTGAGTCTTGATGAGTCGA CAGGCCTAATAAAGAGCTCAGATGCATCGATCAGAGTGTG TTGGTTTTTTGTGTGGCTAGCTGCGGCCGCaggaacccct agtgatggagttggccactccctctctgcgcgctcgctcg ctcactgaggccgggcgaccaaaggtcgcccgacgcccgg gctttgcccgggcggcctcagtgagcgagcgagcgcgcag RGX-DYS6 (full cassette including flanking ITRs, Spc5-12 promoter to polyA and intervening seqs) 4584 bp 104 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGCAGGGTAATGGGGATCCTCTAGAG GCCGTCCGCCCTCGGCACCATCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTAGAGCG GTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAA AATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTG GACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATA TTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGG GCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGC GCCAAGCGgAATTCGCCACCATGCTTTGGTGGGAAGAGGT Structure SEQ ID Nucleic Acid Sequence ITRs shown in lower case GGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGG CAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTGCATGCCC TGAACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAA CAAT GT GGACCT GGT CAATAT T GGCAGCACAGACAT T GT G GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACA TCATCCTGCAC T GGCAAGT GAAGAAT GT GAT GAAGAACAT CATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGT CAGACAGAGCAC CAGAAAC TAC C C T CAAG TGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTG TTTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAC AGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCT GGGAATTGAGAAACTGCTGGACCCTGAGGATGTGGACACC ACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGC CATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTG ACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACT ACT CT CAGCAGAT CACAGT GT CT CT GGCCCAGGGATAT GA GAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCC TAGACACAG GCTGCCTATGTGACCACATCTGACCC CACAA GAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGA CAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAAC CTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCT GGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCAC ACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGG GCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGAT T GGCACAGGCAAGCT GTCT GAGGAT GAAGAGACAGAGGT G CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTC T GAGAGT GGCCAGCAT GGAAAAGCAGAGCAACCT GCACAG AGT GC T CAT GGAC C T GCAGAAT CAGAAAC T GAAAGAAC T G AAT GACTGGCTGAC CAAGACAGAAGAAAGGAC TAGGAAGA T GGAAGAGGAAC CTCTGGGACCAGACC T GGAAGAT C T GAA AAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTT GAGCAAGAGCAAGT GAGAGT GAACAGCCT GACACACAT GG TGGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGC TGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGG GCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGC TCCAGGACATTCTGCTGAAGTGGCAGAGACTGACAGAGGA ACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGAT GCT GT CAACAAGAT CCATACCACAGGCT T CAAGGAT CAGA ATGAGATGCTCAGCTCCCTGCAGAAACTGGCTGTGCTGAA GGCTGACCTGGAAAAGAAAAAGCAGTCCATGGGCAAGCTC TACAGCCTGAAGCAGGACCTGCTGTCTACCCTGAAGAACA AGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTT TGCTAGATGCTGGGACAACCTGGTGCAGAAGCTGGAAAAG Structure SEQ ID Nucleic Acid Sequence TCTACAGCCCAGATCAGCCAGCAACCTGATCTTGCCCCTG GCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTGAC CCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATC AGCAAACTGGAAATGCCCAGCTCTCTGATGCTGGAAGTCC CCACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGA TGAGCTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAA GGCAGCTGGCAGCCAGTTGGGGACCTGCTCATTGATAGCC TGCAGGACCATCTGGAAAAAGTGAAAGCCCTGAGGGGAGA GATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGAC CTGGCCAGACAGCTGACCACACTGGGAATCCAGCTGAGCC CCTACAACCTGAGCACCCTTGAGGACCTGAACACCAGGTG GAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAG CTGCATGAGGCCCACAGAGATTTTGGACCAGCCAGCCAGC ACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGC TATCTCTCCTAACAAGGTGCCCTACTACATCAACCATGAG ACACAGACCACCT GT T GGGAT CACCCCAAGAT GACAGAGC TGTACCAGAGTCTGGCAGACCTCAACAATGTCAGATTCAG TGCCTACAGGACTGCCATGAAGCTCAGAAGGCTCCAGAAA GCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTG AT GC C C T GGAC CAGCACAAT C T GAAGCAGAAT GAC CAGC C TAT GGACAT CC T C CAGAT CAT CAAC TGCCTCACCACCATC TAT GATAGGCT GGAACAAGAGCACAACAAT CT GGT CAAT G TGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAA T GT GTAT GACACAGGCAGAACAGGCAGGAT CAGAGT CCT G TCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACT TGGAGGACAAGTACAGATACCTGTTCAAGCAAGTGGCCTC CAGCACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTC CTGCATGACAGCATTCAGATCCCTAGACAGCTGGGAGAAG TGGCTTCCTTTGGAGGCGCCAAGCACCAGGCCAAGTGCAA CATCTGCAAAGAGTGCCCCATCATTGGCTTCAGATACAGA TCCCTGAAGCACTTCAACTATGATATCTGCCAGAGCTGCT TCTTTAGTGGCAGGGTTGCCAAGGGCCACAAAATGCACTA CCCCATGGTGGAATACTGCACCCCAACAACCTCTGGGGAA GATGTTAGAGACTTTGCCAAGGTGCTGAAAAACAAGTTCA GGACCAAGAGATACTTTGCTAAGCACCCCAGAATGGGCTA CCTGCCTGTCCAGACAGTGCTTGAGGGTGACAACATGGAA ACCCCTGTGACACTGATCAATTTCTGGCCAGTGGACTCTG CCCCTGCCTCAAGTCCACAGCTGTCCCATGATGACACCCA CAGCAGAATTGAGCACTATGCCTCCAGACTGGCAGAGATG GAAAACAGCAATGGCAGCTACCTGAATGATAGCATCAGCC C CAAT GAGAGCAT T GAT GAT GAGCAT C T GC T GAT C CAGCA CTACTGTCAGTCCCTGAACCAGGACTCTCCACTGAGCCAG CCTAGAAGCCCTGCTCAGATCCTGATCAGCCTTGAGTCTG AGGAAAGGG GAGAGC T GGAAAGAAT CCTGGCAGATCTTGA GGAAGAGAACAGAAACCTGCAGGCAGAGTATGACAGGCTC AAACAGCAGCATGAGCACAAGGGACTGAGCCCTCTGCCTT CTCCTCCTGAAATGATGCCCACCTCTCCACAGTCTCCAAG GTGATGACTCGAGAGGCCTAATAAAGAGCTCAGATGCATC GATCAGAGTGTGTTGGTTTTTTGTGTGCCAGGGTAATGGG Structure SEQ ID Nucleic Acid Sequence CTAGCTGCGGCCGCaggaacccctagtgatggagttggcc actccctctctgcgcgctcgctcgctcactgaggccgggc gaccaaaggtcgcccgacgcccgggctttgcccgggcggc ctcagtgagcgagcgagcgcgcag RGX-DYS7 (full cassette including flanking ITRs, Spc5-12 promoter to polyA and intervening seqs) 4746 bp ITRs shown in lower case 105 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGCAGGGTAATGGGGATCCTCTAGAG GCCGTCCGCCCTCGGCACCATCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTAGAGCG GTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAA AATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTG GACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATA TTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGG GCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGC GCCAAGCGgAATTCGCCACCATGCTTTGGTGGGAAGAGGT GGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGG CAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTGCATGCCC TGAACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAA CAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACA TCATCCTGCAC T GGCAAGT GAAGAAT GT GAT GAAGAACAT CATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGT CAGACAGAGCAC CAGAAAC TAC C C T CAAG TGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTG TTTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAC AGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCT GGGAATTGAGAAACTGCTGGACCCTGAGGATGTGGACACC ACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGC CATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTG ACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACT ACT CT CAGCAGAT CACAGT GT CT CT GGCCCAGGGATAT GA GAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCC TAGACACAG GCTGCCTATGTGACCACATCTGACCC CACAA GAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGA CAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAAC CTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCT GGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCAC ACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGG GCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGAT T GGCACAGGCAAGCT GTCT GAGGAT GAAGAGACAGAGGT G Structure SEQ ID Nucleic Acid Sequence CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTC T GAGAGT GGCCAGCAT GGAAAAGCAGAGCAACCT GCACAG AGT GC T CAT GGAC C T GCAGAAT CAGAAAC T GAAAGAAC T G AAT GACTGGCTGAC CAAGACAGAAGAAAGGAC TAGGAAGA T GGAAGAGGAAC CTCTGGGACCAGACC T GGAAGAT C T GAA AAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTT GAGCAAGAGCAAGT GAGAGT GAACAGCCT GACACACAT GG TGGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGC TGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGG GCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGC TCCAGGACATTCTGGAGATCAGCTATGTGCCCAGCACCTA CCTGACAGAGATCACCCATGTGTCTCAGGCCCTGCTGGAA GTGGAACAGCTGCTGAATGCCCCTGACCTGTGTGCCAAGG ACTTTGAGGACCTGTTCAAGCAAGAGGAAAGCCTGAAGAA CATCAAGGACAGCCTGCAGCAGTCCTCTGGCAGAATTGAC ATCATCCACAGCAAGAAAACAGCTGCCCTGCAGTCTGCCA CACCTGTGGAAAGAGTGAAGCTGCAAGAGGCCCTGAGCCA GCTGGACTTCCAGTGGGAGAAAGTGAACAAGATGTACAAG GACAGGCAGGGCAGAT TT GATAGAAGT GT GGAAAAGTGGA GAAGGT T CCACTAT GACAT CAAGAT CT T CAACCAGT GGCT GACAGAGGC TGAGCAGTTCCT GAGAAAGACACAGAT CC C T GAGAACTGGGAGCATGCCAAGTACAAGTGGTATCTGAAAG AACT GCAGGAT GGCAT TGGCCAGAGACAGACAGT T GTCAG AACCCTGAATGCCACAGGGGAAGAGATCATCCAGCAGAGC AGCAAGACAGATGCCAGCATCCTGCAAGAGAAGCTGGGCA GCCTGAACCTGAGATGGCAAGAAGTGTGCAAGCAGCTGTC TGACAGAAAGAAGAGGCTGGAAGAACAGACACTGGAAAGG CTGCAAGAACTTCAAGAGGCCACAGATGAGCTGGACCTGA AGCTGAGACAGGCTGAAGTGATCAAAGGCAGCTGGCAGCC AGTTGGGGACCTGCTCATTGATAGCCTGCAGGACCATCTG GAAAAAGTGAAAGCCCTGAGGGGAGAGATTGCCCCTCTGA AAGAAAATGTGTCCCATGTGAATGACCTGGCCAGACAGCT GACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGAGC ACCCTTGAGGACCTGAACACCAGGTGGAAGCTCCTCCAGG T GGCAGT GGAAGATAGAGT CAGGCAGCT GCAT GAGGCCCA CAGAGAT TTTGGACCAGC CAGC CAGCAC TTTCTGTCTACC TCTGTGCAAGGCCCCTGGGAGAGAGCTATCTCTCCTAACA AGGTGCCCTACTACATCAACCATGAGACACAGACCACCTG TTGGGATCACCCCAAGATGACAGAGCTGTACCAGAGTCTG GCAGACCTCAACAATGTCAGATTCAGTGCCTACAGGACTG CCATGAAGCTCAGAAGGCTCCAGAAAGCTCTGTGCCTGGA CCTGCTTTCCCTGAGTGCAGCTTGTGATGCCCTGGACCAG CACAATCTGAAGCAGAATGACCAGCCTATGGACATCCTCC AGAT CAT CAAC TGCCTCACCAC CAT C TAT GATAGGC T GGA ACAAGAGCACAACAATCTGGTCAATGTGCCCCTGTGTGTG GACAT GT GC CT GAAT T GGCT GCT GAAT GT GTAT GACACAG GCAGAACAGGCAGGATCAGAGTCCTGTCCTTCAAGACAGG CATCATCTCCCTGTGCAAAGCCCACTTGGAGGACAAGTAC AGATACCTGTTCAAGCAAGTGGCCTCCAGCACAGGCTTTT Structure SEQ ID Nucleic Acid Sequence GTGACCAGAGAAGGCTGGGCCTGCTCCTGCATGACAGCAT TCAGATCCCTAGACAGCTGGGAGAAGTGGCTTCCTTTGGA GGCAGCAATATTGAGCCATCAGTCAGGTCCTGTTTTCAGT TTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTCCT GGACTGGATGAGACTTGAGCCTCAGAGCATGGTCTGGCTG CCTGTGCTTCATAGAGTGGCTGCTGCTGAGACTGCCAAGC ACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCAT T GGC T T CAGATACAGAT C C C T GAAGCAC T T CAAC TAT GAT ATCTGCCAGAGCTGCTTCTTTAGTGGCAGGGTTGCCAAGG GCCACAAAATGCACTACCCCATGGTGGAATACTGCACCCC AACAACCTCTGGGGAAGATGTTAGAGACTTTGCCAAGGTG CTGAAAAACAAGTTCAGGACCAAGAGATACTTTGCTAAGC ACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTTGA GGGTGACAACATGGAAACCCCTGTGACACTGATCAATTTC TGGCCAGTGGACTCTGCCCCTGCCTCAAGTCCACAGCTGT CCCATGATGACACCCACAGCAGAATTGAGCACTATGCCTC CAGACT GGCAGAGAT GGAAAACAGCAAT GGCAGCTACCT G AAT GATAGCAT CAGCCCCAAT GAGAGCAT T GAT GAT GAGC ATCTGCTGATCCAGCACTACTGTCAGTCCCTGAACCAGGA CTCTCCACTGAGCCAGCCTAGAAGCCCTGCTCAGATCCTG ATCAGCCTTGAGTCTTGATGAGTCGACAGGCCTAATAAAG AGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTTGTGT GGCTAGCTGCGGCCGCaggaacccctagtgatggagttgg ccactccctctctgcgcgctcgctcgctcactgaggccgg gcgaccaaaggtcgcccgacgcccgggctttgcccgggcg gcctcagtgagcgagcgagcgcgcag RGX-DYS8 (full cassette including flanking ITRs, Spc5-12 promoter to polyA and intervening seqs) 4470 bp ITRs shown in lower case 106 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagc ccgggcgtcgggcgacctttggtcgcccggcctcagtgag cgagcgagcgcgcagagagggagtggccaactccatcact aggggttcctCATATGCAGGGTAATGGGGATCCTCTAGAG GCCGTCCGCCCTCGGCACCATCCTCACGACACCCAAATAT GGCGACGGGTGAGGAATGGTGGGGAGTTATTTTTAGAGCG GTGAGGAAGGTGGGCAGGCAGCAGGTGTTGGCGCTCTAAA AATAACTCCCGGGAGTTATTTTTAGAGCGGAGGAATGGTG GACACCCAAATATGGCGACGGTTCCTCACCCGTCGCCATA TTTGGGTGTCCGCCCTCGGCCGGGGCCGCATTCCTGGGGG CCGGGCGGTGCTCCCGCCCGCCTCGATAAAAGGCTCCGGG GCCGGCGGCGGCCCACGAGCTACCCGGAGGAGCGGGAGGC GCCAAGCGgAATTCGCCACCATGCTTTGGTGGGAAGAGGT GGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCA AGCAGCACATTGAGAACCTGTTCAGTGACCTGCAGGATGG CAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGCAGCACAAGAGTGCATGCCC TGAACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAA CAAT GT GGACCT GGT CAATAT T GGCAGCACAGACAT T GT G GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACA TCATCCTGCAC T GGCAAGT GAAGAAT GT GAT GAAGAACAT Structure SEQ ID Nucleic Acid Sequence CATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGT CAGACAGAGCAC CAGAAAC TAC C C T CAAG TGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACT GGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTG TTTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAC AGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCT GGGAATTGAGAAACTGCTGGACCCTGAGGATGTGGACACC ACCTATCCTGACAAGAAATCCATCCTCATGTACATCACCA GCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGC CATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTG ACCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACT ACT CT CAGCAGAT CACAGT GT CT CT GGCCCAGGGATAT GA GAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCC TAGACACAG GCTGCCTATGTGACCACATCTGACCC CACAA GAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGA CAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAAC CTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCT GGCTGCTGTCTGCTGAGGATACACTGCAGGCTCAGGGTGA AATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCAC ACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGG GCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGAT T GGCACAGGCAAGCT GTCT GAGGAT GAAGAGACAGAGGT G CAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTC T GAGAGT GGCCAGCAT GGAAAAGCAGAGCAACCT GCACAG AGT GC T CAT GGAC C T GCAGAAT CAGAAAC T GAAAGAAC T G AAT GACTGGCTGAC CAAGACAGAAGAAAGGAC TAGGAAGA T GGAAGAGGAAC CTCTGGGACCAGACC T GGAAGAT C T GAA AAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTT GAGCAAGAGCAAGT CAGAGT GAACAGCCT GACACACAT GG TGGTGGTTGTGGATGAGTCCTCTGGGGATCATGCCACAGC TGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGG GCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGC TCCAGGACATTCTGGAGATCAGCTATGTGCCCAGCACCTA CCTGACAGAGATCACCCATGTGTCTCAGGCCCTGCTGGAA GTGGAACAGCTGCTGAATGCCCCTGACCTGTGTGCCAAGG ACTTTGAGGACCTGTTCAAGCAAGAGGAAAGCCTGAAGAA CATCAAGGACAGCCTGCAGCAGTCCTCTGGCAGAATTGAC ATCATCCACAGCAAGAAAACAGCTGCCCTGCAGTCTGCCA CACCTGTGGAAAGAGTGAAGCTGCAAGAGGCCCTGAGCCA GCTGGACTTCCAGTGGGAGAAAGTGAACAAGATGTACAAG GACAGGCAGGGCAGAT TT GATAGAAGT GT GGAAAAGTGGA GAAGGT T CCACTAT GACAT CAAGAT CT T CAACCAGT GGCT GACAGAGGC TGAGCAGTTCCT GAGAAAGACACAGAT CC C T GAGAACTGGGAGCATGCCAAGTACAAGTGGTATCTGAAAG AACT GCAGGAT GGCAT TGGCCAGAGACAGACAGT T GTCAG AACCCTGAATGCCACAGGGGAAGAGATCATCCAGCAGAGC AGCAAGACAGATGCCAGCATCCTGCAAGAGAAGCTGGGCA GCCTGAACCTGAGATGGCAAGAAGTGTGCAAGCAGCTGTC TGACAGAAAGAAGAGGCTGGAAGAACAGACACTGGAAAGG Structure SEQ ID Nucleic Acid Sequence CTGCAAGAACTTCAAGAGGCCACAGATGAGCTGGACCTGA AGCTGAGACAGGCTGAAGTGATCAAAGGCAGCTGGCAGCC AGTTGGGGACCTGCTCATTGATAGCCTGCAGGACCATCTG GAAAAAGTGAAAGCCCTGAGGGGAGAGATTGCCCCTCTGA AAGAAAATGTGTCCCATGTGAATGACCTGGCCAGACAGCT GACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGAGC ACCCTTGAGGACCTGAACACCAGGTGGAAGCTCCTCCAGG T GGCAGT GGAAGATAGAGT CAGGCAGCT GCAT GAGGCCCA CAGAGAT TTTGGACCAGC CAGC CAGCAC TTTCTGTCTACC TCTGTGCAAGGCCCCTGGGAGAGAGCTATCTCTCCTAACA AGGTGCCCTACTACATCAACCATGAGACACAGACCACCTG TTGGGATCACCCCAAGATGACAGAGCTGTACCAGAGTCTG GCAGACCTCAACAATGTCAGATTCAGTGCCTACAGGACTG CCATGAAGCTCAGAAGGCTCCAGAAAGCTCTGTGCCTGGA CCTGCTTTCCCTGAGTGCAGCTTGTGATGCCCTGGACCAG CACAATCTGAAGCAGAATGAGCAGCCTATGGACATCCTCC AGAT CAT CAAC TGCCTCACCAC CAT C TAT GATAGGC T GGA ACAAGAGCACAACAATCTGGTCAATGTGCCCCTGTGTGTG GACAT GT GC CT GAAT T GGCT GCT GAAT GT GTAT GACACAG GCAGAACAGGCAGGATCAGAGTCCTGTCCTTCAAGACAGG CATCATCTCCCTGTGCAAAGCCCACTTGGAGGACAAGTAC AGATACCTGTTCAAGCAAGTGGCCTCCAGCACAGGCTTTT GTGACCAGAGAAGGCTGGGCCTGCTCCTGCATGACAGCAT TCAGATCCCTAGACAGCTGGGAGAAGTGGCTTCCTTTGGA GGCAGCAATATTGAGCCATCAGTCAGGTCCTGTTTTCAGT TTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTCCT GGACTGGATGAGACTTGAGCCTCAGAGCATGGTCTGGCTG CCTGTGCTTCATAGAGTGGCTGCTGCTGAGACTGCCAAGC ACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCAT T GGC T T CAGATACAGAT C C C T GAAGCAC T T CAAC TAT GAT ATCTGCCAGAGCTGCTTCTTTAGTGGCAGGGTTGCCAAGG GCCACAAAATGCACTACCCCATGGTGGAATACTGCACCCC AACAACCTCTGGGGAAGATGTTAGAGACTTTGCCAAGGTG CTGAAAAACAAGTTCAGGACCAAGAGATACTTTGCTAAGC ACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTTGA GGGTGACAACATGGAAACCTGATGAGTCGACAGGCCTAAT AAAGAGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTT GTGTGGCTAGCTGCGGCCGCaggaacccctagtgatggag ttggccactccctctctgcgcgctcgctcgctcactgagg ccgggcgaccaaaggtcgcccgacgcccgggctttgcccg ggcggcctcagtgagcgagcgagcgcgcag 5.3.5 Methods of Making rAAV Particles

[00137] Another aspect of the present invention involves making molecules disclosed herein. In some embodiments, a molecule according to the invention is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein. Such capsid proteins are described in Section 5.3.4, supra. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of a capsid protein molecule described herein and retains (or substantially retains) biological function of the capsid protein and the inserted peptide from a heterologous protein or domain thereof. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, preferably 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the AAV8 capsid protein, while retaining (or substantially retaining) biological function of the AAV8 capsid protein and the inserted peptide.

[00138] The capsid protein, coat, and rAAV particles may be produced by techniques known in the art. In some embodiments, the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector. In some embodiments, the viral genome further comprises a cap gene and / or a rep gene for expression and splicing of the cap gene. In embodiments, the cap and rep genes are provided by a packaging cell and not present in the viral genome.

[00139] In some embodiments, the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap plasmid in place of the existing capsid gene. When introduced together into host cells, this plasmid helps package an rAAV genome into the engineered capsid protein as the capsid coat. Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging.

[00140] Numerous cell culture-based systems are known in the art for production of rAAV particles, any of which can be used to practice a method disclosed herein. The cell culture-based systems include transfection, stable cell line production, and infectious hybrid virus production systems which include, but are not limited to, adenovirus-AAV hybrids, herpesvirus-AAV hybrids and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV virus particles require: (1) suitable host cells, including, for example, human-derived cell lines, mammalian cell lines, or insect-derived cell lines; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature-sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences and optionally regulatory elements; and (5) suitable media and media components (nutrients) to support cell growth / survival and rAAV production.

[00141] Nonlimiting examples of host cells include: A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293 and their derivatives (HEK293T cells, HEK293F cells), Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, myoblast cells, CHO cells or CHO-derived cells, or insect-derived cell lines such as SF-9 (e.g. in the case of baculovirus production systems). For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054, which is incorporated by reference herein in its entirety for manufacturing techniques.

[00142] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising an insect cell; (b) introducing into the cell one or more baculovirus vectors encoding at least one of: i. an rAAV genome to be packaged, ii. an AAV rep protein sufficient for packaging, and iii. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the method comprises using a first baculovirus vector encoding the rep and cap genes and a second baculovirus vector encoding the rAAV genome. In some embodiments, the method comprises using a baculovirus encoding the rAAV genome and an insect cell expressing the rep and cap genes. In some embodiments, the method comprises using a baculovirus vector encoding the rep and cap genes and the rAAV genome. In some embodiments, the insect cell is an Sf-9 cell. In some embodiments, the insect cell is an Sf-9 cell comprising one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[00143] In some embodiments, a method disclosed herein uses a baculovirus production system. In some embodiments the baculovirus production system uses a first baculovirus encoding the rep and cap genes and a second baculovirus encoding the rAAV genome. In some embodiments the baculovirus production system uses a baculovirus encoding the rAAV genome and a host cell expressing the rep and cap genes. In some embodiments the baculovirus production system uses a baculovirus encoding the rep and cap genes and the rAAV genome. In some embodiments, the baculovirus production system uses insect cells, such as Sf-9 cells.

[00144] A skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV. The phrase “adenovirus helper functions” refers to a number of viral helper genes expressed in a cell (as RNA or protein) such that the AAV grows efficiently in the cell. The skilled artisan understands that helper viruses, including adenovirus and herpes simplex virus (HSV), promote AAV replication and certain genes have been identified that provide the essential functions, e.g. the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication. In some embodiments of a method disclosed herein, AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, AAV rep and cap genes, helper genes, and rAAV genomes can be introduced into cells by transduction with viral vectors, for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of a method disclosed herein, one or more of AAV rep and cap genes, helper genes, and rAAV genomes are introduced into the cells by transduction with an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes and the rAAV genome. In some embodiments, the rHSV vector encodes the helper genes and the AAV rep and cap genes.

[00145] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising a host cell; (b) introducing into the cell one or more rHSV vectors encoding at least one of: i. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions. In some embodiments, the rHSV vector comprises one or more endogenous genes that encode helper functions. In some embodiments, the rHSV vector comprises one or more heterogeneous genes that encode helper functions. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions and the rAAV genome. In some embodiments, the rHSV vector encodes helper functions and the AAV rep and cap genes. In some embodiments, the cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[00146] In one aspect, provided herein is a method of producing rAAV particles, comprising (a) providing a cell culture comprising a mammalian cell; (b) introducing into the cell one or more polynucleotides encoding at least one of: i. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles. In some embodiments, the helper functions are encoded by adenovirus genes. In some embodiments, the mammalian cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.

[00147] Molecular biology techniques to develop plasmid or viral vectors encoding the AAV rep and cap genes, helper genes, and / or rAAV genome are commonly known in the art. In some embodiments, AAV rep and cap genes are encoded by one plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one plasmid vector. In some embodiments, the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, AAV rep and cap genes are encoded by one viral vector. In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector. In some embodiments, the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one viral vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the AAV rep and cap genes, the adenovirus helper functions necessary for packaging, and the rAAV genome to be packaged are introduced to the cells by transfection with one or more polynucleotides, e.g., vectors. In some embodiments, a method disclosed herein comprises transfecting the cells with a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is an AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is an AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.PHB, or AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the vector encoding the rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs. In some embodiments, the AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes.

[00148] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and rAAV genome to a cell in which rAAV particles are to be produced or packaged. In some embodiments of a method disclosed herein, a first plasmid vector encoding an rAAV genome comprising a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding AAV rep and cap genes, and a third vector encoding helper genes can be used. In some embodiments, a mixture of the three vectors is co-transfected into a cell. In some embodiments, a combination of transfection and infection is used by using both plasmid vectors as well as viral vectors.

[00149] In some embodiments, one or more of rep and cap genes, and AAV helper genes are constitutively expressed by the cells and does not need to be transfected or transduced into the cells. In some embodiments, the cell constitutively expresses rep and / or cap genes. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses Ela. In some embodiments, the cell comprises a stable transgene encoding the rAAV genome.

[00150] In some embodiments, AAV rep, cap, and helper genes (e.g., Ela gene, Elb gene, E4 gene, E2a gene, or VA gene) can be of any AAV serotype. Similarly, AAV ITRs can also be of any AAV serotype. For example, in some embodiments, AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV cap gene is from AAV8 or AAV9 cap gene. In some embodiments, an AAV cap gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.rh74, AAV.hu31, AAV.hu32, or AAV.hu37 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV rep and cap genes for the production of a rAAV particle are from different serotypes. For example, the rep gene is from AAV2 whereas the cap gene is from AAV8. In another example, the rep gene is from AAV2 whereas the cap gene is from AAV9.

[00151] In some embodiments, the rep gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In other embodiments, the rep and the cap genes are from the same serotype. In still other embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises at least one modified protein domain or modified promoter domain. In certain embodiments, the at least one modified domain comprises a nucleotide sequence of a serotype that is different from the capsid serotype. The modified domain within the rep gene may be a hybrid nucleotide sequence consisting fragments different serotypes.

[00152] Hybrid rep genes provide improved packaging efficiency of rAAV particles, including packaging of a viral genome comprising a microdystrophin transgene greater than 4 kb, greater than 4.1 kb, greater than 4.2 kB, greater than 4.3 kb, greater than 4.4 kB, greater than 4.5 kb, or greater than 4.6 kb. AAV rep genes consist of nucleic acid sequences that encode the non-structural proteins needed for replication and production of virus. Transcription of the rep gene initiates from the p5 or pl 9 promoters to produce two large (Rep78 and Rep68) and two small (Rep52 and Rep40) nonstructural Rep proteins, respectively. Additionally, Rep78 / 68 domain contains a DNA-binding domain that recognizes specific ITR sequences within the ITR. All four Rep proteins have common helicase and ATPase domains that function in genome replication and / or encapsidation (Maurer AC, 2020, DOI: 10.1089 / hum.2020.069). Transcription of the cap gene initiates from a p40 promoter, which sequence is within the C-terminus of the rep gene, and it has been suggested that other elements in the rep gene may induce p40 promoter activity. The p40 promoter domain includes transcription factor binding elements EF1A, MLTF, and ATF, Fos / Jun binding elements (AP-1), Spl-like elements (Spl and GGT), and the TATA element (Pereira and Muzyczka, Journal of Virology, June 1997, 71(6):4300-4309). In some embodiments, the rep gene comprises a modified p40 promoter. In some embodiments, the p40 promoter is modified at any one or more of the EFl A binding element, MLTF binding element, ATF binding element, Fos / Jun binding elements (AP-1), Spl-like elements (Spl or GGT), or the TATA element. In other embodiments, the rep gene is of serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, rh8, rhlO, rh20, rh39, rh.74, RHM4-1, or hu37, and the portion or element of the p40 promoter domain is modified to serotype 2. In still other embodiments, the rep gene is of serotype 8 or 9, and the portion or element of the p40 promoter domain is modified to serotype 2.

[00153] ITRs contain A and A’ complimentary sequences, B and B’ complimentary sequences, and C and C’ complimentary sequences; and the D sequence is contiguous with the ssDNA genome. The complimentary sequences of the ITRs form hairpin structures by self-annealing (Bems KI. The Unusual Properties of the AAV Inverted Terminal Repeat. Hum Gene Ther 2020). The D sequence contains a Rep Binding Element (RBE) and a terminal resolution site (TRS), which together constitute the AAV origin of replication. The ITRs are also required as packaging signals for genome encapsidation following replication. In some embodiments, the ITR sequences and the cap genes are from the same serotype, except that one or more of the A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence may be modified to contain sequences from a different serotype than the capsid. In some embodiments, the modified ITR sequences are from the same serotype as the rep gene. In other embodiments, the ITR sequences and the cap genes are from different serotypes, except that one or more of the ITR sequences selected from A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence are from the same serotype as the capsid (cap gene), and one or more of the ITR sequences are from the same serotype as the rep gene.

[00154] In some embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises a modified Rep78 domain, DNA binding domain, endonuclease domain, ATPase domain, helicase domain, p5 promoter domain, Rep68 domain, p5 promoter domain, Rep52 domain, pl9 promoter domain, Rep40 domain or p40 promoter domain. In other embodiments, the rep and the cap genes are from the same serotype, and the rep gene comprises at least one protein domain or promoter domain from a different serotype. In one embodiment, an rAAV comprises a transgene flanked by AAV2 ITR sequences, an AAV8 cap, and a hybrid AAV2 / 8 rep. In another embodiment, the AAV2 / 8 rep comprises serotype 8 rep except for the p40 promoter domain or a portion thereof is from serotype 2 rep. In other embodiments, the AAV2 / 8 rep comprises serotype 2 rep except for the p40 promoter domain or a portion thereof is from serotype 8 rep. In some embodiments, more than two serotypes may be utilized to construct a hybrid rep!cap plasmid.

[00155] Any suitable method known in the art may be used for transfecting a cell may be used for the production of rAAV particles according to a method disclosed herein. In some embodiments, a method disclosed herein comprises transfecting a cell using a chemical based transfection method. In some embodiments, the chemical-based transfection method uses calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)), lipofection. In some embodiments, the chemical-based transfection method uses cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)). In some embodiments, the chemical-based transfection method uses polyethylenimine (PEI). In some embodiments, the chemical-based transfection method uses DEAE dextran. In some embodiments, the chemical-based transfection method uses calcium phosphate.

[00156] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[00157] Nucleic acid sequences of AAV-based viral vectors, and methods of making recombinant AAV and AAV capsids, are taught, e.g., in US 7,282,199; US 7,790,449; US 8,318,480; US 8,962,332; and PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety.

[00158] In preferred embodiments, the rAAVs provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below. 5.4. Therapeutic Utility

[00159] Provided are methods of assaying the constructs, including recombinant gene therapy vectors, encoding microdystrophins, as disclosed herein, for therapeutic efficacy. Methods include both in vitro and in vivo tests in animal models as described herein or using any other methods known in the art for testing the activity and efficacy of microdystrophins. 5.4.1 In vitro assays 5.4.1.1 In vitro infection system for muscle cells

[00160] Provided are methods of testing of the infectivity of a recombinant vector disclosed herein, for example rAAV particles. For example, the infectivity of recombinant gene therapy vectors in muscle cells can be tested in C2C12 myoblasts as described in Example 2, herein. Several muscle or heart cell lines may be utilized, including but not limited to T0034 (human), L6 (rat), MM14 (mouse), P19 (mouse), G-7 (mouse), G-8 (mouse), QM7 (quail), H9c2(2-1) (rat), Hs 74.Ht (human), and Hs 171.Ht (human) cell lines. Vector copy numbers may be assess using polymerase chain reaction techniques and level of microdystrophin expression may be tested by measuring levels of microdystrophin mRNA in the cells. 5.4.2 Animal Models

[00161] The efficacy of a viral vector containing a transgene encoding a microdystrophin as described herein may be tested by administering to an animal model to replace mutated dystrophin, for example, by using the mdx mouse and / or the golden retriever muscular dystrophy (GRMD) model and to assess the biodistribution, expression and therapeutic effect of the transgene expression. The therapeutic effect may be assessed, for example, by assessing change in muscle strength in the animal receiving the microdystrophin transgene. Animal models using larger mammals as well as nonmammalian vertebrates and invertebrates can also be used to assess pre-clinical therapeutic efficacy of a vector described herein. Accordingly, provided are compositions and methods for therapeutic administration comprising a dose of a microdystrophin encoding vector disclosed herein in an amount demonstrated to be effective according to the methods for assessing therapeutic efficacy disclosed here. 5.4.2.1 Murine Models

[00162] The efficacy of gene therapy vectors may be assessed in murine models of DMD. The mdx mouse model (Yucel, N., et al, Humanizing the mdx mouse model of DMD: the long and the short of it, Regenerative Medicine volume 3, Article number: 4 (2018)), carries a nonsense mutation in exon 23, resulting in an early termination codon and a truncated protein (mdx). Mdx mice have 3-fold higher blood levels of pyruvate kinase activity compared to littermate controls. Like the human DMD disease, mdx skeletal muscles exhibit active myofiber necrosis, cellular infiltration, a wide range of myofiber sizes and numerous centrally nucleated regenerating myofibers. This phenotype is enhanced in the diaphragm, which undergoes progressive degeneration and myofiber loss resulting in an approximately 5-fold reduction in muscle isometric strength. Necrosis and regeneration in hind-limb muscles peaks around 3-4 weeks of age, but plateaus thereafter. In mdx mice and mdx mice crossed onto other mouse backgrounds (for example DBA / 2J), a mild but significant decrease in cardiac ejection fraction is observed (Van Westering, Molecules 2015, 20, 8823-8855). Such DMD model mice with cardiac functional defects may be used to assess the cardioprotective effects or improvement or maintenance of cardiac function or attenuation of cardiac dysfunction of the gene therapy vectors described herein. Example 3 herein details use of the mdx mouse model to assess gene therapy vectors encoding microdystrophins.

[00163] Additional mdx mouse models: A number of alternative versions in different genetic backgrounds have been generated including the mdx2cv, mdx3cv, mdx4cv, and mdx5cv lines (C57BL / 6 genetic background). These models were created by treating mice with N-ethyl-N-nitrosourea, a chemical mutagen. Each strain carries a different point mutation. As a whole, there are few differences in the presentation of disease phenotypes in the mdxcv models compared to the mdx mouse. Additional mouse models have been created by crossing the mdx line to various knock-out mouse models (e.g. Myodi'1', a-Integrin.7'1', a-Dystrobrevin'1', and Utrophin'1'). All mouse models which are currently used to study DMD have been described in detail by Yucel, N., et al, Humanizing the mdx mouse model of DMD: the long and the short of it, npj Regenerative Medicine volume 3, Article number: 4 (2018), which is incorporated herein by reference. 5.4.2.2 Canine

[00164] Most canine studies are conducted in the golden retriever muscular dystrophy (GRMD) model (Korneygay, J.N., et al, The golden retriever model of Duchenne muscular dystrophy. SkeletMuscle. 2017; 7: 9, which is incorporated by reference in its entirety). Dogs with GRMD are afflicted with a progressive, fatal disease with skeletal and cardiac muscle phenotypes and selective muscle involvement - a severe phenotype that more closely mirrors that of DMD. GRMD dogs carry a single nucleotide change that leads to exon skipping and an out-offrame DMD transcript. Phenotypic features in dogs include elevation of serum CK, CRDs on EMG, and histopathologic evidence of grouped muscle fiber necrosis and regeneration. Phenotypic variability is frequently observed in GRMD, as in humans. GRMD dogs develop paradoxical muscle hypertrophy which seems to play a role in the phenotype of affected dogs, with stiffness at gait, decreased joint range of motion, and trismus being common features. Objective biomarkers to evaluate disease progression include tetanic flexion, tibiotarsal joint angle, % eccentric contraction decrement, maximum hip flexion angle, pelvis angle, cranial sartorius circumference, and quadriceps femoris weight. 5.5. Methods of Treatment

[00165] Provided are methods of treating human subjects for any muscular dystrophy disease that can be treated by providing a functional dystrophin. DMD is the most common of such disease, but the gene therapy vectors that express microdystrophin provided herein can be administered to treat Becker muscular dystrophy (BMD), myotonic muscular dystrophy (Steinert’s disease), Facioscapulohumeral disease (FSHD), limb-girdle muscular dystrophy, X-linked dilated cardiomyopathy, or oculopharyngeal muscular dystrophy. The microdystrophin of the present disclosure may be any microdystrophin described herein, including those that have the domains in an N-terminal to C-terminal order of ABD-H1-R1-R2-R3-H3-R24-H4-CR, ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, ABD-H1-R1-R2-R16-R17-R24-H4-CR, or ABD-H1-R1-R2-R16-R17-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, RI is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, RI 6 is a spectrin 16 region of dystrophin, RI 7 is a spectrin 16 region of dystrophin, R24 is a spectrin 24 region of dystrophin, CR is a cysteine-rich region of dystrophin and CT is at least a portion of a C-terminal region of dystrophin comprising a al-syntrophin binding site and / or an a-dystrobrevin binding site. In embodiments, the microdystrophin has an amino acid sequence of SEQ ID Nos: 1, 2, 79, 91, 92, or 93. The vectors encoding the microdystrophin include those having a nucleic acid sequence of SEQ ID NO: 20, 21, 81, 101, 102 or 103, in certain embodiments, operably linked to regulatory elements for constitutive, musclespecific (including skeletal, smooth muscle and cardiac muscle-specific) expression, or CNS specific expression, and other regulatory elements such as poly A sites. Such nucleic acids may be in the context of an rAAV genome, for example, flanked by ITR sequences, particularly, AAV2 ITR sequences. In certain embodiments, the methods and compositions comprising administering to a subject in need thereof, an rAAV comprising the construct having a nucleic acid sequence of SEQ ID NO: 53, 54, 55, 56, 82, 104, 105, or 106. In embodiments, the patient has been diagnosed with and / or has symptom(s) associated with DMD. Recombinant vectors used for delivering the transgene encoding the microdystrophin are described in Section 5.3.4.1. Such vectors should have a tropism for human muscle cells (including skeletal muscle, smooth mucle and / or cardiac muscle) and can include non-replicating rAAV, particularly those bearing an AAV8 capsid. The recombinant vectors, such as those shown in FIG. 1A and FIG. 22, can be administered in any manner such that the recombinant vector enters the muscle tissue or CNS, preferably by introducing the recombinant vector into the bloodstream.

[00166] Subjects to whom such gene therapy is administered can be those responsive to gene therapy mediated delivery of a microdystrophin to muscles. In particular embodiments, the methods encompass treating patients who have been diagnosed with DMD or other muscular dystrophy disease, such as, Becker muscular dystrophy (BMD), myotonic muscular dystrophy (Steinert’s disease), Facioscapulohumeral disease (FSHD), limb-girdle muscular dystrophy, X-linked dilated cardiomyopathy, or oculopharyngeal muscular dystrophy, or have one or more symptoms associated therewith, and identified as responsive to treatment with microdystrophin, or considered a good candidate for therapy with gene mediated delivery of microdystrophin. In specific embodiments, the patients have previously been treated with synthetic version of dystrophin and have been found to be responsive to one or more of synthetic versions of dystrophin. To determine responsiveness, the synthetic version of dystrophin (e.g., produced in human cell culture, bioreactors, etc.) may be administered directly to the subject.

[00167] Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the muscle (e.g., skeletal muscle or cardiac muscle), preferably by introducing the recombinant vector into the bloodstream. In specific embodiments, the vector is administered subcutaneously, intramuscularly or intravenously. Intramuscular, subcutaneous, or intravenous administration should result in expression of the soluble transgene product in cells of the muscle (including skeletal muscle, cardiac muscle, and / or smooth muscle) and / or the CNS. The expression of the transgene product results in delivery and maintenance of the transgene product in the muscle and / or the CNS. Alternatively, the delivery may result in gene therapy delivery and expression of the microdystrophin in the liver, and the soluble microdystrophin product is then carried through the bloodstream to the muscles where it can impart its therapeutic effect. In other embodiments, the recombinant vector may be administered such that it is delivered to the CNS, for example, but not limited to, intrathecally, intracerebroventricularly, intranasally or suprachoroidally.

[00168] The actual dose amount administered to a particular subject can be determined by a clinician, considering parameters such as, but not limited to, physical and physiological factors including body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject, and / or route of administration.

[00169] Doses can range from lx 108 vector genomes per kg (vg / kg) to lx 1015 vg / kg. Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (i.e., days, weeks, months, etc.).

[00170] Pharmaceutical compositions suitable for intravenous, intramuscular, subcutaneous or hepatic administration comprise a suspension of the recombinant vector comprising the transgene encoding microdystrophin in a formulation buffer comprising a physiologically compatible aqueous buffer. The formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.

[00171] The gene therapy vectors provided herein may be administered in combination with other treatments for muscular dystrophy, including corticosteroids, beta blockers and ACE inhibitors. 5.5.1 Muscle degeneration / regene ration

[00172] Deletion of dystrophin results in mechanical instability causing myofibers to weaken and eventually break during contraction. Patients with DMD first display skeletal muscle weakness in early childhood, which progresses rapidly to loss of muscle mass, spinal curvature known as kyphosis, paralysis and ultimately death from cardiorespiratory failure before 30 years of age. Skeletal muscles of DMD patients also develop muscle hypertrophy, particularly of the calf, evidence of focal necrotic myofibers, abnormal variation in myofiber diameter, increased fat deposition and fibrosis, as well as lack of dystrophin staining in immunohistological sections.

[00173] The goal of gene therapy treatment provided herein is to slow or arrest the progression of DMD, or other muscular dystrophy disease, or to reduce the severity of one or more symptoms associated with DMD, or other muscular dystrophy disease. In particular, the goal of gene therapy provided herein is to reduce muscle degeneration, induce / improve muscle regeneration, and / or prevent / reduce downstream pathologies including inflammation and fibrosis that interfere with muscle regeneration and cause loss of movement, orthopedic complications, and, ultimately, respiratory and cardiac failure.

[00174] Efficacy may be monitored by measuring changes from baseline in gross motor function using the North Star Ambulatory Assessment (NSAA) (scale is ordinal with 34 as the maximum score indicating fully-independent function) or an age-appropriate modified assessment, by assessing changes in ambulatory function (e.g. 6-min (distance walked < 300m, between 300 and 400m, or > 400m)), by performing a timed function test to measure changes from baseline in time taken to stand from a supine position (1 to 8s (good), 8 to 20s (moderate), and 20 to 35s (poor)), by performing time to climb (4 steps) and time to run / walk assessments (10 meters), as well as myometry to evaluate changes from baseline in strength of upper and lower extremities [Mazzone et al, North Star Ambulatory Assessment...

Claims

1. A recombinant adeno-associated virus (rAAV) particle comprising a) anucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, or the reverse complement of the nucleotide sequence, and b) a capsid comprising a capsid protein.

2. The rAAV particle of claim 1, wherein the nucleotide sequence encodingthe microdystrophin protein comprises the nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 20 or the reverse complement thereof.

3. The rAAV particle of claim 1 or claim 2, wherein the nucleotide sequenceencoding the microdystrophin protein comprises the nucleotide sequence of SEQ ID NO: 20.

4. The rAAV particle of any one of claims 1-3, wherein the nucleic acidcomprises a transcription regulatory element that promotes expression in muscle, wherein the transcription regulatory element is operably linked to the nucleotide sequence encoding the microdystrophin protein.

5. The rAAV particle of any one of claims 1-4, wherein the transcriptionregulatory element is an SPc5-12 promoter or a transcriptionally active portion thereof.

6. The rAAV particle of any one of claims 1-5, wherein the nucleic acidcomprises from 5' to 3': a first AAV ITR sequence — a transcription regulatory element sequence — the nucleotide sequence encoding the microdystrophin protein — a polyadenylation sequence — a second AAV ITR sequence.2020392243   17 Sep 20267.     The rAAV particle of any one of claims 1-6, wherein the nucleic acidcomprises from 5' to 3' (i) a first AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 73, (ii) an SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39, (iii) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, (iv) a polyadenylation nucleotide sequence of SEQ ID NO: 42, and (v) a second AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 74.

8. The rAAV particle of any one of claims 1-7, wherein the capsid proteincomprises a) an amino acid sequence that is at least 95% identical to SEQ ID NO: 77, b) the amino acid sequence of SEQ ID NO: 77, c) an amino acid sequence that is at least 95% identical to SEQ ID NO: 78, or d) the amino acid sequence of SEQ ID NO: 78.

9. The rAAV particle of claim 8, wherein the capsid protein comprises theamino acid sequence of SEQ ID NO: 77.

10. An rAAV particle comprising a) a nucleic acid comprising 5' to 3': a first AAV ITR sequence — a transcription regulatory element sequence —a nucleotide sequence encoding a microdystrophin protein — a polyadenylation sequence — a second AAV ITR sequence, wherein the microdystrophin protein comprises the amino acid sequence of SEQ ID NO: 1, wherein the transcription regulatory element is an SPc5-12 promoter or a transcriptionally active portion thereof, and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77.

11. An rAAV particle comprising a) a nucleic acid comprising 5' to 3' (i) a first AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 73, (ii) an SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39, (iii) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, (iv) a polyadenylation nucleotide sequence of SEQ ID NO: 42, and (v) a second AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 74 and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77.2020392243   17 Sep 202612. A pharmaceutical composition comprising i) a rAAV particle comprising a) a nucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, or the reverse complement of the nucleotide sequence, and b) a capsid comprising a capsid protein, and ii) a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising:A.     1) an rAAV particle comprising a) a nucleic acid comprising 5' to 3': a firstAAV ITR sequence — a transcription regulatory element sequence — a nucleotide sequence encoding a microdystrophin protein — a polyadenylation sequence — a second AAV ITR sequence, wherein the microdystrophin protein comprises the amino acid sequence of SEQ ID NO: 1, wherein the transcription regulatory element is an SPc5-12 promoter or a transcriptionally active portion thereof, and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77, and 2) a pharmaceutically acceptable carrier; orB.     1) an rAAV particle comprising a) a nucleic acid comprising 5' to 3' (i) afirst AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 73, (ii) an SPc5-12 promoter comprising the nucleotide sequence of SEQ ID NO: 39, (iii) a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, (iv) a polyadenylation nucleotide sequence of SEQ ID NO: 42, and (v) a second AAV2 ITR comprising the nucleotide sequence of SEQ ID NO: 74 and b) a capsid comprising a capsid protein comprising the amino acid sequence of SEQ ID NO: 77 and 2) a pharmaceutically acceptable carrier.

14. A nucleic acid comprising a nucleotide sequence encoding a microdystrophin protein comprising the amino acid sequence of SEQ ID NO: 1, or the reverse complement of the nucleotide sequence.

15. The nucleic acid of claim 14, wherein the microdystrophin protein consists of the amino acid sequence of SEQ ID NO: 1.2020392243   17 Sep 202616. The nucleic acid of claim 14 or claim 15, wherein the nucleotide sequence encoding the microdystrophin protein comprises the nucleotide sequence of SEQ ID NO: 20, or a nucleotide sequence at least 85% identical to the nucleotide sequence of SEQ ID NO: 20, or the reverse complement thereof.

17. The nucleic acid of claim 16, wherein the nucleotide sequence encoding the microdystrophin protein comprises the nucleotide sequence of SEQ ID NO: 20.

18. The rAAV particle of any one of claims 1-11, the pharmaceutical composition of claim 12 or claim 13, or the nucleic acid of any one of claims 14-17, wherein the nucleic acid comprises a transcription regulatory element that promotes expression in muscle,wherein the transcription regulatory element is operably linked to the nucleotide sequence encoding the microdystrophin protein.

19. The nucleic acid of claim 18, wherein the transcription regulatory element is an SPc5-12 promoter or a transcriptionally active portion thereof.

20. The rAAV particle of any one of claims 1-11, the pharmaceutical composition of claim 12 or claim 13, or the nucleic acid of any one of claims 14-19, wherein the nucleic acid comprises a nucleotide sequence comprising from 5’ to 3‘: a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence - the transcription regulatory element sequence - the nucleotide sequence encoding the microdystrophin protein - a polyadenylation sequence - a second AAV ITR sequence.

21. The nucleic acid of claim 20, wherein the AAV ITR sequence is an AAV2 ITR sequence.

22. The rAAV particle of any one of claims 1-11, the pharmaceutical composition of claim 12 or claim 13, or the nucleic acid of any one of claims 14-21, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence that is 85% identical to SEQ ID NO: 53.2020392243   17 Sep 202623. The nucleic acid of claim 22, wherein the nucleotide sequence encoding the microdystrophin protein comprises the nucleotide sequence of SEQ ID NO: 53.

24. A plasmid comprising a nucleic acid comprising the nucleic acid of any one of claims 14-23.

25. A host cell comprising a plasmid comprising the nucleic acid of any one of claims 14-23 or a plasmid of claim 24.

26. A method of treating a dystrophinopathy in a human subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of (i) a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) particle of any one of claims 1-11, (ii) a pharmaceutical composition of claim 12 or claim 13, or (iii) a pharmaceutical composition comprising an rAAV particle wherein the rAAV particle comprises the nucleic acid of any one of claims 14-23, wherein the administration results in expression of a microdystrophin protein in a muscle cell of the subject thereby treating the dystrophinopathy in the subject.

27. The method of claim 26, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy, or the subject is a female carrier of DMD or BMD.

28. The method of claim 26 or claim 27, wherein the dystrophinopathy is DMD.

29. A method of delivering a nucleic acid encoding a microdystrophin protein to a cell, the method comprising contacting the cell with (i) an rAAV particle of any one of claims 1-11, (ii) the pharmaceutical composition of claim 12 or claim 13, or (iii) an rAAV particle wherein the rAAV particle comprises the nucleic acid of any one of claims 14-23, wherein the nucleic acid encoding the microdystrophin protein is delivered to the cell.2020392243   17 Sep 202630. A method of producing recombinant adeno-associated viruses (AAVs) comprising:A. culturing a host cell containing:(i) the nucleic acid of any one of claims 14-23;(ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV rep and capsid protein operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; and(iii) sufficient adenovirus helper genes to permit replication and packaging of the artificial genome by the AAV capsid proteins; andB. recovering recombinant AAV encapsidating the artificial genome from the cell culture.

31. Use of (i) a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) particle of any one of claims 1-11, (ii) a pharmaceutical composition of claim 12 or claim 13, or (iii) a pharmaceutical composition comprising an rAAV particle wherein the rAAV particle comprises the nucleic acid of any one of claims 14-23, in the manufacture of a medicament for treating a dystrophinopathy in a human subject in need thereof, wherein the treating results in expression of a microdystrophin protein in a muscle cell of the subject thereby treating the dystrophinopathy in the subject.

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