Dystrophin constructs and methods of use thereof

Abstract

The present disclosure provides gene constructs that encode a truncated dystrophin protein. The present disclosure also provides systems and methods for delivering a truncated dystrophin protein in a subject, and methods for treating a subject having a dystrophin-associated disease or disorder, e.g., muscular dystrophy, e.g., Duchenne muscular dystrophy (DMD).

Description

DYSTROPHIN CONSTRUCTS AND METHODS OF USE THEREOFRELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 639,556, filed on April 26, 2024, the entire contents of which are incorporated herein by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 23, 2025, is named 135388-00820.xml and is 1,500,856 bytes in size.BACKGROUND OF THE INVENTION

[0003] Muscular’ dystrophy (MD) is a group of genetic diseases characterized by progressive weakness and degeneration of muscle mass. Some forms of MD present symptoms at birth or develop during childhood, while others may not appear until middle age or later. The disorders differ in terms of the distribution and extent of muscle weakness, age of onset, rate of progression, and pattern of inheritance.

[0004] Duchenne muscular dystrophy (DMD) is the most common form of MD. The disease occurs due to a defective DMD gene that results in absence of dystrophin, a protein that is involved in maintaining the integrity of muscle. As a result of this genetic defect, individuals with DMD may have symptoms such as trouble walking and running, falling frequently, fatigue, learning disabilities / difficulties, heart issues as a result of impact on heart muscle functioning, and breathing problems due to weakening of respiratory muscles involved in lung function. Symptoms of muscle weakness associated with DMD typically begin in childhood, often between 3 to 6 years of age. DMD mainly affects males and in rare cases may affect females. About one in every 3,500 boys are affected by this disorder. As the disease progresses, lifethreatening heart and respiratory problems can occur.

[0005] The DMD gene is one of the largest known human genes. Its largest isoform contains 79 exons and encodes for a 427 kDa dystrophin protein. The extremely large size of the genecontributes to a complex mutational spectrum, with >7,000 different mutations and a high spontaneous mutation rate. The most severe phenotype associated with DMD is most often caused by out-of-frame mutations, resulting in complete loss of dystrophin protein expression. In-frame mutations that allow for the synthesis of an internally truncated but partially functional protein are associated with a milder phenotype known as Becker muscular dystrophy (BMD). [00061 Current treatments for DMD focus on managing symptoms, including corticosteroid medications to slow down the progression of muscle weakness, stretching and exercise programs, and use of equipment such as braces or a wheelchair as walking becomes more difficult. Exon-skipping therapies, which use antisense oligonucleotides to restore small amounts of dystrophin in specific subsets of DMD patients with a genetic phenotype amenable to the skipping strategy, are also used but require repeated administration. Furthermore, gene therapy strategies to replace the missing dystrophin have been limited by an inability to efficiently deliver the large and complex dystrophin gene sequence.

[0007] Accordingly, there remains an unmet need to develop new therapies to treat muscular dystrophies and to ameliorate deficiencies in patients afflicted with DMD-associated disorders caused by mutations in the dystrophin gene.SUMMARY OF THE INVENTION

[0008] The present disclosure provides isolated recombinant nucleic acid molecules encoding truncated dystrophin proteins that can be delivered and expressed in a subject using a dual adeno-associated virus (AAV) vector system to allow expression of truncated dystrophin proteins that are otherwise too large to fit into a single AAV system. The truncated dystrophin proteins can be used to restore the expression and function instead of a wild-type dystrophin in a subject in need thereof. The present disclosure also provides systems and methods for expressing or delivering a truncated dystrophin protein in a subject, and methods for treating a subject having a dystrophin-associated disease or disorder, e.g., muscular dystrophy, e.g., Duchenne muscular’ dystrophy (DMD). In some embodiments, the AAV vectors can be delivered to a subject in need thereof, e.g., a subject having a dystrophin-associated disease or disorder, e.g., muscular dystrophy, e.g., DMD, to produce a significant level of a functional truncated dystrophin, and to protect muscle fibers from injury, increase muscle strength, reduce and / orprevent fibrosis, in the subject. The AAV vectors can also be used for increasing muscular force and / or increasing muscle mass in order to address the gene defect observed in DMD patients.

[0009] Accordingly, in one aspect, the present invention is directed to a system for generating a truncated human dystrophin protein, comprising a first recombinant nucleic acid molecule and a second recombinant nucleic acid molecule, wherein the first nucleic acid molecule comprises a first coding region encoding an N-terminal portion of the truncated dystrophin protein and a 3’ ribozyme, wherein the first coding region is operably linked to the 3’ ribozyme at its 3’ end, wherein the second nucleic acid molecule comprising a second coding region encoding a C- terminal portion of the truncated dystrophin protein and a 5 ’ribozyme, wherein the second coding region is operably linked to the 5’ ribozyme at its 5’ end, wherein upon ribozyme- mediated catalytic ligation (“trans-ligation”), the first coding region and the second coding region forms a third coding region encoding for the complete truncated human dystrophin protein, and wherein the truncated human dystrophin protein comprises at least 1640 amino acids.

[0010] In some embodiments, the first coding region is operably linked to two or more 3’ ribozymes at its 3’ end. In some embodiments, the two or more 3’ ribozymes are the same 3’ ribozyme. In some embodiments, the two or more 3’ ribozymes are different 3’ ribozymes.

[0011] In some embodiments, the second coding region is operably linked to two or more 5’ ribozymes at its 5’ end. In some embodiments, the two or more 5’ ribozymes are the same 5’ ribozymes. In some embodiments, the two or more 5’ ribozymes are different 5’ ribozymes.

[0012] In some embodiments, the 5’ ribozyme and the 3’ ribozyme are each independently selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), Twister (Cpa), Twister Sister, Hammerhead (RzB), HDV, Pistol, Varkud Satellite (VS), Hatchet, Hairpin, and Hovlinc (Hov).

[0013] In some embodiments, the 5’ ribozyme and the 3’ ribozyme are each independently selected from the group consisting of SEQ ID NOs: 6 - 20.

[0014] In some embodiments, the first nucleic acid molecule further comprises an intron splice donor sequence, and the second nucleic acid molecule further comprises an intron splice acceptor sequence.

[0015] In some embodiments, the splice donor sequence is positioned between the first coding region and the 3’ ribozyme.

[0016] In some embodiments, the splice donor sequence is selected from the group consisting of SEQ ID NOs: 133 - 136.

[0017] In some embodiments, the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R7 domain, the R8 domain, the R9 domain, the RIO domain, the R11 domain, the R12 domain, the R13 domain, the R14 domain, the R15 domain, the R16 domain, the R17 domain, the R18 domain, the R19 domain, the H3 domain, the R20 domain, the R21 domain, and the R22 domain.

[0018] In some embodiments, the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R8 domain, the R19 domain, the H3 domain, the R20 domain, and the R21 domain.

[0019] In some embodiments, the splice donor sequence is not positioned within the R21 domain.

[0020] In some embodiments, the splice donor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 3’ ribozyme.

[0021] In some embodiments, the splice acceptor sequence is positioned between the 5’ ribozyme and the second coding region.

[0022] In some embodiments, the splice acceptor sequence is selected from the group consisting of SEQ ID Nos: 137-141.

[0023] In some embodiments, the splice acceptor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 5’ ribozyme.

[0024] In some embodiments, the splice donor sequence and the splice acceptor sequence are positioned such that the resulting spliced intron is between 50 - 200 bp in length.

[0025] In some embodiments, the splice donor sequence and splice acceptor sequence are positioned such that the resulting spliced intron encodes a single predominant reading frame.

[0026] In some embodiments, a stop codon sequence is introduced into the splice donor sequence or the splice acceptor sequence.

[0027] In some embodiments, at least one of the first coding region and the second coding region is at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length.

[0028] In some embodiments, the first coding region and the second coding region are each at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length.

[0029] In some embodiments, the third coding region is at least 4920 nucleotides in length, or at least 5100 nucleotides in length, or at least 5300 nucleotides in length.

[0030] In some embodiments, the first coding region and the second coding region do not share a region of substantial sequence identity.

[0031] In some embodiments, the 3’ end of the first coding region does not have a sequence identity to the 5’ end of the second coding region.

[0032] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0033] In some embodiments, the truncated dystrophin protein further comprises Hl Domain (SEQ ID NO: 22).

[0034] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of: a. midi-Dys A R1-R15 (SEQ ID NO: 83), b. midi-Dys A R2-R15 (SEQ ID NO: 84), c. midi-Dys A R3-R15 (SEQ ID NO: 85), d. midi-Dys A H2-R15 (SEQ ID NO: 86), e. midi-Dys A R4-R15 (SEQ ID NO: 87), f. midi-Dys A R5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 13-33 (SEQ ID NO: 93), h. midi-Dys A exon 13-39 (SEQ ID NO: 94), i. midi-Dys A exon 13-41 (SEQ ID NO: 95), j. midi-Dys A exon 13-48 (SEQ ID NO: 96), k. midi-Dys A exon 15-39 (SEQ ID NO: 97), l. midi-Dys A exon 15-41 (SEQ ID NO: 98), m. midi-Dys A exon 15-48 (SEQ ID NO: 99),n. midi-Dys A exon 17-39 (SEQ ID NO: 100), o. midi-Dys A exon 17-41 (SEQ ID NO: 101), p. midi-Dys A exon 17-48 (SEQ ID NO: 102), q. midi-Dys A exon 18-39 (SEQ ID NO: 220), r. midi-Dys A exon 18-41 (SEQ ID NO: 221), s. midi-Dys A exon 18-48 (SEQ ID NO: 222), t. midi-Dys A exon 19-39 (SEQ ID NO: 103), u. midi-Dys A exon 19-41 (SEQ ID NO: 104), v. midi-Dys A exon 19-48 (SEQ ID NO: 105), w. midi-Dys A exon 21-41 (SEQ ID NO: 106), x. midi-Dys A exon 21-42 (SEQ ID NO: 223), and y. midi-Dys A exon 21-48 (SEQ ID NO: 107).

[0035] In some embodiments, the truncated dystrophin protein further comprises R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), and R19 Domain (SEQ ID NO:42).

[0036] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of a. midi-Dys A R1-R15 (SEQ ID NO: 83), b. midi-Dys A R2-R15 (SEQ ID NO: 84), c. midi-Dys A R3-R15 (SEQ ID NO: 85), d. midi-Dys A H2-R15 (SEQ ID NO: 86), e. midi-Dys A R4-R15 (SEQ ID NO: 87), f. midi-Dys A R5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 10-33 (SEQ ID NO: 89), h. midi-Dys A exon 10-39 (SEQ ID NO: 90), i. midi-Dys A exon 10-41 (SEQ ID NO: 91), j. midi-Dys A exon 11-33 (SEQ ID NO: 216), k. midi-Dys A exon 11-39 (SEQ ID NO: 217), l. midi-Dys A exon 11-41 (SEQ ID NO: 218), m. midi-Dys A exon 13-33 (SEQ ID NO: 93), n. midi-Dys A exon 13-39 (SEQ ID NO: 94),o. midi-Dys A exon 13-41 (SEQ ID NO: 95), p. midi-Dys A exon 15-39 (SEQ ID NO: 97), q. midi-Dys A exon 15-41 (SEQ ID NO: 98), r. midi-Dys A exon 17-39 (SEQ ID NO: 100), s. midi-Dys A exon 17-41 (SEQ ID NO: 101), t. midi-Dys A exon 18-39 (SEQ ID NO: 220), u. midi-Dys A exon 18-41 (SEQ ID NO: 221), v. midi-Dys A exon 19-39 (SEQ ID NO: 103), w. midi-Dys A exon 19-41 (SEQ ID NO: 104), x. midi-Dys A exon 21-41 (SEQ ID NO: 106), and y. midi-Dys A exon 21-42 (SEQ ID NO: 223).

[0037] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0038] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0039] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0040] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0041] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0042] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R4 domain (SEQ ID NO:27), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0043] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R11 domain (SEQ ID NO:412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0044] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0045] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0046] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0047] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0048] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R14 Domain (SEQ ID NO: 41 ), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39),R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).[00491 In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0050] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0051] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0052] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41 ),R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0053] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51 ).

[0054] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0055] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0056] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0057] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0058] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0059] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0060] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0061] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0062] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0063] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0064] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0065] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0066] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0067] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO: 27), a partial R5 domain (SEQ ID NO: 411), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0068] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO: 27), a partial R5 domain (SEQ ID NO: 411), a partial R16 Domain (SEQ ID NO: 416), R17 Domain (SEQ ID NO: 40), R 18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0069] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0070] The truncated dystrophin proteins described above are depicted in Table 1 below:Table 1: Truncated Dystrophins and corresponding SEQ ID NOs and subdomains included.

[0071] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:83-107 and 216-223, or an amino acid at least about 90% identical thereto.

[0072] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 86, or an amino acid at least about 90% identical thereto.

[0073] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 95 or an amino acid at least about 90% identical thereto.

[0074] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 101 or an amino acid at least about 90% identical thereto.

[0075] In some embodiments, the amino acid sequence of the truncated dystrophin protein is not identical to an amino acid sequence of SEQ ID NO: 143.

[0076] In some embodiments, the truncated dystrophin protein is not a polypeptide of 2361 amino acids. In some embodiments, the truncated dystrophin protein is less than 2361 amino acid in length. In some embodiments, the truncated dystrophin protein is greater than 2361 amino acid in length.

[0077] In some embodiments, the truncated dystrophin protein is functional.

[0078] In some embodiments, the first nucleic acid molecule is present in a first viral vector, and the second nucleic acid molecule is present in a second viral vector.

[0079] In some embodiments, the first viral vector and the second viral vector are each independently selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a vaccinia vector, a herpes simplex viral vector, and an Epstein- Barr viral vector.

[0080] In some embodiments, the first viral vector is an adeno-associated viral (AAV) vector, and the second viral vector is an AAV vector.

[0081] In some embodiments, the AAV vector is selected from the group consisting of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAV-rhlO, AAV-DJ, AAV-LK03, AAV-MYO, AAV-MYO2, AAV-MYO3, MYO3A-AAV, MYO4A-AAV, and MYO4E-AAV.

[0082] In some embodiments, the first AAV vector further comprises a first promoter operably linked to the first nucleic acid molecule. In some embodiments, the second AAV vector further comprises a second promoter operably linked to the second nucleic acid molecule.

[0083] In some embodiments, the promoter comprises a tissue specific promoter or a ubiquitous promoter. In some embodiments, the promoter comprises a CK8 promoter, an MHCK7 promoter, an SPC5 promoter, or a minimal CKM promoter. In some embodiments, the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 144-150, or a nucleotide sequence at least 95% identical thereto.

[0084] In some embodiments, the first and / or second AAV vectors further comprise an inverted terminal repeat (ITR) sequence. In some embodiments, the ITR sequence comprises a nucleotide sequence of SEQ ID NO: 202 and / or 203, or a nucleotide sequence at least 95% identical thereto.

[0085] In some embodiments, the first and / or second AAV vectors further comprise an intron region. In some embodiments, the intron region comprise a nucleotide sequence of SEQ ID NO: 156 or 157, or a nucleotide sequence at least 95% identical thereto.

[0086] In some embodiments, the first and / or second AAV vectors further comprise a polyadcnylation sequence. In some embodiments, the poly adenylation sequence comprises a nucleotide sequence of SEQ ID NO: 151 or 152, or a nucleotide sequence at least 95% identical thereto.

[0087] In some embodiments, the first and / or second AAV vectors further comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the WPRE comprises a nucleotide sequence of SEQ ID NOs: 153-155, or a nucleotide sequence at least 95% identical thereto.

[0088] In some embodiments, the first and / or second AAV vectors further comprise a Kozak sequence.

[0089] In one aspect, the present invention is directed to a vector system for expressing a truncated human dystrophin protein, comprising a first AAV vector and a second AAV vector, wherein the first AAV vector comprises a first nucleic acid molecule comprising a first coding region encoding an N-terminal portion of the truncated dystrophin protein and a 3’ ribozyme, where the first coding region is operably linked to the 3’ ribozyme at its 3’ end, wherein the second AAV vector comprises a second nucleic acid molecule comprising a second coding region encoding a C-terminal portion of the truncated dystrophin protein and a 5’ ribozyme, where the second coding region is operably linked to the 5’ ribozyme at its 5’ end, wherein upon ribozyme-mediated catalytic ligation, the first coding region and the second coding region formsa third coding region encoding for the truncated human dystrophin protein, and wherein the truncated human dystrophin protein comprises at least 1640 amino acids.

[0090] In some embodiments, the first coding region is operably linked to two or more 3’ ribozymes at its 3’ end. In some embodiments, the two or more 3’ ribozymes are the same 3’ ribozyme. In some embodiments, the two or more 3’ ribozymes are different 3’ ribozymes.

[0091] In some embodiments, the second coding region is operably linked to two or more 5’ ribozymes at its 5’ end. In some embodiments, the two or more 5’ ribozymes are the same 5’ ribozymes. In some embodiments, the two or more 5’ ribozymes are different 5’ ribozymes.

[0092] In some embodiments, the 5’ ribozyme and the 3’ ribozyme are each independently selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), Twister (Cpa), Twister Sister, Hammerhead (RzB), HDV, Pistol, Varkud Satellite (VS), Hatchet, Hairpin, and Hovlinc (Hov).

[0093] In some embodiments, the 5’ ribozyme and the 3’ ribozyme arc each independently selected from the group consisting of SEQ ID NOs: 6 - 20.

[0094] In some embodiments, the first nucleic acid molecule further comprises an intron splice donor sequence, and the second nucleic acid molecule further comprises an intron splice acceptor sequence.

[0095] In some embodiments, the splice donor sequence is positioned between the first coding region and the 3’ ribozyme.

[0096] In some embodiments, the splice donor sequence is selected from the group consisting of SEQ ID NOs: 133 - 136.

[0097] In some embodiments, the splice donor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 3’ ribozyme.

[0098] In some embodiments, the splice acceptor sequence is positioned between the 5’ ribozyme and the second coding region.

[0099] In some embodiments, the splice acceptor sequence is selected from the group consisting of SEQ ID Nos: 137-141.

[0100] In some embodiments, the splice acceptor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 5’ ribozyme.

[0101] In some embodiments, the splice donor sequence and the splice acceptor sequence are positioned such that the resulting spliced intron is between 50 - 200 bp in length.

[0102] In some embodiments, the splice donor sequence and splice acceptor sequence are positioned such that the resulting spliced intron encodes a single predominant reading frame.

[0103] In some embodiments, a stop codon sequence is introduced into the splice donor sequence or the splice acceptor sequence.

[0104] In some embodiments, at least one of the first coding region and the second coding region is at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length.

[0105] In some embodiments, the first coding region and the second coding region are each at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length.

[0106] In some embodiments, the third coding region is at least 4920 nucleotides in length, or at least 5100 nucleotides in length, or at least 5300 nucleotides in length.

[0107] In some embodiments, the first coding region and the second coding region do not share a region of substantial sequence identity.

[0108] In some embodiments, the 3’ end of the first coding region does not have a sequence identity to the 5’ end of the second coding region.

[0109] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0110] In some embodiments, the truncated dystrophin protein further comprises Hl Domain (SEQ ID NO: 22).

[0111] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of: a. midi-Dys A R1-R15 (SEQ ID NO: 83), b. midi-Dys A R2-R15 (SEQ ID NO: 84), c. midi-Dys A R3-R15 (SEQ ID NO: 85), d. midi-Dys A H2-R 15 (SEQ ID NO: 86),e. midi-Dys A R4-R15 (SEQ ID NO: 87), f. midi-Dys A R5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 13-33 (SEQ ID NO: 93), h. midi-Dys A exon 13-39 (SEQ ID NO: 94), i. midi-Dys A exon 13-41 (SEQ ID NO: 95), j. midi-Dys A exon 13-48 (SEQ ID NO: 96), k. midi-Dys A exon 15-39 (SEQ ID NO: 97), l. midi-Dys A exon 15-41 (SEQ ID NO: 98), m. midi-Dys A exon 15-48 (SEQ ID NO: 99), n. midi-Dys A exon 17-39 (SEQ ID NO: 100), o. midi-Dys A exon 17-41 (SEQ ID NO: 101), p. midi-Dys A exon 17-48 (SEQ ID NO: 102), q. midi-Dys A exon 18-39 (SEQ ID NO: 220), r. midi-Dys A exon 18-41 (SEQ ID NO: 221), s. midi-Dys A exon 18-48 (SEQ ID NO: 222), t. midi-Dys A exon 19-39 (SEQ ID NO: 103), u. midi-Dys A exon 19-41 (SEQ ID NO: 104), v. midi-Dys A exon 19-48 (SEQ ID NO: 105), w. midi-Dys A exon 21-41 (SEQ ID NO: 106), x. midi-Dys A exon 21-42 (SEQ ID NO: 223), and y. midi-Dys A exon 21-48 (SEQ ID NO: 107).

[0112] In some embodiments, the truncated dystrophin protein further comprises R16 Domain(SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), and R19Domain (SEQ ID NO:42).

[0113] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of a. midi-Dys A R1-R15 (SEQ ID NO: 83), b. midi-Dys A R2-R15 (SEQ ID NO: 84), c. midi-Dys A R3-R15 (SEQ ID NO: 85), d. midi-Dys A H2-R15 (SEQ ID NO: 86), e. midi-Dys A R4-R 15 (SEQ ID NO: 87),f. midi-Dys A R5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 10-33 (SEQ ID NO: 89), h. midi-Dys A exon 10-39 (SEQ ID NO: 90), i. midi-Dys A exon 10-41 (SEQ ID NO: 91), j. midi-Dys A exon 11-33 (SEQ ID NO: 216), k. midi-Dys A exon 11-39 (SEQ ID NO: 217), l. midi-Dys A exon 11-41 (SEQ ID NO: 218), m. midi-Dys A exon 13-33 (SEQ ID NO: 93), n. midi-Dys A exon 13-39 (SEQ ID NO: 94), o. midi-Dys A exon 13-41 (SEQ ID NO: 95), p. midi-Dys A exon 15-39 (SEQ ID NO: 97), q. midi-Dys A exon L5-41 (SEQ ID NO: 98), r. midi-Dys A exon 17-39 (SEQ ID NO: 100), s. midi-Dys A exon 17-41 (SEQ ID NO: 101), t. midi-Dys A exon 18-39 (SEQ ID NO: 220), u. midi-Dys A exon 18-41 (SEQ ID NO: 221), v. midi-Dys A exon 19-39 (SEQ ID NO: 103), w. midi-Dys A exon 19-41 (SEQ ID NO: 104), x. midi-Dys A exon 21-41 (SEQ ID NO: 106), and y. midi-Dys A exon 21-42 (SEQ ID NO: 223).

[0114] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0115] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ IDNO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0116] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0117] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0118] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0119] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R4 domain (SEQ ID NO:27), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ IDNO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0120] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R11 domain (SEQ ID NO:412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0121] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0122] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0123] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0124] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0125] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0126] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0127] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0128] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain(SEQ ID NO: 406), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0129] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0130] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0131] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0132] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ IDNO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0133] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0134] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0135] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0136] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R15 Domain (SEQ ID NO: 414), R16Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).[01371 In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0138] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0139] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0140] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQID NO: 409), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0141] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0142] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0143] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0144] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ IDNO:27), a partial R5 domain (SEQ ID NO: 411), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0145] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R16 Domain (SEQ ID NO: 416), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0146] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0147] The truncated dystrophin proteins described above are depicted in Table 1 above.

[0148] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:83-107 and 216-223, or an amino acid at least about 90% identical thereto.

[0149] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 86, or an amino acid at least about 90% identical thereto.

[0150] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 95 or an amino acid at least about 90% identical thereto.

[0151] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 101 or an amino acid at least about 90% identical thereto.

[0152] In some embodiments, the amino acid sequence of the truncated dystrophin protein is not identical to an amino acid sequence of SEQ ID NO: 143.

[0153] In some embodiments, the truncated dystrophin protein is not a polypeptide of 2361 amino acids. In some embodiments, the truncated dystrophin protein is less than 2361 amino acid in length. In some embodiments, the truncated dystrophin protein is greater than 2361 amino acid in length.

[0154] In some embodiments, the truncated dystrophin protein is functional.

[0155] In some embodiments, the first coding region comprises the sequence selected from the group consisting of SEQ ID NOs: 284, 286, 288, 290, 291, 293, 295 and 297.

[0156] In some embodiments, the second coding region comprises the sequence selected from the group consisting of SEQ ID NOs: 285, 287, 289, 292, 294 and 296.

[0157] In some embodiments, the first coding sequence comprises SEQ ID NO: 286 and the second coding sequence comprises SEQ ID NO:287. In some embodiments, the first coding sequence comprises SEQ ID NO: 288 and the second coding sequence comprises SEQ ID NO:289. In some embodiments, the first coding sequence comprises SEQ ID NO: 290 and the second coding sequence comprises SEQ ID NO:289. In some embodiments, the first coding sequence comprises SEQ ID NO: 291 and the second coding sequence comprises SEQ ID NO:292. In some embodiments, the first coding sequence comprises SEQ ID NO: 293 and the second coding sequence comprises SEQ ID NO:294. In some embodiments, the first coding sequence comprises SEQ ID NO: 295 and the second coding sequence comprises SEQ ID NO:296. In some embodiments, the first coding sequence comprises SEQ ID NO: 297 and the second coding sequence comprises SEQ ID NO:292. In some embodiments, the first coding sequence comprises SEQ ID NO: 288 and the second coding sequence comprises SEQ ID NO:289. In some embodiments, the first coding sequence comprises SEQ ID NO: 288 and the second coding sequence comprises SEQ ID NO:287.

[0158] In some embodiments, the AAV vector is selected from the group consisting of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV 13, AAVrh74, AAV-rhlO, AAV-DJ, AAV-LK03, AAV-MYO, AAV-MYO2, AAV-MYO3, MYO3A-AAV, MYO4A-AAV, and MYO4E-AAV.

[0159] In some embodiments, the first AAV vector further comprises a first promoter operably linked to the first nucleic acid molecule. In some embodiments, the second AAV vector further comprises a second promoter operably linked to the second nucleic acid molecule.

[0160] In some embodiments, the promoter comprises a tissue specific promoter or a ubiquitous promoter. In some embodiments, the promoter comprises a CK8 promoter, an MHCK7 promoter, an SPC5-12 promoter, or a minimal CKM promoter. In some embodiments, the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 144-150, or a nucleotide sequence at least 95% identical thereto.

[0161] In some embodiments, the first and / or second AAV vectors further comprise an inverted terminal repeat (ITR) sequence. In some embodiments, the ITR sequence comprises a nucleotide sequence of SEQ ID NO: 202 and / or 203, or a nucleotide sequence at least 95% identical thereto.

[0162] In some embodiments, the first and / or second AAV vectors further comprise an intron region. In some embodiments, the intron region comprise a nucleotide sequence of SEQ ID NO: 156 or 157, or a nucleotide sequence at least 95% identical thereto.

[0163] In some embodiments, the first and / or second AAV vectors further comprise a polyadenylation sequence. In some embodiments, the poly adenylation sequence comprises a nucleotide sequence of SEQ ID NO: 151 or 152, or a nucleotide sequence at least 95% identical thereto.

[0164] In some embodiments, the first and / or second AAV vectors further comprise a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the WPRE comprises a nucleotide sequence of SEQ ID NO: 153 or 154, or 155, or a nucleotide sequence at least 95% identical thereto.

[0165] In some embodiments, the first and / or second AAV vectors further comprise a Kozak sequence.

[0166] In some embodiments, the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a first coding region encoding an N-terminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, and a 3’ ITR sequence.

[0167] In some embodiments, the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a first coding region encoding an N-terminal portion of thetruncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, a polyadenylation sequence, and a 3’ ITR sequence.

[0168] In some embodiments, the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, an intron region, a first coding region encoding an N-terminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, a polyadenylation sequence, and a 3’ ITR sequence.

[0169] In some embodiments, the first AAV vector comprises a sequence selected from the group consisting of SEQ ID NO: 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 301, 303, 305, 307, 308, 310, 312, 314, 394, and 395.

[0170] In some embodiments, the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, and a 3’ ITR sequence.

[0171] In some embodiments, the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, a polyadenylation sequence, and a 3’ ITR sequence.

[0172] In some embodiments, the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, a WPRE sequence, a polyadenylation sequence, and a 3’ ITR sequence.

[0173] In some embodiments, the second AAV vector comprises a sequence selected from the group consisting of SEQ ID NO:161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 302, 304, 306, 309, 311, and 313.

[0174] In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 303, and the second AAV vector comprises the sequence of SEQ ID NO: 304. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 305, and the second AAV vector comprises the sequence of SEQ ID NO: 306. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 307, and the second AAV vector comprises the sequence of SEQ ID NO: 306. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 308, and the second AAV vector comprises the sequence of SEQ ID NO: 309. In someembodiments, the first AAV vector comprises the sequence of SEQ ID NO: 310, and the second AAV vector comprises the sequence of SEQ ID NO: 311. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 312, and the second AAV vector comprises the sequence of SEQ ID NO: 313. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 314, and the second AAV vector comprises the sequence of SEQ ID NO: 309. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 394, and the second AAV vector comprises the sequence of SEQ ID NO: 309. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 395, and the second AAV vector comprises the sequence of SEQ ID NO: 306. In some embodiments, the first AAV vector comprises the sequence of SEQ ID NO: 395, and the second AAV vector comprises the sequence of SEQ ID NO: 304.

[0175] In one aspect, the present invention is directed to a pharmaceutical composition comprising a first nucleic acid molecule and a second nucleic acid molecule of the present disclosure, and a pharmaceutically acceptable excipient.

[0176] In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are presented at a ratio of 1:1. 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1.

[0177] In another aspect, the present invention is directed to a pharmaceutical composition comprising a first AAV vector and a second AAV vector of the present disclosure, and a pharmaceutically acceptable excipient.

[0178] In some embodiments, the first AAV vector and the second AAV vector are presented at a ratio of 1:1. 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1.

[0179] In another aspect, the present invention is directed to a pharmaceutical composition comprising an isolated recombinant dystrophin protein as described below, a nucleic acid encoding such dystrophin protein, a viral genome encoding such dystrophin protein, or a host cell incorporating such a nucleic acid or viral genome.

[0180] In one aspect, the present invention is directed to a method for treating a dystrophin- associated disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the first nucleic acid molecule and the second nucleic acid molecule of the present disclosure, or a therapeutically effective amount of the first AAV vector and the second AAV vector of the present disclosure, or the pharmaceutical composition of the present disclosure, thereby treating the dystrophin-associated disorder in the subject.

[0181] In another aspect, the present invention is directed to a method for increasing expression of dystrophin in a subject having or diagnosed with having a dystrophin-associated disorder, comprising administering a therapeutically effective amount of the first nucleic acid molecule and the second nucleic acid molecule of the present disclosure, or a therapeutically effective amount of the first AAV vector and the second AAV vector of the present disclosure, or the pharmaceutical composition of the present disclosure, thereby increasing expression of dystrophin in the subject.

[0182] In a further aspect, the present invention is directed to a method for increasing muscle mass or muscle strength and / or preventing fibrosis in a subject having or diagnosed with having a dystrophin-associated disorder, comprising administering a therapeutically effective amount of the first nucleic acid molecule and the second nucleic acid molecule of the present disclosure, or a therapeutically effective amount of the first AAV vector and the second AAV vector of the present disclosure, or the pharmaceutical composition of the present disclosure, thereby increasing muscle strength and / or preventing fibrosis in the subject.

[0183] In some embodiments, the dystrophin-associated disorder is muscular dystrophy.

[0184] In some embodiments, the dystrophin-associated disorder is Duchenne muscular' dystrophy.

[0185] In some embodiments, the first nucleic acid molecule and the second nucleic acid molecule, the first AAV vector and the second AAV vector, or the pharmaceutical composition is administered by intramuscular injection, or intravenous injection.

[0186] In some embodiments, the first AAV vector and the second AAV vector are administered together.

[0187] In some embodiments, the first AAV vector and the second AAV vector are administered separately.

[0188] In one aspect, the present invention is directed to an isolated recombinant dystrophin protein that has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: 83, 85-87, 89-107, and 216-223.

[0189] In one aspect, the present disclosure provides an isolated nucleic acid molecule encoding a truncated human dystrophin protein that has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 83, 85-87, 89-107, and 216-223.

[0190] In some embodiments, the isolate nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 108-132, 224-231, 260-280, and 396-403, or a nucleotide sequence at least 90% identical thereto. In another embodiment, the isolated nucleic acid molecule is part of an isolated recombinant vector.

[0191] In one aspect, the present invention is directed to an isolated recombinant viral genome comprising a nucleic acid molecule encoding a truncated human dystrophin protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 83-107 and 216- 223.

[0192] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 108-132, 224-231, 260-280, and 396-403, or a nucleotide sequence at least 90% identical thereto.

[0193] In one aspect, the present invention is directed to a host cell comprising the first nucleic acid molecule and / or the second nucleic acid molecule of the present disclosure, or the first AAV vector and / or the second AAV vector of the present disclosure.

[0194] In some embodiments, the cell is a mammalian cell, an insect cell, or a bacterial cell.

[0195] In another aspect, the present invention is directed to a method of making a first recombinant adeno-associated virus (rAAV) particle, the method comprising providing a host cell comprising the first nucleic acid molecule of the present disclosure, and incubating the host cell under conditions suitable to encapsulate the first nucleic acid in an AAV capsid protein; thereby making the first rAAV particle.

[0196] In yet another aspect, the present invention is directed to a method of making a second recombinant adeno-associated virus (rAAV) particle, the method comprising providing a host cell comprising the second nucleic acid molecule of the present disclosure, and incubating the host cell under conditions suitable to encapsulate the second nucleic acid in an AAV capsid protein; thereby making the second rAAV particle.

[0197] In some embodiments, the cell is a mammalian cell, an insect cell, or a bacterial cell.

[0198] In one aspect, the present invention is directed to a system for use in the treatment of a dystrophin-associated disorder.

[0199] In another aspect, the present invention is directed to a first nucleic acid molecule and a second nucleic acid molecule for use in the treatment of a dystrophin-associated disorder.

[0200] In one aspect, the present invention is directed to a first AAV vector and a second AAV vector for use in the treatment of a dystrophin-associated disorder.

[0201] In another aspect, the present invention is directed to a pharmaceutical composition for use in the treatment of a dystrophin-associated disorder.

[0202] In yet another aspect, the present invention is directed to an isolated nucleic acid molecule for use in the treatment of a dystrophin-associated disorder.

[0203] The details of various aspects or embodiments of the present disclosure are set forth below. Other features, objects, and advantages of the disclosure will be apparent from the description and the claims. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of this disclosure. In the case of conflict, the present description will control.BRIEF DESCRIPTION OF DRAWINGS

[0204] FIG. 1 is a schematic depicting the dual-vector system for expressing a dystrophin protein. The system comprises two separate vectors, such as AAV vectors. The first AAV vector comprises a first nucleic acid molecule comprising a coding region for the N-terminal (Nt) portion of a dystrophin protein (e.g., a truncated dystrophin), a splicing donor sequence (SD) and a 3’ ribozyme (3’Rz), under the control of a muscle-specific promoter and flanked by two ITR sequences. The second AAV vector comprises a second nucleic acid molecule comprising a coding region for the C-terminal (Ct) portion of a dystrophin protein (e.g., a truncated dystrophin), a splicing acceptor sequence (SA) and a 5’ ribozyme (5’Rz), under the control of a muscle-specific promoter and flanked by two ITR sequences. Ribozymes are utilized on the 3' end of the Nt vector and 5' end of the Ct vector to create precise RNA termini and scarless trans- ligation, followed by splicing mediated by the splice donor and acceptor sequences, to generate a single RNA molecule containing a single open reading frame encoding the Nt portion and the Ct portion of the dystrophin protein.

[0205] FIG. 2 depicts various truncated dystrophin proteins having deletions of different domains or deletions of regions encoded by specific exons. “Y” indicates the inclusion of the particular domain in the truncated dystrophin protein, with domains as described in Table 1 . “N”indicates the absence of the particular domain in the truncated dystrophin protein. “P” indicates that the particular domain is partially included in the truncated dystrophin protein. Partially included (“P”, “Pl” and “P2”) here is used when the truncated protein includes at least 1 but not all of the amino acid encoded from that particular domain as described in Table 1. Defined domains are exemplary listings of these sub-domain sequences, and the exact boundaries between each sub-domain can be shifted from the example sequences provided here, which could vary the example categorizations provided here, particularly the “P” (Partially included) category.

[0206] FIG. 3 depicts various truncated dystrophin proteins having deletions of different domains or deletions of regions encoded by specific exons. “Y” indicates the inclusion of the particular exon in the truncated dystrophin protein, with exons defined in SEQ IDs 315-393. “N” indicates the absence of the particular exon in the truncated dystrophin protein. “P” indicates that the particular’ exon is partially included in the truncated dystrophin protein, as described in Table 1. Exon sequences are based on NM.004006.3. Exons 1 and 79 include untranslated regions (UTRs) which are not required for encoding midi-Dys amino acids, hence the “U” designation.

[0207] FIG. 4 depicts the results of delivering various truncated midi-Dys expression plasmid combinations to primary human skeletal muscle cells cultured in vitro. FIG. 4 includes Western blot analysis of cell lysates 24-to-48 hours after transfection of DNA expression plasmids demonstrates that dual plasmid delivery of truncated midi-Dys sequences results in varying levels of truncated midi-Dys protein expression.

[0208] FIG. 5 depicts the results of utilizing different split sites (SS) to divide truncated midi- Dys into N-terminal (Nt) and C-terminal (Ct) plasmids which are transfected into primary human skeletal muscle cells. FIG. 5 shows Western blot results of cell lysates 24-to-48 hours after transfection of various Nt and Ct combinations to express truncated midi-Dystrophins.

[0209] FIG. 6 depicts the results of utilizing different codon optimized and CpG augmented midi-Dystrophin sequences to express midi-Dystrophin protein (SEQ ID NO 259) in primary human skeletal muscle cells. FIG. 6 shows Western blot results of cell lysates 24-to-48 hours after transfection of various codon optimized truncated midi-Dystrophins.

[0210] FIG. 7 depicts the results of utilizing two different dual AAV vector designs to express midi-Dystrophin proteins in primary human skeletal muscle cells, utilizing transfected AAV cis- plasmids.DETAILED DESCRIPTION OF THE INVENTION

[0211] The present disclosure provides isolated recombinant nucleic acid molecules encoding truncated dystrophin proteins, that can be delivered and expressed in a subject using a dual adeno-associated virus (AAV) vector system, to allow expression of truncated dystrophin proteins that are otherwise too large to fit into a single AAV system. The truncated dystrophin proteins can be used to restore the expression and function of a wild-type dystrophin in a subject in need thereof. The present disclosure also provides systems and methods for expressing or delivering a truncated dystrophin protein in a subject, and methods for treating a subject having a dystrophin-associated disease or disorder, e.g., muscular dystrophy, e.g., Duchenne muscular dystrophy (DMD).

[0212] Gene therapy is a promising therapeutic approach for the treatment of many diseases, such as genetic disease and / or diseases that can be treated by expression of a therapeutic protein. Recombinant AAV vectors are generally regarded as one of the safest and most effective classes of vectors for gene therapy. However, one challenge that hampers the development and clinical deployment of therapeutic AAV vectors for gene therapy is their packaging capacity, which is restricted to approximately 4.7 kb of DNA. As a result, expressing a full-length dystrophin protein of 3,686 amino acids, using a single AAV vector, could not be possible since the DMD gene encoding the dystrophin protein exceeds the packaging limit of AAV genomes by more than 2-fold. Accordingly, attention has focused on creating smaller versions of dystrophin that eliminate non-essential subdomains while maintaining at least some function of the full-length protein.

[0213] The present disclosure is based, at least in part, on the development of truncated dystrophin proteins that can be delivered using a dual AAV vector system to a subject for expression. Without wishing to be bound by theory, it is believed that the AAV vectors described herein can be used to administer and / or deliver a functional truncated dystrophin protein in order to achieve sustained and high concentrations and / or more consistent levels of the dystrophin protein. The compositions and methods described herein can be used in the treatment of disorders associated with a lack of a dystrophin protein and / or activity, such as muscular dystrophy, e.g., Duchenne muscular dystrophy (DMD).I. Definitions

[0214] As used herein, each of the following terms has the meaning associated with it in this section.

[0215] The articles “a” and “an” are used herein to refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

[0216] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0217] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0218] Adeno-associated virus (AAV): As used herein, the term “adeno-associated virus” or “AAV” refers to members of the Dependoparvovirus genus or a variant, e.g., a functional variant, thereof. In some embodiments, the AAV is wildtypc, or naturally occurring. In some embodiments, the AAV is recombinant.

[0219] AAV Particle'. As used herein, an “AAV particle” refers to a particle or a virion comprising an AAV capsid, e.g., an AAV capsid variant, and a polynucleotide, e.g., a viral genome. In some embodiments, the viral genome of the AAV particle comprises at least one payload region encoding a protein of interest, e.g., a truncated dystrophin protein or portion thereof, and at least one ITR. In some embodiments, the AAV particle is capable of delivering a nucleic acid, e.g., a payload region, encoding a payload to cells, typically, mammalian, e.g., human, cells. In some embodiments, an AAV particle of the present disclosure may be produced recombinantly. In some embodiments, an AAV particle may be derived from any serotype, described herein or known in the art, including combinations of serotypes (e.g., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self-complementary). In some embodiments, the AAV particle may be replication defective, targeted to a specific tissues or subset of tissues, and / or detargeted to a specific tissue or subset of tissues. In some embodiments, the AAV particle may comprise a peptide, e.g., targeting peptide, present, e.g., inserted into, the capsid to enhance tropism for a desired target tissue. It is to be understood that reference to the AAV particle of the disclosure also includes pharmaceutical compositions thereof, even if not explicitly recited.

[0220] AAV vector: As used herein, the term "AAV vector" or “AAV construct” refers to a vector derived from an adeno-associated virus serotype. "AAV vector" refers to a vector that includes AAV nucleotide sequences as well as heterologous nucleotide sequences. AAV vectors require only the 145 base terminal repeats in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans (Muzyczka (1992) Curr. Topics Microbiol. Immunol. 158:97-129). Typically, the recombinant AAV vector genome will only retain the inverted terminal repeat (ITR) sequences so as to maximize the size of the transgene that can be efficiently packaged by the vector. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging.

[0221] Administering: As used herein, the term “administering” to a subject includes dispensing, delivering or applying a composition of the disclosure to a subject by any suitable route for delivery of the composition to the desired location in the subject. Alternatively, or in combination, delivery is by the topical, parenteral or oral route, intracerebral injection, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

[0222] Capsid'. As used herein, the term “capsid” refers to the exterior, e.g., a protein shell, of a virus particle, e.g., an AAV particle, that is substantially (e.g., >50%, >60%, >70%, >80%, >90%, >95%, >99%, or 100%) protein. In some embodiments, the capsid is an AAV capsid comprising an AAV capsid protein described herein, e.g., a VP1, VP2, and / or VP3 polypeptide. The AAV capsid protein can be a wild-type AAV capsid protein or a variant, e.g., a structural and / or functional variant from a wild-type or a reference capsid protein, referred to herein as an “AAV capsid variant.” In some embodiments, the AAV capsid variant described herein has the ability to enclose, e.g., encapsulate, a viral genome and / or is capable of entry into a cell, e.g., a mammalian cell.

[0223] Codon optimization'. As used herein, the term “codon optimization” refers to a process of changing codons of a given gene in such a manner that the polypeptide sequence encoded by the gene remains the same while the changed codons improve the process of expression of the polypeptide sequence. For example, if the polypeptide is of a human protein sequence and expressed in E. coli, expression will often be improved if codon optimization is performed on theDNA sequence to change the human codons to codons that are more effective for expression in E. coli. Human protein-coding nucleic acid sequences delivered by vectors such as AAV can be further codon optimized for more effective expression in human cells and / or specific human cell or tissue types.

[0224] Contacting: As used herein, the term "contacting" (i.e., contacting a cell with an agent) is intended to include incubating the agent and the cell together in vitro (e.g., adding the agent to cells in culture) or administering the agent to a subject such that the agent and cells of the subject are contacted in vivo. The term "contacting" is not intended to include exposure of cells to an agent that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).

[0225] Cpg Motif: As used herein, a “CpG motif’ is a pattern of bases that include a central CpG (“p” refers to the phosphodiester link between consecutive C and G nucleotides) surrounded by at least one base flanking (on the 3' and the 5' side of) the central CpG.

[0226] Certain constructs are described containing the Greek symbol “A”. In other instances, the same constructs may be described as “delta”. As used herein, the word “delta” and the symbol “A” are used interchangeably.

[0227] Dystrophin: As used herein, the term “dystrophin” refers to a sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell51(6):919-28, 1987). The DAPC is composed of multiple proteins at the muscle sarcolemma that form a structural link between the extracellular matrix (ECM) and the cytoskeleton via dystrophin, an actin binding protein, and alpha-dystroglycan, a laminin-binding protein. These structural links act to stabilize the muscle cell membrane during contraction and protect against contraction-induced damage. With dystrophin loss, membrane fragility results in sarcolemmal tears and an influx of calcium, triggering calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol. 10(2): 168-75, 1997). This uncontrolled cycle of muscle degeneration and regeneration ultimately exhausts the muscle stem cell population, resulting in progressive muscle weakness, endomysial inflammation, and fibrotic scarring (Sacco et al., Cell, 2010. 143(7): p. 1059-71). The dystrophin (DMD) gene is 2.2 megabases at locus Xp21 and has 79 exon s encoding a protein which is over 3500 amino acids. Normal skeleton muscle tissue contains only small amounts of dystrophin but its absence or abnormal expression leads to the development of severe and incurable symptoms. Some mutations in the dystrophingene lead to the production of defective dystrophin and severe dystrophic phenotype in affected patients. Some mutations in the dystrophin gene lead to partially-functional dystrophin protein and a much milder dystrophic phenotype in affected patients.

[0228] Functional: As used herein, the term “functional” or “functional protein” refers to a truncated protein that is capable of, partially or completely, restoring a function of an endogenously expressed full-length protein. A functional truncated dystrophin protein is capable of, partially or completely, restoring a function, e.g., supporting a link between the extracellular matrix and the cytoskeleton, of a full-length dystrophin protein in vitro or in vivo, e.g., in a disease model in an animal, or in a human.

[0229] Dystrophin-associated disorder: The terms “Dystrophin-associated disorder,” “Dystrophin-associated disease,” and the like refer to diseases or disorders having a deficiency in the DMD gene, such as a heritable, e.g., X-linked, mutation in DMD resulting in deficient or defective dystrophin protein expression in patient cells. Dystrophin-associated disorders include, but are not limited to muscular dystrophies, e.g., Duchenne muscular dystrophy (DMD), or Becker muscular dystrophy (BMD).

[0230] Isolated: As used herein, the term “isolated” refers to a substance or entity that is altered or removed from the natural state, e.g., altered or removed from at least some of component with which it is associated in the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Such polynucleotides could be part of a vector and / or such polynucleotides or polypeptides could be part of a composition and still be isolated in that such vector or composition is not part of the environment in which it is found in nature. In some embodiments, an isolated nucleic acid is recombinant, e.g., incorporated into a vector.

[0231] Muscle cell / tissue: As used herein, the term “muscle cell” or “muscle tissue” refers to a cell or group of cells derived from muscle of any kind, for example, skeletal muscle and smooth muscle, e.g., from the digestive tract, urinary bladder, blood vessels or cardiac tissue. Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts.

[0232] Muscular dystrophy: As used herein, the term "muscular dystrophy" or "muscular dystrophies" refers to a group of hereditary muscle diseases that weakens skeletal muscles. Muscular dystrophies are characterized by a genetic defect resulting in muscle weakness or loss of muscle tissue which progressively increases over time. Muscular dystrophies include, but are not limited to, Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy facioscapulohumeral muscular dystrophy, limb girdle muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0233] Duchenne muscular dystrophy (DMD): As used herein, the term “Duchenne muscular dystrophy (DMD)” refers to a fatal, X-linked genetic disease caused by mutations in the dystrophin gene and a complete loss of a functional dystrophin protein. As a result of this genetic defect, individuals with DMD may have symptoms such as trouble walking and running, falling frequently, fatigue, learning disabilitics / difficultics, heart issues as a result of impact on heart muscle functioning, and breathing problems due to weakening of respiratory muscles involved in lung function. Symptoms of muscle weakness associated with DMD typically begin in childhood, often between 3 to 6 year’s of age. Most individuals with DMD require full-time wheelchair use in their early teens and eventually lose their ability to do daily tasks, such as using restrooms, washing and eating, independently. The disease progresses to life-threatening heart and respiratory failure, often resulting in premature death in the 20s or 30s.

[0234] Becker muscular dystrophy (BMD): As used herein, the term “Becker muscular dystrophy (BMD)” refers to an X-linked muscle disease caused by in-frame mutations of the dystrophin gene. These BMD-causing mutations result in the production of a truncated isoform of dystrophin protein that is partially functional and expressed at reduced amounts. The reduced levels of a truncated dystrophin protein lead to progressive skeletal and cardiac muscle dysfunction. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD).

[0235] Mutation As used herein, the term “mutation” refers to a change and / or alteration. In some embodiments, mutations may be changes and / or alterations to proteins (including peptides and polypeptides) and / or nucleic acids (including polynucleic acids). In some embodiments, mutations comprise changes and / or alterations to a protein and / or nucleic acid sequence. Such changes and / or alterations may comprise the addition, substitution and or deletion of one or moreamino acids (in the case of proteins and / or peptides) and / or nucleotides (in the case of nucleic acids and or polynucleic acids). In embodiments wherein mutations comprise the addition and / or substitution of amino acids and / or nucleotides, such additions and / or substitutions may comprise 1 or more amino acid and / or nucleotide residues and may include modified amino acids and / or nucleotides. One or more mutations may result in a “mutant,” “derivative,” or “valiant,” e.g., of a nucleic acid sequence or polypeptide or protein sequence.

[0236] Naturally occurring: As used herein, “naturally occurring” or “wild-type” means existing in nature without artificial aid, or involvement of the hand of man. “Naturally occurring” or “wild-type” may refer to a native form of a biomolecule, sequence, or entity.

[0237] Nucleic acid: As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” refer to any nucleic acid polymers composed of either polydeoxyribonucleotides (containing 2-deoxy-D-ribose), or polyribonucleotides (containing D- ribosc), or any other type of polynucleotide that is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases. There is no intended distinction in length between the term “nucleic acid,” “polynucleotide,” and “oligonucleotide,” and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA.

[0238] Operably linked: As used herein, the phrase “operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.

[0239] Particle'. As used herein, a “particle” is a virus comprised of at least two components, a protein capsid and a polynucleotide sequence enclosed within the capsid.

[0240] Payload: As used herein, “payload” or “payload region” or “transgene” refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome or an expression product of such polynucleotide or polynucleotide region, e.g., a transgene, a polynucleotide encoding a polypeptide.

[0241] Peptide: As used herein, the term “peptide” refers to a chain of amino acids that is less than or equal to about 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0242] Pharmaceutically acceptable'. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0243] Pharmaceutically acceptable excipients: As used herein, the term “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic and non-inflammatory in subjects. In some embodiments, pharmaceutically acceptable excipients are vehicles capable of suspending and / or dissolving active agents. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmcllosc, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and / or xylitol.

[0244] Polypeptide: As used herein, the term “polypeptide” refers to an organic polymer consisting of a large number of amino-acid residues bonded together in a chain. A monomeric protein molecule is a polypeptide.

[0245] Preventing'. As used herein, the term “preventing” refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0246] Promoter: As used herein, the term “promoter” refers to a nucleic acid site to which a polymerase enzyme will bind to initiate transcription (DNA to RNA) or reverse transcription (RNA to DNA).

[0247] Recombinant nucleic acid molecule'. As used herein, the term “recombinant nucleic acid molecule” or “recombinant polynucleotide” refers to a nucleic acid molecule or a polynucleotide having sequences that are not naturally joined together. An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable host cell. A recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.

[0248] Regulatory sequence: As used herein, the term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells, those which are constitutively active, those which are inducible, and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue- specific regulatory sequences). The expression vectors of the disclosure can be introduced into host cells to thereby produce proteins or portions thereof, including fusion proteins or portions thereof, encoded by nucleic acids as described herein.

[0249] Ribozyme: As used herein, the term “ribozyme” refers to an RNA molecule capable of acting as an enzyme. For example, some ribozymes are capable of cleaving RNA molecules. RNA cleaving ribozymes typically consist at least of a catalytic domain and a recognition sequence that is recognized by the catalytic domain. The catalytic domain can be a part of the same RNA molecule as the recognition sequence, and thus mediate cis- cleavage. Alternatively, the catalytic domain can be a separate RNA molecule from the RNA molecule comprising the recognition sequence, and thus mediate trans-cleavage.

[0250] Sequence identity: As used herein, the term “sequence identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Sequence identity of polymeric molecules to one another can be calculated as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical to the nucleotides or amino acid residuesin a given polymeric molecule, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as pail of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the ail, for instance, using publicly available computer software such as BLAST, BLAST-2, or ALIGN. Those skilled in the ail can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0251] Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as are understood in the art.

[0252] Split Site: As used herein, the term “Split Site” refers to the location in the nucleic acid sequence where a truncated midi-Dystrophin transgenic sequence is divided into two portions, which will then be delivered to cells by an N-terminal (Nt) vector (or plasmid) and a C-terminal (Ct) vector or plasmid. As the ribozyme sequences and adjoining splice donor sequences in the Nt vector (or plasmid) or splice acceptor sequences in the Ct vector (or plasmid) are influenced by the 5’ and 3’ protein coding sequences (in the Nt and Ct vectors, respectively), Split Sites must be selected carefully based on their sequence properties and can be empirically optimized for enhanced ability to join the Nt and Ct sequences for midi-Dys protein expression.

[0253] Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Similarly, “subject” or “patient” refers to an organism who may seek, who may require, who is receiving, or who will receive treatment or who is under care by a trained professional for a particular disease or condition. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, dogs, non-human primates, and humans). In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. In certain embodiments, the subject is a human. In some embodiments, the subject is a child. In other embodiments, the subject is an adult. In certain embodiments, a subject or patient may besusceptible to or suspected of having a dystrophin-associated disorder, e.g., muscular dystrophy, e.g., DMD. In certain embodiments, a subject or patient may be diagnosed with a dystrophin- associated disorder, e.g., muscular dystrophy, e.g., DMD.

[0254] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0255] Therapeutic Agent: The term “therapeutic agent” refers to any agent that, when administered to a subject has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect.

[0256] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses. Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of such a dosage regimen.

[0257] Treating: As used herein, the term “treating” refers to partially or completely alleviating, ameliorating, improving, relieving, reversing, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0258] Truncated Dystrophin'. As used herein, a “truncated dystrophin” is any protein shorter than the full length 427 kDa Dystrophin protein isoform. A truncated dystrophin refers to any transgenic Dystrophin protein delivered by either a single AAV vector or reconstituted by a dual vector system, since in both cases, these delivery technologies are incapable of delivering the full-length 427 kDa dystrophin protein. In some embodiments, a truncated dystrophin protein, as described herein, is functional, i.e., the truncated dystrophin protein is capable of restoring, partially or completely, a function of a full-length dystrophin protein in vitro (e.g., supporting a link between the extracellular matrix and the cytoskeleton) or in vivo, e.g., in a disease model in an animal, or in a human.

[0259] Vector: As used herein, a “vector” is any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule. Vectors of the present disclosure may be produced recombinantly and may be based on and / or may comprise adeno-associated virus (AAV) parent or reference scqucncc(s). Such parent or reference AAV sequences may serve as an original, second, third or subsequent sequence for engineering vectors. In non-limiting examples, such parent or reference AAV sequences may comprise any one or more of the following sequences: a polynucleotide sequence encoding a polypeptide or multi-polypeptide, having a sequence that may be wild-type or modified from wild-type and which sequence may encode full-length or partial sequence of a protein, protein domain, or one or more subunits of dystrophin protein and valiants thereof; a polynucleotide encoding dystrophin protein and variants thereof, having a sequence that may be wild-type or modified from wild-type; and a transgene encoding dystrophin protein and variants thereof that may or may not be modified from wild-type sequence.

[0260] Viral genome: As used herein, a “viral genome” or “vector genome” is a polynucleotide comprising at least one inverted terminal repeat (ITR) and at least one encoded payload. A viral genome encodes at least one copy of the payload.

[0261] Wild-type: As used herein, “wild-type” is a native form of a biomolecule, sequence, or entity. The term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or valiant forms.II. Truncated Dystrophin

[0262] The present disclosure provides isolated recombinant nucleic acid molecules encoding a dystrophin protein or portion thereof, e.g., a truncated dystrophin protein or portion thereof.

[0263] Dystrophin is a cytoplasmic protein encoded by the DMD gene, which is a part of a protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. 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.

[0264] The full-length dystrophin muscle isoform (Dp427m) 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 (ACBD), a central so-called “rod” domain, a cystcinc-rich (CR) domain and lastly a carboxy-terminal (CT) domain. The rod domain is comprised of 4 proline-rich hinge domains (abbreviated H) and 24 spectrin-like repeats (abbreviated R) in the following order: a first hinge domain (Hl), 3 spectrin-like repeats (Rl, R2, R3), a second hinge domain (H2), 16 more spectrin-like repeats (R4, R5, R6, R7, R8, R9, RIO, Rl 1, R12, R13, R14, R15, R16, R17, R18, R19), a third hinge domain (H3), 5 more spectrin-like repeats (R20, R21, R22, R23, R24), and finally a fourth hinge domain (H4). Subdomains toward the carboxy-terminus of the protein are involved in connecting to the dystrophin-associated glycoprotein complex (DGC), a large protein complex that forms a critical link between the cytoskeleton and the extra-cellular matrix. The amino acid sequences of the various domains are provided in Table 2 below.Table 2. Domains of Human Dystrophin

[0265] The DMD gene is one of the largest known human genes at approximately 2.2 Mb. The gene is located on the X chromosome at position Xp21 and contains 79 exons. The most common mutations that cause Duchenne muscular dystrophy (DMD), or Becker muscular dystrophy (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, singlebase changes, and splice site changes accounting for approximately 25%-35% of pathogenicvariants in males with DMD and about 10%-20% of males with BMD) can also cause pathogenic dystrophin valiants.

[0266] 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. The BMD genotype is similar to DMD in that deletions are present in the dystrophin gene. However, these deletions leave the reading frame intact. Thus an internally truncated but partially functional dystrophin protein is created. Thus, changing a DMD genotype to a BMD genotype is a common strategy to correct dystrophin. There are many strategies to correct dystrophin, many of which rely on restoring the reading frame of the endogenous dystrophin. This shifts the disease genotype from DMD to BMD.

[0267] The present disclosure provides truncated dystrophin gene sequences and expression vectors containing the same. Such genes and expression vectors arc useful gene therapy to prevent or treat dystrophin-associated disorders, e.g., muscular dystrophies, e.g.. Duchenne muscular dystrophy (DMD), in subjects in need thereof. Expression of functional truncated proteins in transduced muscle cells is able to replicate and replace at least some of the function normally attributable to full-length dystrophin, such as supporting a mechanically strong link between the extracellular matrix and the cytoskeleton.

[0268] In one aspect, the present disclosure provides an isolated, recombinant nucleic acid molecule encoding a truncated human dystrophin protein, wherein the truncated dystrophin protein comprises an ABCD domain (SEQ ID NO: 21), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0269] In some embodiments, the truncated dystrophin protein further comprises an Hl Domain (SEQ ID NO: 22). In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of: a. midi-Dys AR1-R15 (SEQ ID NO: 83), b. midi-Dys AR2-R15 (SEQ ID NO: 84), c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), e. midi-Dys AR4-R15 (SEQ ID NO: 87), f . midi-Dys AR5-R 15 (SEQ ID NO : 88 ) ,g. midi-Dys A exon 13-33 (SEQ ID NO: 93), h. midi-Dys A exon 13-39 (SEQ ID NO: 94), i. midi-Dys A exon 13-41 (SEQ ID NO: 95), j. midi-Dys A exon 13-48 (SEQ ID NO: 96), k. midi-Dys A exon 15-39 (SEQ ID NO: 97), l. midi-Dys A exon 15-41 (SEQ ID NO: 98), m. midi-Dys A exon 15-48 (SEQ ID NO: 99), n. midi-Dys A exon 17-39 (SEQ ID NO: 100), o. midi-Dys A exon 17-41 (SEQ ID NO: 101), p. midi-Dys A exon 17-48 (SEQ ID NO: 102), q. midi-Dys A exon 18-39 (SEQ ID NO: 220), r. midi-Dys A exon 18-41 (SEQ ID NO: 221), s. midi-Dys A exon 18-48 (SEQ ID NO: 222), t. midi-Dys A exon 19-39 (SEQ ID NO: 103), u. midi-Dys A exon 19-41 (SEQ ID NO: 104), v. midi-Dys A exon 19-48 (SEQ ID NO: 105), w. midi-Dys A exon 21-41 (SEQ ID NO: 106), x. midi-Dys A exon 21-42 (SEQ ID NO: 223), and y. midi-Dys A exon 21-48 (SEQ ID NO: 107).

[0270] In some embodiments, the truncated dystrophin protein further comprises an R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), and R19 Domain (SEQ ID NO:42).

[0271] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of a. midi-Dys AR1-R15 (SEQ ID NO: 83), b. midi-Dys AR2-R15 (SEQ ID NO: 84), c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), e. midi-Dys AR4-R15 (SEQ ID NO: 87), f. midi-Dys AR5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 10-33 (SEQ ID NO: 89), h. midi-Dys A exon 10-39 (SEQ ID NO: 90), i. midi-Dys A exon 10-41 (SEQ ID NO: 91), j. midi-Dys A exon 11-33 (SEQ ID NO: 216), k. midi-Dys A exon 11-39 (SEQ ID NO: 217), l. midi-Dys A exon 11-41 (SEQ ID NO: 218), m. midi-Dys A exon 13-33 (SEQ ID NO: 93), n. midi-Dys A exon 13-39 (SEQ ID NO: 94), o. midi-Dys A exon 13-41 (SEQ ID NO: 95), p. midi-Dys A exon 15-39 (SEQ ID NO: 97), q. midi-Dys A exon 15-41 (SEQ ID NO: 98), r. midi-Dys A exon 17-39 (SEQ ID NO: 100), s. midi-Dys A exon 17-41 (SEQ ID NO: 101),t. midi-Dys A exon 18-39 (SEQ ID NO: 220), u. midi-Dys A exon 18-41 (SEQ ID NO: 221), v. midi-Dys A exon 19-39 (SEQ ID NO: 103), w. midi-Dys A exon 19-41 (SEQ ID NO: 104), x. midi-Dys A exon 21-41 (SEQ ID NO: 106), and y. midi-Dys A exon 21-42 (SEQ ID NO: 223).

[0272] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0273] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0274] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0275] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ IDNO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0276] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), RI8 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0277] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R4 domain (SEQ ID NO:27), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0278] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R11 domain (SEQ ID NO:412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0279] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46),R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0280] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0281] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0282] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0283] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51 ).

[0284] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0285] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0286] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0287] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0288] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0289] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0290] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0291] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0292] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0293] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0294] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0295] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0296] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQID NO: 409), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0297] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0298] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0299] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0300] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0301] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0302] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0303] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R16 Domain (SEQ ID NO: 416), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0304] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0305] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-107 and 216-223, or an amino acid sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0306] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 83, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0307] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 84, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0308] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 85, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0309] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 86, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0310] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0311] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0312] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0313] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0314] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 91, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0315] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 92, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0316] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 93, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0317] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 94, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0318] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 95, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0319] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 96, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0320] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0321] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 98, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0322] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 99, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0323] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 100, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0324] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 101, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0325] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 102, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0326] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 103, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0327] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0328] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0329] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0330] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 107, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0331] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 216, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0332] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 217, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0333] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 218, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0334] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 219, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0335] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 220, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0336] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 221, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0337] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 222, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0338] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 223, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0339] In some embodiments, the amino acid sequence of the truncated dystrophin protein is not identical to the amino acid sequence of SEQ ID NO: 143. In some embodiments, the truncated dystrophin protein is not a polypeptide of 2361 amino acids. In some embodiments, the truncated dystrophin protein is less than 2361 amino acid in length. In some embodiments, the truncated dystrophin protein is greater than 2361 amino acid in length.

[0340] In some embodiments, the recombinant nucleic acid molecule encoding the truncated dystrophin protein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 108-132, 224-231, 260-280, and 396-403, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0341] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 108, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0342] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 109, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0343] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 110, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0344] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 111, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0345] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 112, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0346] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 113, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0347] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 114, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0348] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 115, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0349] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 116, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0350] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 117, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0351] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 118, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0352] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 119, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0353] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 120, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0354] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 121, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0355] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 122, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0356] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 123, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0357] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 124, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0358] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 125, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0359] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 126, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0360] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 127, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0361] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 128, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0362] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 129, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0363] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 130, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0364] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 131, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0365] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 132, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0366] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 224, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0367] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 225, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0368] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 226, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0369] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 227, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0370] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 228, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0371] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 229, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0372] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 230, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0373] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 231, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0374] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 260, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0375] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 261, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0376] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 262, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0377] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 263, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0378] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 264, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0379] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 265, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0380] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 266, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0381] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 267, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0382] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 268, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0383] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 269, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0384] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 270, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0385] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 271, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0386] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 272, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0387] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 273, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0388] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 274, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0389] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 275, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0390] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 276, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0391] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 277, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0392] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 278, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0393] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 279, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0394] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 280, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0395] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 396, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0396] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 397, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0397] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 398, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0398] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 399, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0399] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 400, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0400] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 401, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0401] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 402, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0402] In some embodiments, the recombinant nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO: 403, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0403] The present disclosure also provides expression vectors and cells comprising the isolated nucleic acid molecules encoding the truncated dystrophin proteins, as described herein in Tables 3 and 4, or a portion (e.g., a 3’ portion or 5’ portion) of the isolated nucleic acid molecules. In some embodiments, the host cell is a mammalian cell, an insect cell, or a bacterial cell.Table 3. Truncated Dystrophin Proteins - Subdomain Components and OrganizationTable 4. Truncated Dystrophin SequencesIII. Systems of the Disclosure

[0404] The present disclosure also provides systems for efficiently and reliably generating a large single nucleic acid molecule that encodes a protein of interest, e.g., a truncated dystrophin protein, as described herein, whose coding sequence is too large to package into a single expression vector, e.g., an adeno-associated virus (AAV) vector.

[0405] The present disclosure also provides systems for delivery and expression of a protein of interest, e.g., a truncated dystrophin protein, as described herein. Specifically, the invention utilizes ribozyme-mediated zran -ligation of two or more nucleic acid molecules to assemble a single nucleic acid molecule encoding a protein of interest.

[0406] Ribozymes are small catalytic RNA sequences capable of nucleotide-specific selfcleavage found widespread in nature. Ribozyme cleavage generates unique 2', 3 '-phosphate and 5'-hydroxyl termini, and mammalian cells have an inherent capacity to catalyze the / ra -ligation of independent RNAs that have been cleaved by ribozymes. The efficient and precise nature of ribozyme cleavage, which produce precise and unique nucleotide termini, allow for a trans- ligated RNA to be scarless and able to maintain a protein-coding open reading frame. The ligated mRNAs can behave essentially indistinguishably from their natural full-length counterparts, in that they can be spliced using conventional introns and translated into functional proteins.

[0407] For example, provided herein arc a first nucleic acid molecule comprising a first coding region encoding a first portion of the dystrophin protein (e.g., an N-terminal portion of the truncated dystrophin protein), and a second nucleic acid molecule comprising a second coding region encoding a second portion of the dystrophin protein (e.g., a C-terminal portion of the truncated dystrophin protein). Upon ribozyme- mediatedligation of the two nucleic acid molecules, a single nucleic acid molecule is assembled comprising a third (i.e., combined), coding region which encodes the truncated dystrophin protein, hi some embodiments, the first nucleic acid molecule and the second nucleic acid molecule are included in separate viral vectors for delivery to target cells, e.g., muscle cells. In one embodiment, the viral vector is an adeno- associated viral vector (AAV). This dual AAV strategy allows for efficient delivery andexpression of large therapeutic proteins, e.g.. dystrophin, in order to correct disease pathology and treat a subject having a dystrophin-associated disease or disorder, e.g., muscular’ dystrophy, e.g., Duchenne muscular- dystrophy (DMD).

[0408] Accordingly, in one aspect, the present disclosure provides a system for generating a truncated human dystrophin protein, comprising a first recombinant nucleic acid molecule and a second recombinant nucleic acid molecule, wherein the first nucleic acid molecule comprises a first coding region encoding an N-terminal portion of the truncated dystrophin protein and a 3’ ribozyme, where the first coding region is operably linked to the 3’ ribozyme at its 3’ end, wherein the second nucleic acid molecule comprising a second coding region encoding a C- terminal portion of the truncated dystrophin protein and a 5 ’ribozyme, where the second coding region is operably linked to the 5’ ribozyme at its 5’ end, wherein upon ribozyme-mediated catalytic ligation, the first coding region and the second coding region forms a third coding region encoding for the truncated human dystrophin protein. In some embodiments, the systems of the disclosure are capable of producing a significant level of functional truncated dystrophin in transduced cells.

[0409] In some embodiments, the first coding region is operably linked to two or more 3’ ribozymes at its 3’ end. In some embodiments, the second coding region is operably linked to two or more 5’ ribozymes at its 5’ end.

[0410] In some embodiments, the 3’ ribozyme in the first nucleic acid molecule is able to catalyze itself out of the nucleic acid molecule leaving a 3’P or 2’ 3’ cyclic phosphate (cP) end. In some embodiments, the 5’ ribozyme in the second nucleic acid molecule is able to catalyze itself out of the nucleic acid molecule leaving a 5’ OH end. The 3’P or 2’ 3’ cP end and the 5’ OH end of nucleic acid molecules that have undergone ribozyme-mediated cleavage can be ligated together. As such, the coding region of the first nucleic acid molecule, which encodes the N-terminal portion of the truncated dystrophin protein, can be ligated to the coding region of the second nucleic acid molecule, which encodes the C-terminal portion of the truncated dystrophin protein, to form a longer nucleic acid molecule encoding the functional truncated dystrophin protein. In one embodiment, the functional truncated dystrophin is able to restore the expression and function, partially or completely, of an endogenously expressed full-length dystrophin protein.

[0411] In some embodiments, the 3’ ribozyme is selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), Twister (Cpa), Twister Sister, Hammerhead (HH), Hepatitis Delta Virus (HDV), Pistol, Varkud Satellite (VS), Hatchet, Hairpin, and Hovlinc (Hov), or a variant or fragment thereof.

[0412] In some embodiments, the 3’ ribozyme comprises a sequence selected from the group consisting of SEQ ID NOs: 6 - 20.

[0413] In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 6. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO:7. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO:8. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO:9. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 10. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 11 . In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 12. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 13. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 14. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 15. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 16. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 17. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 18. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO: 19. In some embodiments, the 3’ ribozyme comprises a sequence of SEQ ID NO:20.

[0414] In some embodiments, the 5’ ribozyme is selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), Twister (Cpa), Twister Sister, Hammerhead (HH), Hepatitis Delta Virus (HDV), Pistol, Varkud Satellite (VS), Hatchet, Hairpin, and Hovlinc (Hov), or a variant or fragment thereof.

[0415] In some embodiments, the 5’ ribozyme comprises a sequence selected from the group consisting of SEQ ID NOs: 6 - 20.

[0416] In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 6. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO:7. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO:8. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO:9. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 10. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 11. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 12.In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 13. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 14. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 15. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 16. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 17. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 18. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO: 19. In some embodiments, the 5’ ribozyme comprises a sequence of SEQ ID NO:20.

[0417] In some embodiments, the 3’ ribozyme comprises an HDV ribozyme. In other embodiments, the 5’ ribozyme comprises an HH ribozyme. In some embodiments, the HDV ribozyme is selected from the group consisting of HDV, HDV68, HDV67, HDV56, genHDV, and antiHDV, or a variant or fragment thereof. In some embodiment, the HH ribozyme is RzB ribozyme.

[0418] In some embodiments, the 3’ ribozyme comprises a Twister ribozyme. In other embodiments, the 5’ ribozyme comprises an HH ribozyme. In some embodiments, the Twister ribozyme is selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), and Twister (Cpa). In some embodiment, the HH ribozyme is RzB ribozyme.

[0419] In some embodiments, the 3’ ribozyme comprises a Twister ribozyme. In other embodiments, the 5’ ribozyme comprises a Twister ribozyme. In some embodiment, the Twister ribozyme is selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), and Twister (Cpa). In some embodiments, the Twister ribozyme is Twister (Osa).

[0420] Pre-mRNA splicing by the spliceosome has been shown to enhance mRNA translation, either through deposition of factors which promote a pioneer round of translation or through promoting RNA processing and export to the cytoplasm. The addition of a chimeric cis-splicing intron within a transgene has also been shown to promote transgene protein expression. Thus, in some embodiments, the addition of intron splice donor and intron splice acceptor sites that are recognized and cis-spliced by the spliceosome may enhance protein expression from split precursor RNA molecules. Moreover, the inclusion of an intron splice donor and an intron splice acceptor sequence allows for the use of ribozymes that are not completely scarless, since any remaining ribozyme sequences will be removed through splicing of the intron.

[0421] In some embodiments, the first nucleic acid molecule further comprises an intron splice donor sequence, and the second nucleic acid molecule further comprises an intron splice acceptor sequence.

[0422] In some embodiments, the first nucleic acid molecule comprises a first coding region encoding an N-terminal portion of the truncated dystrophin protein, an intron splice donor sequence, and a 3’ ribozyme, and the second nucleic acid molecule comprises a second coding region encoding a C-terminal portion of the truncated dystrophin protein, an intron splice acceptor sequence, and a 5’ ribozyme.

[0423] In some embodiments, the splice donor sequence is positioned between the first coding region and the 3’ ribozyme, hi some embodiments, the splice donor sequence is selected from the group consisting of SEQ ID NOs: 133 - 136. In some embodiments, the splice donor sequence comprises a sequence of SEQ ID NO: 133. In some embodiments, the splice donor sequence comprises a sequence of SEQ ID NO: 134. hi some embodiments, the splice donor sequence comprises a sequence of SEQ ID NO: 135. In some embodiments, the splice donor sequence comprises a sequence of SEQ ID NO: 136. In some embodiments, the splice donor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 3’ ribozyme.

[0424] In some embodiments, the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R7 domain, the R8 domain, the R9 domain, the R10 domain, the R11 domain, the R12 domain, the R13 domain, the R 14 domain, the R15 domain, the R16 domain, the R17 domain, the R18 domain, the R19 domain, the H3 domain, the R20 domain, the R21 domain, and the R22 domain. In some embodiments, the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R8 domain, the R19 domain, the H3 domain, the R20 domain, and the R21 domain, hi some embodiments, the splice donor sequence is not positioned within the R21 domain.

[0425] In some embodiments, the splice acceptor sequence is positioned between the 5’ ribozyme and the second coding region. In some embodiments, the splice acceptor sequence is selected from the group consisting of SEQ ID Nos: 137-141. In some embodiments, the splice acceptor sequence comprises a sequence of SEQ ID NO: 137. In some embodiments, the spliceacceptor sequence comprises a sequence of SEQ ID NO: 138. In some embodiments, the splice acceptor sequence comprises a sequence of SEQ ID NO: 139. In some embodiments, the splice acceptor sequence comprises a sequence of SEQ ID NO: 140. In some embodiments, the splice acceptor sequence comprises a sequence of SEQ ID NO: 141. In some embodiments, the splice acceptor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 5’ ribozyme.

[0426] In some embodiments, the splice donor sequence and the splice acceptor sequence are positioned such that the resulting spliced intron is between 50 - 200 bp in length. In some embodiments, the splice donor sequence and splice acceptor sequence are positioned such that the resulting spliced intron encodes a single predominant reading frame. In some embodiments, a stop codon sequence is introduced into the splice donor sequence or the splice acceptor sequence.

[0427] In some embodiments, the first nucleic acid molecule and the nucleic acid RNA molecule are ligated together by an endogenous ligase that exists in the native cell or tissue in which the nucleic acid assembly is taking place. In some embodiments, the systems of the present invention comprises an exogenous ligase to induce the ligation of the processed nucleic acid molecules together. In one embodiment, the ligase is RNA 2',3'-Cyclic Phosphate and 5'-OH (RtcB) ligase.

[0428] The coding region of the first nucleic acid molecule, which encodes the N-terminal portion of the truncated dystrophin protein, and the coding region of the second nucleic acid molecule, which encodes the C-terminal portion of the truncated dystrophin protein, forms a longer nucleic acid molecule comprising a third coding region which encodes for the truncated dystrophin protein.

[0429] In some embodiments, at least one of the first coding region and the second coding region is at least 2000 nucleotides in length. In some embodiments, at least one of the first coding region and the second coding region is at least 2100 nucleotides in length. In some embodiments, at least one of the first coding region and the second coding region is at least 2200 nucleotides in length. In some embodiments, at least one of the first coding region and the second coding region is at least 2300 nucleotides in length. In some embodiments, at least one of the first coding region and the second coding region is at least 2400 nucleotides in length. In some embodiments, at least one of the first coding region and the second coding region is at least 2500 nucleotides inlength. In some embodiments, at least one of the first coding region and the second coding region is at least 2600 nucleotides in length.

[0430] In some embodiments, the first coding region and the second coding region are each at least 2000 nucleotides in length. In some embodiments, the first coding region and the second coding region are each at least 2100 nucleotides in length. In some embodiments, the first coding region and the second coding region are each at least 2200 nucleotides in length. In some embodiments, the first coding region and the second coding region are each at least 2300 nucleotides in length. In some embodiments, the first coding region and the second coding region are each at least 2400 nucleotides in length. In some embodiments, the first coding region and the second coding region are each at least 2500 nucleotides in length. In some embodiments, the first coding region and the second coding region are each at least 2600 nucleotides in length.

[0431] In some embodiments, the first coding region and the second coding region do not share a region of substantial sequence identity, i.e., the sequence identity of the first coding region and the second coding region is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, or less than 30%. In some embodiments, the 3’ end of the first coding region does not have a sequence identity to the 5’ end of the second coding region.

[0432] In some embodiments, the third coding region, i.e., the combination of the first coding and the second coding region, is at least 4920 nucleotides in length. In some embodiments, the third coding region is at least 5000 nucleotides in length. In some embodiments, the third coding region is at least 5100 nucleotides in length. In some embodiments, the third coding region is at least 5200 nucleotides in length. In some embodiments, the third coding region is at least 5300 nucleotides in length.

[0433] In some embodiments, the truncated human dystrophin proteins are functional.

[0434] In some embodiments, the truncated human dystrophin protein comprises at least 1640 amino acids.

[0435] In some embodiments, the truncated human dystrophin protein comprises ABCD domain (SEQ ID NO: 21), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51 ).

[0436] In some embodiments, the truncated dystrophin protein further comprises Hl Domain (SEQ ID NO: 22). In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of: a. midi-Dys AR1-R15 (SEQ ID NO: 83), b. midi-Dys AR2-R15 (SEQ ID NO: 84), c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), c. midi-Dys AR4-R15 (SEQ ID NO: 87), f. midi-Dys AR5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 13-33 (SEQ ID NO: 93), h. midi-Dys A exon 13-39 (SEQ ID NO: 94), i. midi-Dys A exon 13-41 (SEQ ID NO: 95), j. midi-Dys A exon 13-48 (SEQ ID NO: 96), k. midi-Dys A exon 15-39 (SEQ ID NO: 97), l. midi-Dys A exon 15-41 (SEQ ID NO: 98), m. midi-Dys A exon 15-48 (SEQ ID NO: 99), n. midi-Dys A exon 17-39 (SEQ ID NO: 100), o. midi-Dys A exon 17-41 (SEQ ID NO: 101), p. midi-Dys A exon 17-48 (SEQ ID NO: 102), q. midi-Dys A exon 18-39 (SEQ ID NO: 220), r. midi-Dys A exon 18-41 (SEQ ID NO: 221), s. midi-Dys A exon 18-48 (SEQ ID NO: 222), t. midi-Dys A exon 19-39 (SEQ ID NO: 103), u. midi-Dys A exon 19-41 (SEQ ID NO: 104), v. midi-Dys A exon 19-48 (SEQ ID NO: 105), w. midi-Dys A exon 21-41 (SEQ ID NO: 106), x. midi-Dys A exon 21-42 (SEQ ID NO: 223), and y. midi-Dys A exon 21-48 (SEQ ID NO: 107).

[0437] In some embodiments, the truncated dystrophin protein further comprises R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), and R19Domain (SEQ ID NO:42).

[0438] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of a. midi-Dys AR1-R15 (SEQ ID NO: 83), b. midi-Dys AR2-R15 (SEQ ID NO: 84), c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), e. midi-Dys AR4-R15 (SEQ ID NO: 87), f. midi-Dys AR5-R15 (SEQ ID NO: 88) g. midi-Dys A exon 10-33 (SEQ ID NO: 89), h. midi-Dys A exon 10-39 (SEQ ID NO: 90),i. midi-Dys A exon 1041 (SEQ ID NO: 91), j. midi-Dys A exon 11 33 (SEQ ID NO: 216), k. midi-Dys A exon 11 39 (SEQ ID NO: 217), l. midi-Dys A exon 11 41 (SEQ ID NO: 218), m. midi-Dys A exon 13 33 (SEQ ID NO: 93), n. midi-Dys A exon 13 39 (SEQ ID NO: 94), o. midi-Dys A exon 13 41 (SEQ ID NO: 95), p. midi-Dys A exon 15 39 (SEQ ID NO: 97), q. midi-Dys A exon 15 41 (SEQ ID NO: 98), r. midi-Dys A exon 17- 39 (SEQ ID NO: 100), s. midi-Dys A exon 17- 41 (SEQ ID NO: 101), t. midi-Dys A exon 18 39 (SEQ ID NO: 220), u. midi-Dys A exon 18 41 (SEQ ID NO: 221), v. midi-Dys A exon 19 39 (SEQ ID NO: 103), w. midi-Dys A exon 19 41 (SEQ ID NO: 104), x. midi-Dys A exon 21- 41 (SEQ ID NO: 106), and y. midi-Dys A exon 21- 42 (SEQ ID NO: 223).

[0439] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0440] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0441] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ IDNO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0442] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), RI8 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0443] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0444] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R4 domain (SEQ ID NO:27), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0445] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R11 domain (SEQ ID NO:412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46),R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0446] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0447] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41 ), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0448] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0449] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0450] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0451] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0452] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0453] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0454] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0455] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0456] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0457] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0458] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0459] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0460] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0461] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0462] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R19 Domain (SEQ ID NO:415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0463] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0464] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0465] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0466] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0467] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0468] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0469] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO: 27), a partial R5 domain (SEQ ID NO: 411), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0470] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO: 27), a partial R5 domain (SEQ ID NO: 411), a partial R16 Domain (SEQ ID NO: 416), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0471] In some embodiments, the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

[0472] In some embodiments, the truncated human dystrophin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-107 amd 216-223, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0473] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 83, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0474] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 84, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0475] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 85, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0476] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 86, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0477] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 87, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0478] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0479] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0480] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0481] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 91, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0482] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 92, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0483] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 93, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0484] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 94, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0485] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 95, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0486] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 96, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0487] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 97, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0488] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 98, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0489] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 99, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0490] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 100, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0491] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 101, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0492] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 102, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0493] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 103, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0494] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0495] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0496] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0497] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 107, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0498] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 216, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0499] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 217, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0500] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 218, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0501] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 219, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0502] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 220, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0503] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 221, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0504] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 222, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0505] In some embodiments, the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 223, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0506] In some embodiments, the truncated human dystrophin protein does not comprise 2361 amino acids. In some embodiments, the truncated human dystrophin protein is greater than 2361 amino acids in length. In some embodiments, the truncated human dystrophin protein is less than 2361 amino acids in length. In some embodiments, the truncated human dystrophin protein is not identical to the sequence of SEQ ID NO: 143.

[0507] In some embodiments, the third coding region (created by trans-ligation of the first and second coding regions) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 108-132, 224-231, 260-280, and 396-403, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0508] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 108, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:108, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO: 108, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0509] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 109, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:109, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO: 109, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0510] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 110, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:110, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO:110, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0511] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 111, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 111, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:111, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0512] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 1 12, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:112, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 112, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0513] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 113, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:113, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:113, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0514] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 114, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:114, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the secondcoding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 114, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0515] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 115, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:115, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:115, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0516] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 116, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:116, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:116, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0517] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 117, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:117, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second portion of the nucleotide sequence of SEQ ID NO: 117, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0518] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 118, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 1 18, or a nucleotide sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 118, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0519] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 119, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:119, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:119, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0520] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 120, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:120, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 120, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0521] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 121, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:121, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:121, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0522] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 122, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first,e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 122, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:122, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.[05231 In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 123, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 123, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO: 123, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0524] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 124, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 124, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO: 124, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0525] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 125, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 125, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO: 125, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0526] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 126, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:126, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 126, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0527] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 127, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 127, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 127, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0528] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 128, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:128, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 128, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0529] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 129, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:129, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 129, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0530] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 130, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:130, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:130, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0531] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 131, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:131 , or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:131, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0532] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 132, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO: 132, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO: 132, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0533] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 224, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:224, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO:224, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0534] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 225, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:225, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:225, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0535] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 226, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:226, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:226, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0536] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 227, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:227, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:227, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0537] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 228, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:228, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the secondcoding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:228, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0538] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 229, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:229, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:229, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0539] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 230, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:230, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:230, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0540] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 231, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:231, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:231, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0541] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 260, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:260, or a nucleotide sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:260, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0542] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 261, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:261, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:261, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0543] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 262, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:262, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:262, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0544] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 263, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:263, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:263, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0545] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 264, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first,e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:264, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:264, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.[05461 In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 265, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:265, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:265, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0547] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 266, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:266, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:266, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0548] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 267, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:267, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:267, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0549] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 268, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:268, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:268, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0550] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 269, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:269, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:269, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0551] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 270, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:270, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:270, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0552] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 271, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:271, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:271, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0553] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 272, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:272, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:272, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0554] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 273, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:273, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:273, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0555] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 274, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:274, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:274, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0556] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 275, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:275, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ IDNO:275, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0557] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 276, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:276, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:276, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0558] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 277, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:277, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:277, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0559] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 278, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:278, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:278, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0560] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 279, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:279, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the secondIl lcoding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:279, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0561] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 280, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:280, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:280, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0562] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 396, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:396, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:396, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0563] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 397, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:397, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:397, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0564] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 398, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:398, or a nucleotide sequence at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:398, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0565] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 399, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:399, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:399, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0566] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 400, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:400, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:400, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0567] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 401, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:401, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:401, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0568] In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 402, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first,e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:402, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:402, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.[05691 In some embodiments, the third coding region comprises a nucleotide sequence of SEQ ID NO: 403, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the first coding region comprises a first, e.g., 5’, portion of the nucleotide sequence of SEQ ID NO:403, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the second coding region comprises a second, e.g., 3’, portion of the nucleotide sequence of SEQ ID NO:403, or a nucleotide sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0570] Provided herein, in Table 5, are exemplary first, second, and third coding regions of different truncated dystrophin proteins.Table 5. Exemplary First, Second and Third Coding Regions

[0571] The first isolated nucleic acid molecule and the second isolated nucleic acid molecule of the system can also be introduced into a vector. In some embodiments, the first isolated nucleic acid molecule and the second isolated nucleic acid molecule are encoded in separate vectors.

[0572] The isolated nucleic acid molecules of the invention can be cloned into a number of types of vectors. For example, the nucleic acid molecules can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors ofparticular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0573] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0574] In one embodiment, the isolated nucleic acid molecule is introduced into a vector derived from an adeno-associated virus (AAV) particle. AAV belonging to the genus Dependovirus of the Parvoviridae family and, as used herein, include any serotype of the over 100 serotypes of AAV known. In general, serotypes of AAV have genomic sequences with a significant homology at the level of amino acids and nucleic acids, provide an identical scries of genetic functions, produce virions that are essentially equivalent in physical and functional terms, and replicate and assemble through practically identical mechanisms. Peptide insertions into any of these serotypes may also enhance the tissue- specific tropism and therefore also be used to introduce isolated nucleic acid molecules (For examples, see: Yu, CY., Yuan, Z., Cao, Z. et al. A muscle-targeting peptide displayed on AAV2 improves muscle tropism on systemic delivery.Gene Ther 16, 953-962 (2009). https: / / doi.org / 10.1038 / gt.20Q9.59; Weinmann J, Weis S, Sippel J, Tulalamba W, Remes A, El Andari J, Herrmann AK, et al. Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants. Nat Commun. 2020 Oct 28; 11(1):5432. doi: 10.1038 / s41467-020-19230-w.; Jihad El Andari et al.,Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders. Sei. Adv. 8, eabn4704(2022). DOI:10.1126 / sciadv.abn4704; Tabebordbar M, Lagerborg KA, Stanton A, King EM, et al. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell. 2021 Sep 16;184(19):4919-4938.e22. doi: 10.1016 / j.cell.2021.08.028.)

[0575] The AAV genome is approximately 4.7 kilobases long and is composed of singlestranded deoxyribonucleic acid (ssDNA) which may be either positive- or negative- sensed. The genome comprises two open reading frames (ORFs) encoding the proteins responsible for replication (Rep) and the structural protein of the capsid (Cap). The open reading frames areflanked by two inverted terminal repeats (ITRs), which serve as the origin of replication of the viral genome. The rep frame is made of four overlapping genes encoding Rep proteins ((Rep78, Rep68, Rep52, Rep40). The cap frame contains overlapping nucleotide sequences of three capsid proteins: VP1, VP2 and VP3. The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid. See Carter B, Adeno-associated virus and adeno- associated virus vectors for gene delivery, Lassie D, et ah, Eds., "Gene Therapy: Therapeutic Mechanisms and Strategies" (Marcel Dekker, Inc., New York, NY, US, 2000) and Gao G, et al, J. Virol. 2004; 78( 12):6381-6388.

[0576] AAV have been explored as vectors for delivery of gene therapeutics because of several unique features. Non-limiting examples of the features include (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host range for infectivity, including human cells;(iii) wild-type AAV has not been associated with any disease and has not been shown to replicate in infected cells; (iv) the lack of ccll-mcdiatcd immune response against the vector, and (v) the non-integrative nature in a host chromosome thereby reducing potential for long-term genetic alterations. Moreover, infection with AAV vectors has minimal influence on changing the pattern of cellular gene expression (Stilwell and Samulski et al., Biotechniques, 2003, 34, 148, the contents of which are herein incorporated by reference in their entirety).

[0577] Typically, AAV vectors for protein delivery may be recombinant viral vectors which are replication defective as they lack sequences encoding functional Rep and Cap proteins within the viral genome. In some cases, the defective AAV vectors may lack most or all coding sequences and essentially only contain one or two AAV ITR sequences and a payload sequence.

[0578] AAV vectors may be modified to enhance the efficiency of delivery. Such modified AAV vectors of the present disclosure can be packaged efficiently and can be used to successfully infect the target cells at high frequency and with minimal toxicity.

[0579] The term "AAV vector" means a vector derived from an adeno-associated virus serotype, including without limitation, serotype 1 (AAV 1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), serotype 13 (AAV13), AAVrh74, AAV-rhlO, AAV-DJ, AAV-LK03, AAV-MYO, AAV-MY02, AAV-MY03, MY03A-AAV, MY04A-AAV, or MY04E-AAV. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more typesof cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.Dual-Vector System

[0580] The present disclosure provides a dual- vector system, where a transgene, e.g., a truncated dystrophin protein, is split into two separate vectors, e.g., AAV vectors. Co-infection of a cell with these two AAV vectors result in the transcription of an assembled RNA that could not be encoded by a single AAV vector because of the packaging limits of AAV.

[0581] In one aspect, the present disclosure provides a vector system for expressing a truncated human dystrophin protein, comprising a first AAV vector and a second AAV vector, wherein the first AAV vector comprises a first nucleic acid molecule comprising a first coding region encoding an N-terminal portion of the truncated dystrophin protein and a 3’ ribozyme, and the second AAV vector comprises a second nucleic acid molecule comprising a second coding region encoding a C-terminal portion of the truncated dystrophin protein and a 5’ ribozyme. Upon ribozyme-mediated catalytic ligation, the first coding region and the second coding region forms a third coding region encoding for the truncated human dystrophin protein. Upon delivery into a target cell, the truncated dystrophin protein is able to restore the expression and function of an endogenously expressed full-length dystrophin protein. It will be understood that, in various embodiments, the first coding region and second coding region comprised in the first AAV and second AAV vectors may be the sequences of any first coding region and second coding regions described herein.

[0582] In some embodiments, the first nucleic acid molecule within the first AAV vector further comprises an intron splice donor sequence, and the second nucleic acid molecule within the second AAV vector further comprises an intron splice acceptor sequence.

[0583] In some embodiments, the splice donor sequence is positioned between the first coding region and the 3’ ribozyme, hi some embodiments, the splice acceptor sequence is positioned between the 5’ ribozyme and the second coding region.

[0584] In certain embodiments, the vectors also include conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and / or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, “operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest (e.g., a truncated dystrophin protein or portion thereof) and expression control sequences that act in trans or at a distance to control the gene of interest.

[0585] Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (z.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the ail and may be utilized.

[0586] In some embodiments, the first AAV vector comprises a first promoter operably linked to the first nucleic acid molecule comprising a first coding region encoding an N-terminal portion of the truncated dystrophin protein. In some embodiments, the second AAV vector comprises a second promoter operably linked to the second nucleic acid molecule comprising a second coding region encoding a C-terminal portion of the truncated dystrophin protein. In some embodiments the first and second promoters are identical. In some embodiments the first and second promoters are different.

[0587] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters and mammalian promoters. In some embodiments, the promoters may be human promoters. In some embodiments, the first promoter and / or the second promoter is a ubiquitous promoter or a tissue specific promoter.

[0588] In some embodiments, the promoter is a ubiquitous promoter that results in expression in one or more, e.g., multiple, cells and / or tissues. In some embodiments, a promoter which drives or promotes expression in most mammalian tissues includes, but is not limited to, human elongation factor la-subunit (EFla), cytomegalovirus (CMV) immediate-early enhancer and / or promoter, chicken P-actin (CBA) and its derivative CAG, glucuronidase (GUSB), and ubiquitin C (UBC).

[0589] In some embodiments, the promoter is a tissue specific promoter, e.g., a muscle specific promoter, e.g., an actin promoter, a myosin promoter, and a creatine kinase promoter. In some embodiments, the promoter is a CK8 promoter, an MHCK7 promoter, an SPC5-12 promoter, a MCK promoter, a desmin promoter, or a Calpain3 promoter.

[0590] In some embodiments, the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 144-150, or a nucleotide sequence at least 95% identical thereto.

[0591] In some embodiments, the promoter comprises a nucleotide sequence of SEQ ID NO: 144, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the promoter comprises a nucleotide sequence of SEQ ID NO: 145, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the promoter comprises a nucleotide sequence of SEQ ID NO: 146, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the promoter comprises a nucleotide sequence of SEQ ID NO: 147, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the promoter comprises a nucleotide sequence of SEQ ID NO: 148, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the promoter comprises a nucleotide sequence of SEQ ID NO: 149, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the promoter comprises a nucleotide sequence of SEQ ID NO: 150, or a nucleotide sequence at least 95% identical thereto.

[0592] In some embodiments, the first AAV vector and / or the second AAV vector further comprise an enhancer. Enhancer sequences found on a vector regulate the expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.

[0593] In some embodiments, the first AAV vector and / or the second AAV vector further comprise an intron or a fragment or derivative thereof. In some embodiments, the intron may enhance expression of a truncated dystrophin protein or portion thereof, as described herein.

[0594] In some embodiments, the first AAV vector and / or the second AAV vector may comprise a human beta-globin intron or a fragment or variant thereof. In some embodiments, the intron comprises one or more human beta- globin sequences (e.g.. including fragments / variants thereof). In some embodiments, the first AAV vector and / or the second AAV vector may comprise an SV40 intron or others known in the art.

[0595] In some embodiments, the intron region comprises a nucleotide sequence of SEQ ID NO: 156, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the intronregion comprise a nucleotide sequence of SEQ ID NO: 157, or a nucleotide sequence at least 95% identical thereto.

[0596] In some embodiments, the first AAV vector and / or the second AAV vector further comprise an inverted terminal repeat (ITR) sequence. The ITR sequence is positioned either 5’ or 3’ relative to the transgene, e.g., the truncated dystrophin protein or portion thereof. In some embodiments, the first AAV vector and / or the second AAV vector have two ITRs. These two ITRs flank the payload region, e.g., the truncated dystrophin protein or portion thereof, at the 5’ and 3’ ends. In some embodiments, the ITR functions as an origin of replication comprising a recognition site for replication. In some embodiments, the ITR comprises a sequence region which can be complementary and symmetrically arranged. In some embodiments, the ITR incorporated into a viral vector described herein may be comprised of a naturally occurring polynucleotide sequence or a recombinantly derived polynucleotide sequence.

[0597] In a non-limiting example, the AAV vector comprises two ITRs. In some embodiments, the ITRs are of the same serotype as one another. In another embodiment, the ITRs are of different serotypes. Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid. In some embodiments both ITRs of the AAV vectors are AAV2 ITRs.

[0598] Independently, each ITR may be about 100 to about 150 nucleotides in length. In some embodiments, the ITR comprises 100-180 nucleotides in length, e.g., about 100-115, about 100- 120, about 100-130, about 100-140, about 100-150, about 100-160, about 100-170, about 100-180, about 110-120, about 110-130, about 110-140, about 110-150, about 110-160, about 110-170, about 110-180, about 120-130, about 120-140, about 120-150, about 120-160, about 120-170, about 120-180, about 130-140, about 130-150, about 130-160, about 130-170, about 130-180, about 140-150, about 140-160, about 140-170, about 140-180, about 150-160, about 150-170, about 150-180, about 160-170, about 160-180, or about 170-180 nucleotides in length. Non-limiting examples of ITR length are 120, 130, 140, 141, 142, 145 nucleotides in length.

[0599] In some embodiments, the ITR sequence comprises a nucleotide sequence of SEQ ID NO: 202, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the ITR sequence comprises a nucleotide sequence of SEQ ID NO: 203, or a nucleotide sequence at least 95% identical thereto.

[0600] In some embodiments, the first AAV vector and / or the second AAV vector further comprise a polyadenylation (polyA) sequence.

[0601] In some embodiments, the polyA sequence comprises a length of about 40-600 nucleotides, e.g., about 40-300 nucleotides, about 40-250 nucleotides, about 100-400 nucleotides, about 100-300 nucleotides, about 100-200 nucleotides, about 200-600 nucleotides, about 200-500 nucleotides, about 200-400 nucleotides, about 200-300 nucleotides, about 300- 600 nucleotides, about 300-500 nucleotides, about 300-400 nucleotides, about 400-600 nucleotides, about 400-500 nucleotides, or about 500-600 nucleotides.

[0602] In some embodiments, the polyadenylation sequence is a bovine growth hormone (bGH) polyA sequence. In some embodiments, the polyadenylation sequence comprises a nucleotide sequence of SEQ ID NO: 151, or a nucleotide sequence at least 95% identical thereto.

[0603] In some embodiments, the polyadenylation sequence is a synthetic bovine growth hormone (bGH) polyA sequence. In some embodiments, the polyadenylation sequence comprises a nucleotide sequence of SEQ ID NO: 152, or a nucleotide sequence at least 95% identical thereto.

[0604] In some embodiments, the first AAV vector and / or the second AAV vector further comprise an untranslated region (UTR). Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription. Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production.

[0605] Any UTR from any gene known in the art may be incorporated into the AAV vectors. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected or they may be altered in orientation or location. In some embodiments, the UTR used in the AAV vector may be inverted, shortened, lengthened, or made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term “altered,” as it relates to a UTR, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.

[0606] In some embodiments, the first AAV vector and / or the second AAV vector further comprise a Kozak sequence. Kozak sequences, which are commonly known to be involved in theprocess by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs.

[0607] In some embodiments, the first AAV vector and / or the second AAV vector further comprise a post transcriptional regulatory element, e.g., a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), e.g., in the 3’ UTR.

[0608] In some embodiments, the WPRE comprises a nucleotide sequence of SEQ ID NO: 153, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the WPRE comprises a nucleotide sequence of SEQ ID NO: 154, or a nucleotide sequence at least 95% identical thereto. In some embodiments, the WPRE comprises a nucleotide sequence of SEQ ID NO: 155, or a nucleotide sequence at least 95% identical thereto.

[0609] In some embodiments, the first AAV vector and / or the second AAV vector further comprise one or more filler sequences. The filler sequence may be a wild-type sequence or an engineered sequence. A filler sequence may be a variant of a wild-type sequence.

[0610] The AAV vector comprise one or more filler sequences in order to have the optional length for packaging. In some embodiments, the AAV vector comprises any portion of a filler sequence, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of a filler sequence. The filler sequences can be located within any position within the AAV vector, for example, 3’ to the 5’ ITR sequence, 5’ to the promoter sequence, 3’ to the poly adenylation sequence, or 5’ to the 3’ ITR sequence.

[0611] In order to assess the expression of a truncated dystrophin protein of the invention, the vectors to be introduced into a cell may also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic -resistance genes, such as neo and the like. Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easilydetectable property, e.g.. enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.Exemplary AAV vectors

[0612] In some embodiments, the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a first coding region encoding an N-terminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, and a 3’ ITR sequence.

[0613] In some embodiments, the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a first coding region encoding an N-tcrminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, a polyadenylation sequence, and a 3’ ITR sequence.

[0614] In some embodiments, the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, an intron region, a first coding region encoding an N-teiminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, a polyadenylation sequence, and a 3’ ITR sequence.

[0615] In some embodiments, the first AAV vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 301, 303, 305, 307, 308, 310, 312, 314, 394, and 395, or a nucleotide sequence at least 90% identical thereto.

[0616] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 160, or a nucleotide sequence at least 90% identical thereto.

[0617] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 162, or a nucleotide sequence at least 90% identical thereto.

[0618] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 164, or a nucleotide sequence at least 90% identical thereto.

[0619] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 166, or a nucleotide sequence at least 90% identical thereto.

[0620] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 168, or a nucleotide sequence at least 90% identical thereto.

[0621] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 170, or a nucleotide sequence at least 90% identical thereto.

[0622] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 172, or a nucleotide sequence at least 90% identical thereto.

[0623] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 174, or a nucleotide sequence at least 90% identical thereto.

[0624] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 176, or a nucleotide sequence at least 90% identical thereto.

[0625] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 178, or a nucleotide sequence at least 90% identical thereto.

[0626] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 180, or a nucleotide sequence at least 90% identical thereto.

[0627] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 182, or a nucleotide sequence at least 90% identical thereto.

[0628] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence at least 90% identical thereto.

[0629] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 186, or a nucleotide sequence at least 90% identical thereto.

[0630] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 188, or a nucleotide sequence at least 90% identical thereto.

[0631] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 190, or a nucleotide sequence at least 90% identical thereto.

[0632] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 192, or a nucleotide sequence at least 90% identical thereto.

[0633] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 194, or a nucleotide sequence at least 90% identical thereto.

[0634] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 196, or a nucleotide sequence at least 90% identical thereto.

[0635] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 198, or a nucleotide sequence at least 90% identical thereto.

[0636] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 200, or a nucleotide sequence at least 90% identical thereto.

[0637] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 301, or a nucleotide sequence at least 90% identical thereto.

[0638] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 303, or a nucleotide sequence at least 90% identical thereto.

[0639] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 305, or a nucleotide sequence at least 90% identical thereto.

[0640] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 307, or a nucleotide sequence at least 90% identical thereto.

[0641] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 308, or a nucleotide sequence at least 90% identical thereto.

[0642] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 310, or a nucleotide sequence at least 90% identical thereto.

[0643] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 312, or a nucleotide sequence at least 90% identical thereto.

[0644] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 314, or a nucleotide sequence at least 90% identical thereto.

[0645] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 394, or a nucleotide sequence at least 90% identical thereto.

[0646] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 395, or a nucleotide sequence at least 90% identical thereto.

[0647] In some embodiments, the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, and a 3’ ITR sequence.

[0648] In some embodiments, the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a secondcoding region encoding a C-terminal portion of the truncated dystrophin protein, a polyadenylation sequence, and a 3’ ITR sequence.

[0649] In some embodiments, the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, a WPRE sequence, a polyadenylation sequence, and a 3’ ITR sequence.

[0650] In some embodiments, the second AAV vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 302, 304, 306, 309, 311, and 313, or a nucleotide sequence at least 90% identical thereto.

[0651] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 161 , or a nucleotide sequence at least 90% identical thereto.

[0652] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 163, or a nucleotide sequence at least 90% identical thereto.

[0653] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 165, or a nucleotide sequence at least 90% identical thereto.

[0654] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 167, or a nucleotide sequence at least 90% identical thereto.

[0655] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 169, or a nucleotide sequence at least 90% identical thereto.

[0656] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 171, or a nucleotide sequence at least 90% identical thereto.

[0657] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 173, or a nucleotide sequence at least 90% identical thereto.

[0658] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 175, or a nucleotide sequence at least 90% identical thereto.

[0659] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 177, or a nucleotide sequence at least 90% identical thereto.

[0660] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 179, or a nucleotide sequence at least 90% identical thereto.

[0661] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO:181, or a nucleotide sequence at least 90% identical thereto.

[0662] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 183, or a nucleotide sequence at least 90% identical thereto.

[0663] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 185, or a nucleotide sequence at least 90% identical thereto.

[0664] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 187, or a nucleotide sequence at least 90% identical thereto.

[0665] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 189, or a nucleotide sequence at least 90% identical thereto.

[0666] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 191 , or a nucleotide sequence at least 90% identical thereto.

[0667] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 193, or a nucleotide sequence at least 90% identical thereto.

[0668] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 195, or a nucleotide sequence at least 90% identical thereto.

[0669] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 197, or a nucleotide sequence at least 90% identical thereto.

[0670] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 199, or a nucleotide sequence at least 90% identical thereto.

[0671] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO:201, or a nucleotide sequence at least 90% identical thereto.

[0672] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO:302, or a nucleotide sequence at least 90% identical thereto.

[0673] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO:304, or a nucleotide sequence at least 90% identical thereto.

[0674] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 306, or a nucleotide sequence at least 90% identical thereto.

[0675] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO:309, or a nucleotide sequence at least 90% identical thereto.

[0676] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO:311, or a nucleotide sequence at least 90% identical thereto.

[0677] In some embodiments, the second AAV vector comprises a nucleotide sequence of SEQ ID NO:313, or a nucleotide sequence at least 90% identical thereto.

[0678] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 160, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 161, or a nucleotide sequence at least 90% identical thereto.

[0679] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 162, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 163, or a nucleotide sequence at least 90% identical thereto.

[0680] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 164, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 165, or a nucleotide sequence at least 90% identical thereto.

[0681] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 166, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 167, or a nucleotide sequence at least 90% identical thereto.

[0682] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 168, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 169, or a nucleotide sequence at least 90% identical thereto.

[0683] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 170, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 171, or a nucleotide sequence at least 90% identical thereto.

[0684] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 172, or a nucleotide sequence at least 90% identical thereto, and the second AAV vectorcomprises a nucleotide sequence of SEQ ID NO: 173, or a nucleotide sequence at least 90% identical thereto.

[0685] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 174, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 175, or a nucleotide sequence at least 90% identical thereto.

[0686] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 176, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 177, or a nucleotide sequence at least 90% identical thereto.

[0687] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 178, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 179, or a nucleotide sequence at least 90% identical thereto.

[0688] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 180, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 181, or a nucleotide sequence at least 90% identical thereto.

[0689] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 182, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 183, or a nucleotide sequence at least 90% identical thereto.

[0690] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 185, or a nucleotide sequence at least 90% identical thereto.

[0691] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 186, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 187, or a nucleotide sequence at least 90% identical thereto.

[0692] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 188, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 189, or a nucleotide sequence at least 90% identical thereto.

[0693] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 190, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 191, or a nucleotide sequence at least 90% identical thereto.

[0694] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 192, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 193, or a nucleotide sequence at least 90% identical thereto.

[0695] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 194, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 195, or a nucleotide sequence at least 90% identical thereto.

[0696] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 196, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 197, or a nucleotide sequence at least 90% identical thereto.

[0697] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO: 198, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO: 199, or a nucleotide sequence at least 90% identical thereto.

[0698] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:200, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:201, or a nucleotide sequence at least 90% identical thereto.

[0699] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:301, or a nucleotide sequence at least 90% identical thereto, and the second AAV vectorcomprises a nucleotide sequence of SEQ ID NO:302, or a nucleotide sequence at least 90% identical thereto.

[0700] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:303, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:304, or a nucleotide sequence at least 90% identical thereto.

[0701] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:305, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:306, or a nucleotide sequence at least 90% identical thereto.

[0702] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:307, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:306, or a nucleotide sequence at least 90% identical thereto.

[0703] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:308, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:309, or a nucleotide sequence at least 90% identical thereto.

[0704] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NQ:310, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:311, or a nucleotide sequence at least 90% identical thereto.

[0705] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:312, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:313, or a nucleotide sequence at least 90% identical thereto.

[0706] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:314, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:309, or a nucleotide sequence at least 90% identical thereto.

[0707] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:394, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:309, or a nucleotide sequence at least 90% identical thereto.

[0708] In some embodiments, the first AAV vector comprises a nucleotide sequence of SEQ ID NO:395, or a nucleotide sequence at least 90% identical thereto, and the second AAV vector comprises a nucleotide sequence of SEQ ID NO:306, or a nucleotide sequence at least 90% identical thereto.IV. Viral Production

[0709] Adeno-associated viral (AAV) production includes processes and methods for producing AAV particles and vectors which can contact a target cell to deliver a payload, e.g. a recombinant viral construct, which includes a nucleic acid molecule encoding a payload molecule, e.g., a truncated dystrophin protein or portion thereof.

[0710] In some embodiments, disclosed herein is a method of making a recombinant AAV particle of the present disclosure, the method comprising (i) providing a host cell comprising a viral genome described herein, e.g., a nucleic acid comprising a coding region encoding a truncated dystrophin protein or portion thereof, and incubating the host cell under conditions suitable to encapsulate the viral genome in a capsid protein, thereby making the recombinant AAV particle. In some embodiments, the method comprises prior to step (i), introducing a first nucleic acid comprising the viral genome into a cell. In some embodiments, the host cell comprises a second nucleic acid encoding the capsid protein. In some embodiments, the second nucleic acid is introduced into the host cell prior to, concurrently with, or after the first nucleic acid molecule. In some embodiments, the host cell is a bacterial cell, a mammalian cell (e.g., a HEK293 cell), or an insect cell (e.g., an Sf9 cell).

[0711] In some embodiments, disclosed herein is a method for making a first recombinant AAV particle, the method comprises providing a host cell comprising a first nucleic acid molecule encoding an N-terminal portion of a truncated dystrophin protein, and incubating the host cell under conditions suitable to encapsulate the first nucleic acid in an AAV capsid protein; thereby making the first recombinant AAV particle.

[0712] In some embodiments, disclosed herein is a method for making a second recombinant AAV particle, the method comprises providing a host cell comprising a second nucleic acid molecule encoding a C-terminal portion of a truncated dystrophin protein, and incubating the host cell under conditions suitable to encapsulate the second nucleic acid in an AAV capsid protein; thereby making the second recombinant AAV particle.

[0713] In various embodiments, methods are provided herein of producing AAV particles or vectors by (a) contacting a viral production cell with one or more viral expression constructs encoding at least one AAV capsid protein, and one or more payload constructs encoding a payload molecule, e.g.. a truncated dystrophin protein or portion thereof, e.g., an N-terminal portion of a truncated dystrophin protein, or a C-terminal portion of a truncated dystrophin protein; (b) culturing the viral production cell under conditions such that at least one AAV particle or vector is produced, and (c) isolating t...

Claims

CLAIMS1. A system for generating a truncated human dystrophin protein, comprising a first recombinant nucleic acid molecule and a second recombinant nucleic acid molecule, wherein the first nucleic acid molecule comprises a first coding region encoding an N-terminal portion of the truncated dystrophin protein and a 3’ ribozyme, wherein the first coding region is operably linked to the 3’ ribozyme at its 3’ end, wherein the second nucleic acid molecule comprising a second coding region encoding a C- terminal portion of the truncated dystrophin protein and a 5’ ribozyme, wherein the second coding region is operably linked to the 5’ ribozyme at its 5’ end, wherein upon ribozyme-mediated catalytic ligation, the first coding region and the second coding region forms a third coding region encoding for the truncated human dystrophin protein, and wherein the truncated human dystrophin protein comprises at least 1640 amino acids.

2. The system of claim 1, wherein the first coding region is operably linked to two or more 3’ ribozymes at its 3’ end.

3. The system of claim 2, wherein the two or more 3’ ribozymes are the same 3’ ribozyme.

4. The system of claim 2, wherein the two or more 3’ ribozymes are different 3’ ribozymes.

5. The system of any one of claims 1-4, wherein the second coding region is operably linked to two or more 5’ ribozymes at its 5’ end.

6. The system of claim 5, wherein the two or more 5’ ribozymes are the same 5’ ribozymes.

7. The system of claim 5, wherein the two or more 5’ ribozymes are different 5’ ribozymes.

8. The system of any one of claims 1-7, wherein the 5’ ribozyme and the 3’ ribozyme are each independently selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), Twister (Cpa), Twister Sister, Hammerhead (RzB)c, HDV, Pistol, Varkud Satellite (VS), Hatchet, Hairpin, and Hovlinc (Hov).

9. The system of any one of claims 1-8, wherein the 5’ ribozyme and the 3’ ribozyme are each independently selected from the group consisting of SEQ ID NOs: 6 - 20.

10. The system of any one of claim 1-9, wherein the first nucleic acid molecule further comprises an intron splice donor sequence, and the second nucleic acid molecule further comprises an intron splice acceptor sequence.

11. The system of claim 10, wherein the splice donor sequence is positioned between the first coding region and the 3’ ribozyme.

12. The system of claim 11, wherein the splice donor sequence is selected from the group consisting of SEQ ID NOs: 133 - 136.

13. The system of any of claims 10-12, wherein the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R7 domain, the R8 domain, the R9 domain, the RIO domain, the R11 domain, the R12 domain, the R13 domain, the R14 domain, the R15 domain, the R16 domain, the R17 domain, the R18 domain, the R19 domain, the H3 domain, the R20 domain, the R21 domain, and the R22 domain.

14. The system of any of claims 10-13, wherein the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R8 domain, the R19 domain, the H3 domain, the R20 domain, and the R21 domain.

15. The system of any one of claims 10-14, wherein the splice donor sequence is not positioned within the R21 domain.

16. The system of any one of claims 10-15, wherein the splice donor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 3’ ribozyme.

17. The system of claim 10, wherein the splice acceptor sequence is positioned between the 5’ ribozyme and the second coding region.

18. The system of claim 17, wherein the splice acceptor sequence is selected from the group consisting of SEQ ID Nos: 137-141.

19. The system of any one of claims 10, 17 and 18, wherein the splice acceptor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 5’ ribozyme.

20. The system of any one of claims 10-19, wherein the splice donor sequence and the splice acceptor sequence are positioned such that the resulting spliced intron is between 50 - 200 bp in length.

21. The system of any one of claims 10-20, wherein the splice donor sequence and splice acceptor sequence are positioned such that the resulting spliced intron encodes a single predominant reading frame.

22. The system of any one of claims 10-21, wherein a stop codon sequence is introduced into the splice donor sequence or the splice acceptor sequence.

23. The system of any one of claims 1-22, wherein at least one of the first coding region and the second coding region is at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length!24. The system of any one of claims 1-23, wherein the first coding region and the second coding region are each at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length.

25. The system of any one of claims 1-24, wherein the third coding region is at least 4920 nucleotides in length, or at least 5100 nucleotides in length, or at least 5300 nucleotides in length.

26. The system of any one of claims 1-25, wherein the third coding region comprises 300 or fewer CpG motifs.

27. The system of any one of claims 1-26, wherein the third coding region comprises 290 or fewer CpG motifs.

28. The system of any one of claims 1-27, wherein the third coding region comprises 67 or fewer CpG motifs.

29. The system of any one of claims 1-28, wherein the first coding region and the second coding region do not share a region of substantial sequence identity.

30. The system of any one of claims 1-29, wherein the 3’ end of the first coding region does not have a sequence identity to the 5’ end of the second coding region.

31. The system of any one of claims 1-30, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

32. The system of any one of claims 1-31, wherein the truncated dystrophin protein further comprises Hl Domain (SEQ ID NO: 22).

33. The system of claim 32, wherein the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of: a. midi-Dys AR1-R15 (SEQ ID NO: 83), b . midi-Dys AR2-R 15 (SEQ ID NO : 84) , c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), e. midi-Dys AR4-R15 (SEQ ID NO: 87), f. midi-Dys AR5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 13 -33 (SEQ ID NO: 93), h. midi-Dys A exon 13 -39 (SEQ ID NO: 94), i. midi-Dys A exon 13 -41 (SEQ ID NO: 95), j. midi-Dys A exon 13 -48 (SEQ ID NO: 96), k. midi-Dys A exon 15 -39 (SEQ ID NO: 97), l. midi-Dys A exon 15 -41 (SEQ ID NO: 98), m. midi-Dys A exon 15 -48 (SEQ ID NO: 99), n. midi-Dys A exon 17 -39 (SEQ ID NO: 100), o. midi-Dys A exon 17 -41 (SEQ ID NO: 101), p. midi-Dys A exon 17 -48 (SEQ ID NO: 102), q. midi-Dys A exon 18 -39 (SEQ ID NO: 220), r. midi-Dys A exon 18 -41 (SEQ ID NO: 221), s. midi-Dys A exon 18 -48 (SEQ ID NO: 222), t. midi-Dys A exon 19 -39 (SEQ ID NO: 103), u. midi-Dys A exon 19 -41 (SEQ ID NO: 104), v. midi-Dys A exon 19 -48 (SEQ ID NO: 105), w. midi-Dys A exon 21 -41 (SEQ ID NO: 106), x. midi-Dys A exon 21 -42 (SEQ ID NO: 223), and y. midi-Dys A exon 21 -48 (SEQ ID NO: 107).

34. The system of any one of claims 1-33, wherein the truncated dystrophin protein further comprises R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), and R19 Domain (SEQ ID NO:42).

35. The system of claim 34, wherein the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of a. midi-Dys AR1-R15 (SEQ ID NO: 83), b . midi-Dys AR2-R 15 (SEQ ID NO : 84) , c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), e. midi-Dys AR4-R15 (SEQ ID NO: 87), f. midi-Dys AR5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 10-33 (SEQ ID NO: 89), h. midi-Dys A exon 10-39 (SEQ ID NO: 90), i. midi-Dys A exon 10-41 (SEQ ID NO: 91), j. midi-Dys A exon 11-33 (SEQ ID NO: 216), k. midi-Dys A exon 11-39 (SEQ ID NO: 217), l. midi-Dys A exon 11-41 (SEQ ID NO: 218), m. midi-Dys A exon 13-33 (SEQ ID NO: 93), n. midi-Dys A exon 13-39 (SEQ ID NO: 94), o. midi-Dys A exon 13-41 (SEQ ID NO: 95), p. midi-Dys A exon 15-39 (SEQ ID NO: 97), q. midi-Dys A exon 15-41 (SEQ ID NO: 98), r. midi-Dys A exon 17-39 (SEQ ID NO: 100), s. midi-Dys A exon 17-41 (SEQ ID NO: 101), t. midi-Dys A exon 18-39 (SEQ ID NO: 220), u. midi-Dys A exon 18-41 (SEQ ID NO: 221), v. midi-Dys A exon 19-39 (SEQ ID NO: 103), w. midi-Dys A exon 19-41 (SEQ ID NO: 104), x. midi-Dys A exon 21-41 (SEQ ID NO: 106), and y. midi-Dys A exon 21-42 (SEQ ID NO: 223).

36. The system of any one ol ' claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

37. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), RI6 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

38. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

39. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

40. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ IDNO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

41. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R4 domain (SEQ ID NO:27), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

42. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial Rl l domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

43. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO:39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

44. The systems of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO:40), R18 Domain (SEQ ID NO: 41 ), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO:43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

45. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

46. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

47. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

48. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45),R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

49. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

50. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

51. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

52. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

53. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

54. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

55. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

56. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

57. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

58. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

59. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

60. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R 16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

61. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

62. The systems of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

63. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

64. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ IDNO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

65. The systems of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

66. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

67. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R16 Domain (SEQ ID NO: 416), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

68. The system of any one of claims 1-35, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

69. The system of any one of claims 1-68, wherein the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:83-107 and 216-223, or an amino acid at least about 90% identical thereto.

70. The system of any one of claims 1-69 wherein the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 86, or an amino acid at least about 90% identical thereto.

71. The system of any one of claims 1-69, wherein the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 95 or an amino acid at least about 90% identical thereto.

72. The system of any one of claims 1-69, wherein the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 101 or an amino acid at least about 90% identical thereto.

73. The system of any one of claims l-69wherein the amino acid sequence of the truncated dystrophin protein is not identical to an amino acid sequence of SEQ ID NO: 143.

74. The system of any one of claims 1-73, wherein the truncated dystrophin protein is not a polypeptide of 2361 amino acids.

75. The system of any one of claims 1-74, wherein the truncated dystrophin protein is less than 2361 amino acid in length.

76. The system of any one of claims 1-75, wherein the truncated dystrophin protein is greater than 2361 amino acid in length.

77. The system of any one of claims 1-76, wherein the truncated dystrophin protein is functional.

78. The system of any one of claims 1-77, wherein the first nucleic acid molecule is present in a first viral vector, and the second nucleic acid molecule is present in a second viral vector.

79. The system of claim 78, wherein the first viral vector and the second viral vector are each independently selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a vaccinia vector, a herpes simplex viral vector, and an Epstein- Barr viral vector.

80. The system of claim 78 or 79, wherein the first viral vector is an adeno-associated viral (AAV) vector, and the second viral vector is an AAV vector.

81. The system of claim 80, wherein the AAV vector is selected from the group consisting of an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVrh74, AAV-rhlO, AAV-DJ, AAV-LK03, AAV-MYO, AAV-MY02, AAV-MY03, MYO3A-AAV, MYO4A-AAV, and MYO4E-AAV.

82. The system of any one of claims 78-81, wherein the first AAV vector further comprises a first promoter operably linked to the first nucleic acid molecule.

83. The system of any one of claims 78-82, wherein the second AAV vector further comprises a second promoter operably linked to the second nucleic acid molecule.

84. The system of claim 82 or 83, wherein the promoter comprises a tissue specific promoter or a ubiquitous promoter.

85. The system of any one of claims 82-84, wherein the promoter comprises a CK8 promoter, an MHCK7 promoter, an SPC5-12 promoter, or a minimal CKM promoter.

86. The system of any one of claims 82-85, wherein the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 144-150, or a nucleotide sequence at least 95% identical thereto.

87. The system of any one of claims 83-86, wherein the first and / or second AAV vectors further comprise an inverted terminal repeat (ITR) sequence.

88. The system of claim 87, wherein the ITR sequence comprises a nucleotide sequence of SEQ ID NO: 202 and / or 203, or a nucleotide sequence at least 95% identical thereto.

89. The system of any one of claims 83-88, wherein the first and / or second AAV vectors further comprise an intron region.

90. The system of claim 89, wherein the intron region comprises a nucleotide sequence of SEQ ID NO: 156 or 157, or a nucleotide sequence at least 95% identical thereto.

91. The system of any one of claims 83-90, wherein the first and / or second AAV vectors further comprise a polyadenylation sequence.

92. The system of claim 91, wherein the polyadenylation sequence comprises a nucleotide sequence of SEQ ID NO: 151 or 152, or a nucleotide sequence at least 95% identical thereto.

93. The system of any one of claims 83-92, wherein the first and / or second AAV vectors further comprise a Woodchuck Hepatitis Vims Posttranscriptional Regulatory Element (WPRE).

94. The system of claim 93, wherein the WPRE comprises a nucleotide sequence of SEQ ID NOs: 153-155, or a nucleotide sequence at least 95% identical thereto.

95. The system of any one of claims 83-94, wherein the first and / or second AAV vectors further comprise a Kozak sequence.

96. A vector system for expressing a truncated human dystrophin protein, comprising a first AAV vector and a second AAV vector, wherein the first AAV vector comprises a first nucleic acid molecule comprising a first coding region encoding an N-terminal portion of the truncated dystrophin protein and a 3’ ribozyme, wherein the first coding region is operably linked to the 3’ ribozyme at its 3’ end, wherein the second AAV vector comprises a second nucleic acid molecule comprising a second coding region encoding a C-terminal portion of the truncated dystrophin protein and a 5’ ribozyme, wherein the second coding region is operably linked to the 5’ ribozyme at its 5’ end, wherein upon ribozyme-mediated catalytic ligation, the first coding region and the second coding region forms a third coding region encoding for the truncated human dystrophin protein, and wherein the truncated human dystrophin protein comprises at least 1640 amino acids.

97. The system of claim 96, wherein the first coding region is operably linked to two or more 3’ ribozymes at its 3’ end.

98. The system of claim 97, wherein the two or more 3’ ribozymes are the same 3’ ribozyme.

99. The system of claim 98, wherein the two or more 3’ ribozymes are different 3’ ribozymes.

100. The system of any one of claims 96-99, wherein the second coding region is operably linked to two or more 5’ ribozymes at its 5’ end.

101. The system of claim 100, wherein the two or more 5’ ribozymes are the same 5’ ribozyme.

102. The system of claim 100, wherein the two or more 5’ ribozymes are different 5’ ribozymes.

103. The system of any one of claims 96-102, wherein the 5’ ribozyme and the 3’ ribozyme are each independently selected from the group consisting of Twister (Osa), Twister (Dre), Twister (Nvi), Twister (Sbi), Twister (Envl), Twister (Spu), Twister (Cpa), Twister Sister, Hammerhead (RzB), HDV, Pistol, Varkud Satellite (VS), Hatchet, Hairpin, and Hovlinc (Hov).

104. The system of any one of claims 96-103, wherein the 5’ ribozyme and the 3’ ribozyme are each independently selected from the group consisting of SEQ ID NOs: 6 - 20.

105. The system of any one of claims 96-104, wherein the first nucleic acid molecule further comprises an intron splice donor sequence, and the second nucleic acid molecule further comprises an intron splice acceptor sequence.

106. The system of claim 105, wherein the splice donor sequence is positioned between the first coding region and the 3’ ribozyme.

107. The system of claim 105 or 106, wherein the splice donor sequence is selected from the group consisting of SEQ ID NOs: 133 - 136.

108. The system of any of claims 105-107, wherein the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R7 domain, the R8 domain, the R9 domain, the R10 domain, the R11 domain, the R12 domain, the R13 domain, the R14 domain, the R15 domain, the R16 domain, the R17 domain, the R18 domain, the R19 domain, the H3 domain, the R20 domain, the R21 domain, and the R22 domain.

109. The system of any of claims 105-108, wherein the splice donor sequence is positioned within a region of the truncated dystrophin protein coding for a region selected from the group consisting of the R8 domain, the R19 domain, the H3 domain, the R20 domain, and the R21 domain.

110. The system of any one of claims 105-109, wherein the splice donor sequence is not positioned within the R21 domain.

111. The system of any one of claims 105- 110, wherein the splice donor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 3’ ribozyme.

112. The system of any one of claims 105-111, wherein the splice acceptor sequence is positioned between the 5’ ribozyme and the second coding region.

113. The system of any one of claims 105-112, wherein the splice acceptor sequence is selected from the group consisting of SEQ ID Nos: 137-141.

114. The system of any one of claims 105-113, wherein the splice acceptor sequence is positioned at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or more from the 5’ ribozyme.

115. The system of any one of claims 105-114, wherein the splice donor sequence and the splice acceptor sequence are positioned such that the resulting spliced intron is between 50 - 200 bp in length.

116. The system of any one of claims 105-115, wherein the splice donor sequence and splice acceptor sequence are positioned such that the resulting spliced intron encodes a single predominant reading frame.

117. The system of any one of claims 105-116, wherein a stop codon sequence is introduced into the splice donor sequence or the splice acceptor sequence.

118. The system of any one of claims 96-117, wherein at least one of the first coding region and the second coding region is at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length. The third coding region is at least 4920 nucleotides in length.

119. The system of any one of claims 96-118, wherein the first coding region and the second coding region are each at least 2000 nucleotides in length, or at least 2200 nucleotides in length, or at least 2400 nucleotides in length, or at least 2600 nucleotides in length the third coding region is at least 5100 nucleotides in length.

120. The system of any one of claims 96-119, wherein the third coding region is at least 4920 nucleotides in length, or at least 5100 nucleotides in length, or at least 5300 nucleotides in length.

121. The system of any one of claims 96-120, wherein the third coding region comprises 300 or fewer CpG motifs.

122. The system of any one of claims 96-120, wherein the third coding region comprises 290 or fewer CpG motifs.

123. The system of any one of claims 96 - 120, wherein the third coding region comprises 67 or fewer CpG motifs.

124. The system of any one of claims 96-123, wherein the first coding region and the second coding region do not share a region of substantial sequence identity.

125. The system of any one of claims 96-124, wherein the 3’ end of the first coding region does not have a sequence identity to the 5’ end of the second coding region.

126. The system of any one of claims 96-125, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

127. The system of any one of claims 96-126, wherein the truncated dystrophin protein further comprises Hl Domain (SEQ ID NO: 22).

128. The system of claim 127, wherein the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of: a. midi-Dys AR1-R15 (SEQ ID NO: 83), b . midi-Dys AR2-R 15 (SEQ ID NO : 84) , c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), e. midi-Dys AR4-R15 (SEQ ID NO: 87), f. midi-Dys AR5-R15 (SEQ ID NO: 88), g. midi-Dys A exon 13-33 (SEQ ID NO: 93), h. midi-Dys A exon 13-39 (SEQ ID NO: 94), i. midi-Dys A exon 13-41 (SEQ ID NO: 95), j. midi-Dys A exon 13-48 (SEQ ID NO: 96), k. midi-Dys A exon 15-39 (SEQ ID NO: 97), l. midi-Dys A exon 15-41 (SEQ ID NO: 98), m. midi-Dys A exon 15-48 (SEQ ID NO: 99), n. midi-Dys A exon 17-39 (SEQ ID NO: 100),o. midi-Dys A exon 17-41 (SEQ ID NO: 101), p. midi-Dys A exon 17-48 (SEQ ID NO: 102), q. midi-Dys A exon 18-39 (SEQ ID NO: 220), r. midi-Dys A exon 18-41 (SEQ ID NO: 221), s. midi-Dys A exon 18-48 (SEQ ID NO: 222), t. midi-Dys A exon 19-39 (SEQ ID NO: 103), u. midi-Dys A exon 19-41 (SEQ ID NO: 104), v. midi-Dys A exon 19-48 (SEQ ID NO: 105), w. midi-Dys A exon 21-41 (SEQ ID NO: 106), x. midi-Dys A exon 21-42 (SEQ ID NO: 223), and y. midi-Dys A exon 21-48 (SEQ ID NO: 107).

129. The system of any one of claims 96- 128, wherein the truncated dystrophin protein further comprises R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), and R19 Domain (SEQ ID NO:42).

130. The system of claim 129, wherein the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of a. midi-Dys AR1-R15 (SEQ ID NO: 83), b . midi-Dys AR2-R 15 (SEQ ID NO : 84) , c. midi-Dys AR3-R15 (SEQ ID NO: 85), d. midi-Dys AH2-R15 (SEQ ID NO: 86), e. midi-Dys AR4-R15 (SEQ ID NO: 87), f. midi-Dys AR5-R15 (SEQ ID NO: 88) g. midi-Dys A exon 10-33 (SEQ ID NO: 89), h. midi-Dys A exon 10-39 (SEQ ID NO: 90), i. midi-Dys A exon 10-41 (SEQ ID NO: 91), j. midi-Dys A exon 11-33 (SEQ ID NO: 216), k. midi-Dys A exon 11-39 (SEQ ID NO: 217), l. midi-Dys A exon 11-41 (SEQ ID NO: 218), m. midi-Dys A exon 13-33 (SEQ ID NO: 93), n. midi-Dys A exon 13-39 (SEQ ID NO: 94), o. midi-Dys A exon 13-41 (SEQ ID NO: 95),p. midi-Dys A exon 15-39 (SEQ ID NO: 97), q. midi-Dys A exon 15-41 (SEQ ID NO: 98), r. midi-Dys A exon 17-39 (SEQ ID NO: 100), s. midi-Dys A exon 17-41 (SEQ ID NO: 101), t. midi-Dys A exon 18-39 (SEQ ID NO: 220), u. midi-Dys A exon 18-41 (SEQ ID NO: 221), v. midi-Dys A exon 19-39 (SEQ ID NO: 103), w. midi-Dys A exon 19-41 (SEQ ID NO: 104), x. midi-Dys A exon 21-41 (SEQ ID NO: 106), and y. midi-Dys A exon 21-42 (SEQ ID NO: 223).

131. The system of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

132. The system of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

133. The system of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51 ).

134. The system of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

135. The system of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

136. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO:24), R3 domain (SEQ ID NO:25), H2 domain (SEQ ID NO:26), R4 domain (SEQ ID NO:27), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

137. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial Rl l domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48),H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

138. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO:39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

139. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO:40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

140. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), a partial Hl domain (SEQ ID NO: 404), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

141. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47),R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

142. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

143. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

144. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), a partial R1 domain (SEQ ID NO: 405), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

145. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R11 domain (SEQ ID NO: 412), R12 Domain (SEQ ID NO: 35), R13 Domain (SEQ ID NO: 36), R14 Domain (SEQ ID NO: 37), R15Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

146. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

147. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

148. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), a partial R2 domain (SEQ ID NO: 406), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

149. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48),H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

150. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

151. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 407), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

152. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

153. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43),R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

154. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), a partial R3 domain (SEQ ID NO: 408), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

155. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

156. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 409), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

157. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO:26), a partial R4 domain (SEQ ID NO: 409), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

158. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R14 Domain (SEQ ID NO: 413), R15 Domain (SEQ ID NO: 38), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

159. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

160. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), a partial R4 domain (SEQ ID NO: 410), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

161. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ IDNO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R15 Domain (SEQ ID NO: 414), R16 Domain (SEQ ID NO: 39), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

162. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R16 Domain (SEQ ID NO: 416), R17 Domain (SEQ ID NO: 40), R18 Domain (SEQ ID NO: 41), R19 Domain (SEQ ID NO:42), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

163. The systems of any one of claims 96-130, wherein the truncated dystrophin protein comprises ABCD domain (SEQ ID NO: 21), Hl domain (SEQ ID NO: 22), R1 domain (SEQ ID NO: 23), R2 domain (SEQ ID NO: 24), R3 domain (SEQ ID NO: 25), H2 domain (SEQ ID NO: 26), R4 domain (SEQ ID NO:27), a partial R5 domain (SEQ ID NO: 411), a partial R19 Domain (SEQ ID NO: 415), H3 Domain (SEQ ID NO: 43), R20 Domain (SEQ ID NO: 44), R21 Domain (SEQ ID NO: 45), R22 Domain (SEQ ID NO: 46), R23 Domain (SEQ ID NO: 47), R24 Domain (SEQ ID NO: 48), H4 Domain (SEQ ID NO: 49), CR Domain (SEQ ID NO: 50), and CT Domain (SEQ ID NO: 51).

164. The system of any one of claims 96-163, wherein the truncated dystrophin protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:83-107, or an amino acid at least about 90% identical thereto.

165. The system of any one of claims 96-164, wherein the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 86, or an amino acid at least about 90% identical thereto.

166. The system of any one of claims 96-165, wherein the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 95 or an amino acid at least about 90% identical thereto.

167. The system of any one of claims 96-166, wherein the truncated dystrophin protein comprises an amino acid sequence of SEQ ID NO: 101 or an amino acid at least about 90% identical thereto.

168. The system of any one of claims 96-167, wherein the amino acid sequence of the truncated dystrophin protein is not identical to an amino acid sequence of SEQ ID NO: 143.

169. The system of any one of claims 96-168, wherein the truncated dystrophin protein is not a polypeptide of 2361 amino acids.

170. The system of any one of claims 96-169, wherein the truncated dystrophin protein is less than 2361 amino acid in length.

171. The system of any one of claims 96-170, wherein the truncated dystrophin protein is greater than 2361 amino acid in length.

172. The system of any one of claims 96-171, wherein the truncated dystrophin protein is functional.

173. The system of any one of claims 96-172, wherein the first coding region comprises the sequence selected from the group consisting of SEQ ID NOs: 284, 286, 288, 290, 291, 293, 295 and 297.

174. The system of any one of claims 96-173, wherein the second coding region comprises the sequence selected from the group consisting of SEQ ID NOs: 285, 287, 289, 292, 294 and 296.

175. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 286 and the second coding sequence comprises SEQ ID NO:287.

176. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 288 and the second coding sequence comprises SEQ ID NO:289.

177. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 290 and the second coding sequence comprises SEQ ID NO:289.

178. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 291 and the second coding sequence comprises SEQ ID NO:292.

179. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 293 and the second coding sequence comprises SEQ ID NO:294.

180. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 295 and the second coding sequence comprises SEQ ID NO:296.

181. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 297 and the second coding sequence comprises SEQ ID NO:292.

182. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 288 and the second coding sequence comprises SEQ ID NO:289.

183. The system of any one of claims 96-174, wherein the first coding sequence comprises SEQ ID NO: 288 and the second coding sequence comprises SEQ ID NO:287.

184. The system of any one of claims 96-183, wherein the AAV vector is selected from the group consisting of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAV-rhlO, AAV-DJ, AAV-LK03, AAV-MYO, AAV-MYO2, AAV-MYO3, MYO3A-AAV, MYO4A-AAV, and MYO4E-AAV.

185. The system of any one of claims 96-184, wherein the first AAV vector further comprises a first promoter operably linked to the first nucleic acid molecule.

186. The system of any one of claims 96-185, wherein the second AAV vector further comprises a second promoter operably linked to the second nucleic acid molecule.

187. The system of claim 185 or 186, wherein the promoter comprises a tissue specific promoter or a ubiquitous promoter.

188. The system of any one of claims 185-187, wherein the promoter comprises a CK8 promoter, an MHCK7 promoter, an SPC5 promoter, or a minimal CKM promoter.

189. The system of any one of claims 185-188, wherein the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 144-150, or a nucleotide sequence at least 95% identical thereto.

190. The system of any one of claims 96-189, wherein the first and / or second AAV vectors further comprise an inverted terminal repeat (ITR) sequence.

191. The system of claim 190, wherein the ITR sequence comprises a nucleotide sequence of SEQ ID NO: 202 and / or 203, or a nucleotide sequence at least 95% identical thereto.

192. The system of any one of claims 96-191, wherein the first and / or second AAV vectors further comprise an intron region.

193. The system of claim 192, wherein the intron region comprises a nucleotide sequence of SEQ ID NO: 156 or 157, or a nucleotide sequence at least 95% identical thereto.

194. The system of any one of claims 96-193, wherein the first and / or second AAV vectors further comprise a polyadenylation sequence.

195. The system of claim 194, wherein the polyadenylation sequence comprises a nucleotide sequence of SEQ ID NO: 151 or 152, or a nucleotide sequence at least 95% identical thereto.

196. The system of any one of claims 96-195, wherein the first and / or second AAV vectors further comprise a Woodchuck Hepatitis Vims Posttranscriptional Regulatory Element (WPRE).

197. The system of claim 196, wherein the WPRE comprises a nucleotide sequence of SEQ ID NO: 153 or 154, or 155, or a nucleotide sequence at least 95% identical thereto.

198. The system of any one of claims 96-197, wherein the first and / or second AAV vectors further comprise a Kozak sequence.

199. The system of any one of claims 96-198, wherein the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a first coding region encoding an N-terminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, and a 3’ ITR sequence.

200. The system of any one of claims 96-199, wherein the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a first coding region encoding an N-terminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, a polyadenylation sequence, and a 3’ ITR sequence.

201. The system of any one of claims 96-200, wherein the first AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, an intron region, a first coding region encoding an N-terminal portion of the truncated dystrophin protein, an intron splice donor sequence, a 3’ ribozyme, a polyadenylation sequence, and a 3’ ITR sequence.

202. The system of any one of claims 96-201, wherein the first AAV vector comprises a sequence selected from the group consisting of SEQ ID NO: 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 301, 303, 305, 307, 308, 310, 312, 314, 394, and 395.

203. The system of any one of claims 96-202, wherein the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, and a 3’ ITR sequence.

204. The system of any one of claims 96-203, wherein the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, a polyadenylation sequence, and a 3’ ITR sequence.

205. The system of any one of claims 96-204, wherein the second AAV vector comprises, in 5’ to 3’ order, a 5’ ITR sequence, a promoter sequence, a 5’ ribozyme, an intron splice acceptor sequence, a second coding region encoding a C-terminal portion of the truncated dystrophin protein, a WPRE sequence, a polyadenylation sequence, and a 3’ ITR sequence.

206. The system of any one of claims 96-205, wherein the second AAV vector comprises a sequence selected from the group consisting of SEQ ID NO:161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 302, 304, 306, 309, 311, and 313.

207. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 303, and the second AAV vector comprises the sequence of SEQ ID NO: 304.

208. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 305, and the second AAV vector comprises the sequence of SEQ ID NO: 306.

209. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 307, and the second AAV vector comprises the sequence of SEQ ID NO: 306.

210. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 308, and the second AAV vector comprises the sequence of SEQ ID NO: 309.

211. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 310, and the second AAV vector comprises the sequence of SEQ ID NO: 311.

212. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 312, and the second AAV vector comprises the sequence of SEQ ID NO: 313.

213. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 314, and the second AAV vector comprises the sequence of SEQ ID NO: 309.

214. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 394, and the second AAV vector comprises the sequence of SEQ ID NO: 309.

215. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 395, and the second AAV vector comprises the sequence of SEQ ID NO: 306.

216. The system of any one of claims 96-206, wherein the first AAV vector comprises the sequence of SEQ ID NO: 395, and the second AAV vector comprises the sequence of SEQ ID NO: 304.

217. An isolated truncated dystrophin protein that has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: 83, 85-87, 89-107, and 216-223.

218. An isolated nucleic acid encoding a truncated dystrophin protein that has at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 83, 85-87, 89-107, and 216-223.

219. The nucleic acid of claim 218, wherein the nucleic acid sequence is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 108, 110-112, 114-132, 224- 231, 260-280, and 396-403.

220. An isolated recombinant vector comprising the nucleic acid of claims 218-219.

221. An isolated recombinant viral genome comprising a nucleic acid molecule encoding a truncated human dystrophin protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 83, 85-87, 89-107, and 216-223.

222. The isolated recombinant viral genome of claim 221, wherein the isolate nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 108, 110-112, 114-132, 224-231, 260-280, and 396-403, or a nucleotide sequence at least 90% identical thereto.

223. A host cell comprising the first nucleic acid molecule and / or the second nucleic acid molecule of any one of claims 1-95, the first AAV vector and / or the second AAV vector of anyone of claims 96-216, the nucleic acid of claim 218 or claim 219, the vector of claim 220, or the recombinant viral genome of claim 221 or claim 222.

224. The host cell of claim 223, wherein the cell is a mammalian cell, an insect cell, or a bacterial cell.

225. A pharmaceutical composition comprising a first nucleic acid molecule and a second nucleic acid molecule of any one of claims 1-95, and a pharmaceutically acceptable excipient.

226. The pharmaceutical composition of claim 225, wherein the first nucleic acid molecule and the second nucleic acid molecule are presented at a ratio of 1:

1. 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:l, or 5:l.

227. A pharmaceutical composition comprising a first AAV vector and a second AAV vector of any one of claims 96-216, and a pharmaceutically acceptable excipient.

228. The pharmaceutical composition of claim 227, wherein the first AAV vector and the second AAV vector arc presented at a ratio of 1:

1. 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, or 5:1.

229. A pharmaceutical composition comprising the protein of claim 217, the nucleic acid of claims 218-219, the vector of claim 220, the viral genome of claim 221 or 222, or the host cell of claim 223 or 224.

230. A method for treating a dystrophin-associated disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the first nucleic acid molecule and the second nucleic acid molecule of any one of claims 1-95, the first AAV vector and the second AAV vector of any one of claims 96-216, the protein of claim 217, the nucleic acid of claims 218-219, the vector of claim 220, the viral genome of claim 221 or 222, or the host cell of claim 223 or 224, thereby treating the dystrophin-associated disorder in the subject.

231. A method for increasing expression of dystrophin in a subject having or diagnosed with having a dystrophin-associated disorder, comprising administering a therapeutically effective amount of the first nucleic acid molecule and the second nucleic acid molecule of any one of claims 1-95, the first AAV vector and the second AAV vector of any one of claims 96-216, the protein of claim 217, the nucleic acid of claims 218-219, the vector of claim 220, the viral genome of claim 221 or claim 222, or the host cell of claim 223 or 224, thereby increasing expression of dystrophin in the subject.

232. A method for increasing muscle mass or muscle strength and / or preventing fibrosis in a subject having or diagnosed with having a dystrophin-associated disorder, comprisingadministering a therapeutically effective amount of the first nucleic acid molecule and the second nucleic acid molecule of any one of claims 1-95, the first AAV vector and the second AAV vector of any one of claims 96-216, the protein of claim 217, the nucleic acid of claims 218-219, the vector of claim 220, the viral genome of claim 221 or claim 222, or the host cell of claim 223 or 224, thereby increasing muscle strength and / or preventing fibrosis in the subject.

233. The method of any one of claims 231-232, wherein the dystrophin-associated disorder is muscular dystrophy.

234. The method of any one of claims 231-233, wherein the dystrophin-associated disorder is Duchenne muscular dystrophy.

235. The method of any one of claims 231-234, wherein the pharmaceutical composition is administered by intramuscular injection, or intravenous injection.

236. The method of any one of claims 231 -235 wherein the first AAV vector and the second AAV vector arc administered together.

237. The method of any one of claims 231-236, wherein the first AAV vector and the second AAV vector are administered separately.

238. A method of making a first recombinant adeno-associated virus (rAAV) particle, the method comprising providing a host cell comprising the first nucleic acid molecule of any one of claims 1-95, and incubating the host cell under conditions suitable to encapsulate the first nucleic acid in an AAV capsid protein; thereby making the first rAAV particle.

239. A method of making a second recombinant adeno-associated virus (rAAV) particle, the method comprising providing a host cell comprising the second nucleic acid molecule of any one of claims 1-95, and incubating the host cell under conditions suitable to encapsulate the first nucleic acid in an AAV capsid protein; thereby making the second rAAV particle.

240. The method of claim 238 or 239, wherein the cell is a mammalian cell, an insect cell, or a bacterial cell.

241. The system of any one of claims 1-216 for use in the treatment of a dystrophin-associated disorder.

242. The first nucleic acid molecule and the second nucleic acid molecule of any one of claims 1-95 for use in the treatment of a dystrophin-associated disorder.

243. The first AAV vector and the second AAV vector of any one of claims 93-216 for use in the treatment of a dystrophin-associated disorder.

244. The pharmaceutical composition of any one of claims 242-243 for use the treatment of a dystrophin-associated disorder.

245. The isolated nucleic acid molecule of any one of claims 242-244 for use the treatment of a dystrophin-associated disorder.

Images