Compositions and methods for treating duchenne muscular dystrophy

Abstract

Provided herein are dual adeno-associated virus (AAV) particle compositions for delivering a mid-length dystrophin coding sequence over two AAV vectors resulting in reconstitution of mid-length dystrophin protein in a cell. This method results in the expression of a larger dystrophin protein that cannot be expressed using a traditional single AAV strategy. Such methods can be used for the treatment of Duchenne muscular dystrophy.

Description

COMPOSITIONS AND METHODS FOR TREATING DUCHENNE MUSCULAR DYSTROPHYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No. 63 / 716,012 filed on November 4, 2024, and U.S. Provisional Application No. 63 / 787,645 filed on April 11, 2025; the disclosure of each of which are incorporated by reference in their entireties.FIELD OF THE DISCLOSURE

[0002] The disclosure generally relates to dual adeno-associated virus (AAV) particle compositions for delivering a mid-length dystrophin coding sequence over two AAV vectors, methods of producing the AAV particles, cells producing the AAV particles, and methods of using the AAV particles for the treatment of dystrophinopathies, such as Duchenne muscular dystrophy.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (INMD_217_02WO_SequenceListing.xml; Size: Approximately 71,226 bytes; and Date of Creation: October 7, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0004] Duchenne muscular dystrophy (DMD) is a debilitating X-linked recessive disorder affecting 1 in 5,000 newborn males and is caused by mutations that lead to loss of the large cytoskeletal protein, dystrophin. Patients are generally wheelchair bound by the age of 12 years and die in their late 20s or early 30s due to respiratory-cardiac failure.

[0005] Systemic delivery of dystrophin to skeletal and cardiac muscle using recombinant adeno-associated virus (rAAV) is a promising strategy but has faced the challenge of the large size of dystrophin (~14 kilobases) with the limited packaging capacity of rAAV (~5 kilobases).

[0006] Thus, there is an urgent unmet need in the art to overcome the packaging constraints of dystrophin gene therapy for the treatment of DMD.SUMMARY

[0007] The present disclosure provides a composition comprising: (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a deoxyribonucleic acid (DNA) from 5’ to 3’: (i) a first 5’-ITR sequence; (ii) a first promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; (v) a splicing donor sequence; (vi) a first dimerization domain sequence; (vii) a first poly(A) signal sequence; and (viii) a first 3’-ITR sequence; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’: (i) a second 5’-ITR sequence; (ii) a second promoter sequence; (iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter; (iv) a splicing acceptor sequence; (v) a C-terminal coding sequence encoding a C-terminal portion of the mid-length dystrophin protein; (vi) a second poly(A) signal sequence; and (vii) a second 3 ’ -ITR sequence; wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form a ribonucleic acid (RNA) coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence that encodes the mid-length dystrophin protein; and wherein the mid-length dystrophin protein comprises: (i) an N-terminal region; (ii) a central rod domain comprising: one or more hinge regions and ten spectrin-like repeats; (iii) a cysteine-rich domain; and (iv) a C-terminal domain.

[0008] In some embodiments, the present disclosure provides a composition comprising: (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a DNA from 5’ to 3’: (i) a first 5 ’-ITR sequence; (ii) a first promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; (v) a splicing donor sequence; (vi) a first dimerization domain sequence; (vii) a first poly(A) signal sequence; and (viii) a first 3 ’-ITR sequence; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5 ’ to 3 ’: (i) a second 5 ’ -ITR sequence; (ii) a second promoter sequence; (iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter; (iv) a splicing acceptor sequence; (v) a C-terminal coding sequence encoding a C-terminal portionof the mid-length dystrophin protein; (vi) a second poly(A) signal sequence; and (vii) a second 3’-ITR sequence; wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form an RNA coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence that encodes the mid-length dystrophin protein; and wherein the mid-length dystrophin protein comprises: (i) an N-terminal region; (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrinlike repeat 17, a hinge region 3, a spectrin-like repeat 20, a spectrin-like repeat 21, a spectrinlike repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24, and a hinge region 4; (iii) a cysteine-rich domain; and (iv) a C-terminal domain.

[0009] In some embodiments, the present disclosure provides a composition comprising: (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a DNA from 5’ to 3’: (i) a first 5’-ITR sequence; (ii) a first promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; (v) a splicing donor sequence; (vi) a first dimerization domain sequence; (vii) a first poly(A) signal sequence; and (viii) a first 3’-ITR sequence; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5 ’ to 3 ’: (i) a second 5 ’ -ITR sequence; (ii) a second promoter sequence; (iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter; (iv) a splicing acceptor sequence; (v) a C-terminal coding sequence encoding a C-terminal portion of the mid-length dystrophin protein; (vi) a second poly(A) signal sequence; and (vii) a second 3 ’-ITR sequence; wherein the N-terminal coding sequence encoding the N-terminal portion of the mid-length dystrophin protein comprises: (i) an N-terminal region; and (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, and a spectrin-like repeat 21; and wherein the C-terminal coding sequence encoding the C-terminal portion of the mid-length dystrophin protein comprises: (i) a central rod domain comprising: a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrinlike repeat 24; and a hinge region 4; (ii) a cysteine rich domain; and (iii) a C-terminal domain.

[0010] In some embodiments, the N-terminal coding sequence encoding the N-terminal portion of the mid-length dystrophin protein comprises: (i) an N-terminal region; and (ii) acentral rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, and a spectrin-like repeat 21. In some embodiments, the N-terminal coding sequence is codon-optimized. In some embodiments, the N-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the N-terminal coding sequence comprises a nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the N-terminal coding sequence consists of the nucleic acid sequence of SEQ ID NO: 9.

[0011] In some embodiments, the C-terminal coding sequence encoding the C-terminal portion of the mid-length dystrophin protein comprises: (i) a central rod domain comprising: a spectrinlike repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24; and a hinge region 4; (ii) a cysteine rich domain; and (iii) a C-terminal domain. In some embodiments, the C-terminal coding sequence is codon-optimized. In some embodiments, the C-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, the C-terminal coding sequence comprises a nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the C-terminal coding sequence consists of the nucleic acid sequence of SEQ ID NO: 21.

[0012] In some embodiments, a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form an RNA coding sequence encoding the mid-length dystrophin protein, the RNA coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence.

[0013] In some embodiments, the mid-length dystrophin protein comprises: (i) an N-terminal region; (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, a spectrin-like repeat 21, a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24, and a hinge region 4; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the spectrin-like repeat 16 and / or the spectrin-like repeat 17 bind to neuronal nitric oxide synthase.

[0014] In some embodiments, the RNA coding sequence encoding the mid-length dystrophin protein is codon-optimized. In some embodiments, the RNA coding sequence encoding themid-length dystrophin protein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the RNA coding sequence encoding the mid-length dystrophin protein comprises a nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the RNA coding sequence encoding the mid-length dystrophin protein consists of the nucleic acid sequence of SEQ ID NO: 22.

[0015] In some embodiments, the mid-length dystrophin protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23. In some embodiments, the mid-length dystrophin protein comprises an amino acid sequence of SEQ ID NO: 23. In some embodiments, the mid-length dystrophin protein consists of the amino acid sequence of SEQ ID NO: 23.

[0016] In some embodiments, the N-terminal coding sequence comprises an intervening intron sequence. In some embodiments, the intervening intron sequence is a modified mouse betaactin (Actb) intron 2 sequence. In some embodiments, the intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7.

[0017] In some embodiments, the C-terminal coding sequence comprises a first intervening intron sequence and a second intervening intron sequence. In some embodiments, the first intervening intron sequence is a modified mouse beta-actin intron 2 sequence and the second intervening intron sequence is a modified mouse Gapdh intron 5 sequence. In some embodiments, the first intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the second intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19.

[0018] In some embodiments, the splicing donor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.

[0019] In some embodiments, the splicing acceptor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

[0020] In some embodiments, the first dimerization domain and the second dimerization domain encode RNA sequences that are complementary to each other. In some embodiments, the first dimerization domain and the second dimerization domain encode RNA sequences with complementary RNA stem loops. In some embodiments, the complementary RNA stem loops form a central kissing loop interaction with four or more loop base pairs for intermolecular pairing. In some embodiments, the positioning of the complementary RNA stem loops is offset by at least 1 nt, at least 2 nt, at least 3 nt, at least 4 nt, at least 5 nt, at least 6 nt, at least 7 nt, at least 8 nt, at least 9 nt, or at least 10 nt so that the respective stem regions of the complementary RNA stem loops base pair in trans through strand invasion. In some embodiments, the stem regions of the complementary RNA stem loops contain about 1% to about 30% mismatches, but match to the other dimerization domain. In some embodiments, the RNA sequences encoding the first dimerization domain and the second dimerization domain form an extended duplex through strand invasion. In some embodiments, the first dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, the first dimerization domain comprises a nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the first dimerization domain consists of the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the second dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, the second dimerization domain comprises a nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the second dimerization domain consists of the nucleic acid sequence of SEQ ID NO: 15.

[0021] In some embodiments, the first promoter is a muscle tissue-specific promoter. In some embodiments, the muscle tissue-specific promoter is a muscle- and heart-specific enhancer 7 (MHCK7) promoter. In some embodiments, the MHCK7 promoter comprises a nucleic acid sequence of SEQ ID NO: 2.

[0022] In some embodiments, the second promoter is a muscle tissue-specific promoter. In some embodiments, the muscle tissue-specific promoter is a MHCK7 promoter. In some embodiments, the MHCK7 promoter comprises a nucleic acid sequence of SEQ ID NO: 2.

[0023] In some embodiments, the first 5’-ITR is a 5’ AAV2 ITR. In some embodiments, the first 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the first 3 ’-ITR is a 3’ AAV2 ITR. In some embodiments, the first 3 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 14.

[0024] In some embodiments, the second 5’-ITR is a 5’ AAV2 ITR. In some embodiments, the second 5 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the second 3’-ITR is a 3’ AAV2 ITR. In some embodiments, the second 3’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 14.

[0025] In some embodiments, the first poly(A) signal sequence comprises a late SV40 poly(A) signal sequence. In some embodiments, the first poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 13.

[0026] In some embodiments, the second poly(A) signal sequence comprises a late SV40 poly(A) signal sequence. In some embodiments, the second poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 13.

[0027] In some embodiments, the Kozak sequence comprises a nucleic acid sequence of any one of SEQ ID NOs: 3 and 24-26. In some embodiments, the Kozak sequence comprises a nucleic acid sequence of SEQ ID NO: 3.

[0028] In some embodiments, the first transgene comprises at least one nucleic acid element to suppress expression of a first unjoined transcript. In some embodiments, the at least one nucleic acid element is the absence of a stop codon in the N-terminal coding sequence. In someembodiments, the at least one nucleic acid element is a micro-RNA target site 3’ to the splicing donor. In some embodiments, the at least one nucleic acid element is two micro-RNA target sites 3’ to the splicing donor. In some embodiments, the at least one micro-RNA target site is a micro-RNA- 16 target site. In some embodiments, the micro-RNA- 16 target site comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to a sequence of SEQ ID NO: 12.

[0029] In some embodiments, the second transgene comprises at least one nucleic acid element to suppress expression of a second unjoined transcript. In some embodiments, the at least one nucleic acid element is an out-of-frame start codon 5’ of the splicing acceptor. In some embodiments, the at least one nucleic acid element is a modified mouse Gapdh intron 5 sequence in the C-terminal coding sequence.

[0030] In some embodiments, the present disclosure provides a composition comprising: (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’: (i) a 5’-ITR sequence of SEQ ID NO: 1; (ii) a promoter sequence of SEQ ID NO: 2; (iii) a Kozak sequence of SEQ ID NO: 3; (iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter; (v) a splicing donor sequence of SEQ ID NO: 10; (vi) a dimerization domain sequence of SEQ ID NO: 11; (vii) a poly(A) signal sequence of SEQ ID NO: 13; and (viii) a 3’-ITR sequence of SEQ ID NO: 14; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’: (i) a 5’-ITR sequence of SEQ ID NO: 1; (ii) a promoter sequence of SEQ ID NO: 2; (iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter; (iv) a splicing acceptor sequence of SEQ ID NO: 16; (v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C-terminal portion of mid-length dystrophin protein; (vi) a poly(A) signal sequence of SEQ ID NO: 13; and (vii) a 3’-ITR sequence of SEQ ID NO: 14.

[0031] In some embodiments, the first AAV particle and / or the second AAV particle comprises an AAV capsid encapsidating the first transgene. In some embodiments, the AAV capsid of the first AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, AAVMyo, MyoAAV, or AAV13 capsid proteins. In some embodiments, the first orsecond AAV particle is an AAV9 particle and the AAV capsid of the first AAV particle comprises one or more AAV9 capsid proteins. In some embodiments, the one or more AAV9 capsid proteins of the first and / or second AAV particle comprise AAV9 capsid protein VP1. In some embodiments, the AAV9 capsid protein VP1 comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the AAV9 capsid protein VP1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to the amino acid sequence of SEQ ID NO:33. In some embodiments, the one or more AAV9 capsid proteins of the first and / or second AAV particle comprise AAV9 capsid protein VP2. In some embodiments, the AAV9 capsid protein VP2 comprises amino acids 138-736 of SEQ ID NO:33. In some embodiments, the AAV9 capsid protein VP2 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to amino acids 138-736 of SEQ ID NO:33. In some embodiments, the one or more AAV9 capsid proteins of the first and / or second AAV particle comprise AAV9 capsid protein VP3. In some embodiments, the AAV9 capsid protein VP3 comprises amino acids 203-736 of SEQ ID NO:33. In some embodiments, the AAV9 capsid protein VP3 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to amino acids 203-736 of SEQ ID NO:33. In some embodiments, the AAV capsid of the first AAV particle comprises one or more engineered variants of an AAV9 capsid protein. In some embodiments, the one or more engineered variants of the AAV9 capsid protein of the first and / or second AAV particle comprises an engineered variant of AAV9 capsid protein VP1. In some embodiments, the one or more engineered variants of the AAV9 capsid protein of the first AAV particle comprises an engineered variant of AAV9 capsid protein VP2. In some embodiments, the one or more engineered variants of the AAV9 capsid protein of the first and / or second AAV particle comprises an engineered variant of AAV9 capsid protein VP3. In some embodiments, the one or more engineered variants of the AAV9 capsid protein of the first AAV particle are one or more deimmunized variants of the AAV9 capsid protein.

[0032] In some embodiments, the first AAV particle comprises an AAV capsid encapsidating the first transgene; and the second AAV particle comprises an AAV capsid encapsidating the second transgene. In some embodiments, the AAV capsid of the first AAV particle comprises one or more AAV9 capsid proteins; and wherein the AAV capsid of the second AAV particlecomprises one or more AAV9 capsid proteins. In some embodiments, the one or more AAV9 capsid proteins of the first AAV particle comprise AAV9 capsid protein VP1, AAV9 capsid protein VP2, and / or AAV9 capsid protein VP3; and wherein the one or more AAV9 capsid proteins of the second AAV particle comprise AAV9 capsid protein VP 1, AAV9 capsid protein VP2, and / or AAV9 capsid protein VP3.

[0033] In some embodiments, the present disclosure provides a composition comprising: (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’: (i) a 5’-ITR sequence of SEQ ID NO: 1; (ii) a promoter sequence of SEQ ID NO: 2; (iii) a Kozak sequence of SEQ ID NO: 3; (iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter; (v) a splicing donor sequence of SEQ ID NO: 10; (vi) a dimerization domain sequence of SEQ ID NO: 11; (vii) a poly(A) signal sequence of SEQ ID NO: 13; and (viii) a 3’-ITR sequence of SEQ ID NO: 14; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’: (i) a 5’-ITR sequence of SEQ ID NO: 1; (ii) a promoter sequence of SEQ ID NO: 2; (iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter; (iv) a splicing acceptor sequence of SEQ ID NO: 16; (v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C-terminal portion of mid-length dystrophin protein; (vi) a poly(A) signal sequence of SEQ ID NO: 13; and (vii) a 3’-ITR sequence of SEQ ID NO: 14; wherein the first AAV particle comprises AAV9 capsid proteins; and wherein the second AAV particle comprises AAV9 capsid proteins.

[0034] In some embodiments, the present disclosure provides a composition comprising: (a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises the nucleic acid sequence of Table 1; and (b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises the nucleic acid sequence of Table 2. In a further embodiment, the AAV capsid of the first AAV particle comprises AAV9 capsid proteins. In a further embodiment, the AAV capsid of the second AAV particle comprises AAV9 capsid proteins. In even a further embodiment, the composition comprises the first AAV particle and the second AAV particle combined at a 1:1 ratio.

[0035] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of any of the compositions described herein and a pharmaceutically acceptable carrier, excipient, diluent, or buffer.

[0036] In some embodiments, the composition comprises the same number of vector genomes of the first AAV particle and the second AAV particle. In some embodiments, the composition comprises the first AAV particle and the second AAV particle combined at a 1: 1 ratio. In some embodiments, the 1: 1 ratio is a 1: 1 ratio of encapsidated transgenes. In some embodiments, the 1:1 ratio is a 1:1 ratio of first AAV particles to second AAV particles. In some embodiments, the effective amount of the first AAV particle and the second AAV particle is at a 1:1 ratio.

[0037] In some embodiments, the present disclosure provides a method of treating a dystrophinopathy in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition described herein.

[0038] In some embodiments, the subject is a male subject from about 6 months to about 7 years old. In some embodiments, the subject is a male subject from about 1 year to about 7 years old. In some embodiments, the subject is a male subject from about 2 years to about 7 years old. In some embodiments, the subject is a male subject from about 2 years to about 5 years old.

[0039] In some embodiments, the dystrophinopathy is Duchenne muscular dystrophy (DMD). In some embodiments, the dystrophinopathy is Becker muscular dystrophy. In some embodiments, the dystrophinopathy is DMD-associated dilated cardiomyopathy (DCM).

[0040] In some embodiments, the subject is placed in the Trendelenburg position during the treating. In some embodiments, the composition is administered intrathecally to the subject. In some embodiments, the composition is administered intravenously to the subject.

[0041] In some embodiments, the composition increases mid-length dystrophin expression in skeletal and / or cardiac muscle compared to the pre-treatment level. In some embodiments, the treating comprises increasing the mid-length dystrophin protein level in the subject to at least 200%, about 195%, about 190%, about 185%, about 180%, about 175%, about 170%, about 165%, about 160%, about 155%, about 150%, about 145%, about 140%, about 135%, about 130% about 125%, about 120%, about 115%, about 110%, about 105%, about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30% about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, or about 1% of the pre-treatment level.

[0042] In some embodiments, the effective amount of the administered AAV particles reduces the severity of one or more dystrophinopathy symptoms in the subject. In some embodiments, the one or more dystrophinopathy symptoms comprise muscle weakness, muscle stiffness, muscle pain, reduced motor skills, difficulty swallowing, difficulty breathing, or irregular heartbeat.

[0043] In some embodiments, the effective amount of the AAV particles in the composition is from about l><109to about l><1016vector genomes (vg) per first and second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1 x 109to about 1 x 1015total vg of the first AAV vector and from about 1 x 109to about 1 x 1015total vg of the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1×1012to about 1×1016vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1×1012to about 1×1015vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1×1012to about 1×1014vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1×1012to about 1×1013vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1.0×1013to about 1×1016vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1.0×1013to about 1×1015vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is from about 1.0×1013to about 1×1014vg of each the first AAV vector and the second AAV vector.

[0044] In some embodiments, the effective amount of the AAV particles in the composition is about 1×1013vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is about 5×1013vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is about 1×1014vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is about 5×1014vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is about 1×1015vg of each the first AAV vector and the second AAV vector. Insome embodiments, the effective amount of the AAV particles in the composition is about 5* 1014vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is about l><1015vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is about 5×1015vg of each the first AAV vector and the second AAV vector. In some embodiments, the effective amount of the AAV particles in the composition is about 1 x 1016vg of each the first AAV vector and the second AAV vector.

[0045] In some embodiments, the effective amount of the AAV particles is administered as a single dose. In some embodiments, the effective amount of the AAV particles is administered as two doses. In some embodiments, the effective amount of the AAV particles is administered as divided doses. In some embodiments, the effective amount of the AAV particles is administered in multiple doses.

[0046] In some embodiments, treating comprises increasing the subject’s NorthStar Ambulatory Assessment (NSAA) score subsequent to treatment, compared to a baseline NSAA score of the subject. In some embodiments, increasing the score comprises increasing the score from about 5 points to about 25 points, about 5 points to about 20 points, about 5 points to about 15 points, or about 5 points to about 10 points. In some embodiments, the baseline NSAA score of the subject is measured prior to the subject undergoing the treatment method. In some embodiments, the NSAA score subsequent to treatment is measured about 6 months, about 12 months, about 18 months, or about 24 months after administration of the composition.

[0047] In some embodiments, treating comprises increasing the number of meters walked by the subj ect in the six-minute walk test (6MWT) subsequent to treatment, compared to a baseline number of meters walked by the subject in the 6MWT. In some embodiments, the baseline number of meters walked by the subject in the 6MWT is measured prior to the subject undergoing the treatment method. In some embodiments, the 6MWT subsequent to treatment is assessed about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, or about 60 months after administration of the composition. In some embodiments, increasing the number of meters walked by the subject in the 6MWT comprises increasing by about 5 meters to about 50 meters, about 5 meters to about 45 meters, about 5 meters to about 40 meters, about 5 meters to about 35 meters, about 5 meters to about 30 meters, about 5 meters to about 25 meters, about 5 meters to about 20 meters, about 5 to about 15 meters, or about 5 meters to about 10 meters.

[0048] In some embodiments, treating comprises decreasing the time to complete the 4-stair climb test (4SC) for the subject subsequent to treatment, compared to a baseline time of the subject to complete the 4SC. In some embodiments, the baseline time for the subject to complete the 4SC is measured prior to the administration of the dual vector composition. In some embodiments, the 4SC subsequent to treatment is measured about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, or about 60 months after administration of the composition. In some embodiments, treating comprises decreasing the time to complete the 4SC for the subject by about 1 second to about 8 seconds, by about 1 second to about 7 seconds, by about 1 second to about 6 seconds, by about 1 second to about 5 seconds, by about 1 second to about 4 seconds, by about 1 second to about 3 seconds, or by about 1 second to about 2 seconds, as compared to the time for the subject to complete the 4SC for the subject prior to administration of the composition.

[0049] In some embodiments, treating comprises decreasing a time to stand (TTSTAND) for the subject, compared to a baseline TTSAND measured for the subject. In some embodiments, the baseline TTSTAND for the subject is measured prior to administration of the composition. In some embodiments, the TTSTAND subsequent to treatment is measured about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, or about 60 months after administration of the composition. In some embodiments, treating comprises decreasing the TTSTAND for the subject by about 1 second to about 10 seconds, by 1 to about 9 seconds, by about 1 second to about 8 seconds, by about 1 second to about 7 seconds, by about 1 second to about 6 seconds, by about 1 second to about 5 seconds, by about 1 second to about 4 seconds, by about 1 second to about 3 seconds, or by about 1 second to about 2 seconds as compared to the TTSTAND for the subject prior to administration of the composition.

[0050] In some embodiments, treating comprises increasing a Gross Motor Outcome (GRO) score of the subject subsequent to treatment, compared to a baseline GRO score measured for the subject prior to administration of the composition.

[0051] In some embodiments, the present disclosure provides a method for delivering a middystrophin transgene to skeletal and / or cardiac muscle of a subject, comprising: intrathecally or intravenously administering to a subject an effective amount of the pharmaceutical composition described herein.

[0052] In some embodiments, the present disclosure provides a DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’: (i) a 5’-ITR sequence; (ii) a promoter sequence; (iii) a Kozak sequence; (iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter; (v) a splicing donor sequence; (vi) a dimerization domain sequence; (vii) a poly(A) signal sequence; and (viii) a 3’-ITR sequence; wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the splicing donor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, the promoter is muscle tissue-specific promoter. In some embodiments, the muscle tissuespecific promoter is MHCK7 promoter. In some embodiments, the MHCK7 promoter comprises the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the 5’-ITR is a 5’ AAV2 ITR. In some embodiments, the 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the 3’-ITRis a 3’ AAV2 ITR. In some embodiments, the 3 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, the Kozak sequence comprises a nucleic acid sequence of SEQ ID NO: 3.

[0053] In some embodiments, the present disclosure provides a DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’: (i) a 5 ’-ITR sequence of SEQ ID NO: 1; (ii) a promoter sequence of SEQ ID NO: 2; (iii) a Kozak sequence of SEQ ID NO: 3; (iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter; (v) a splicing donor sequence of SEQID NO: 10; (vi) a dimerization domain sequence of SEQ ID NO: 11; (vii) a poly(A) signal sequence of SEQ ID NO: 13; and (viii) a 3’-ITR sequence of SEQ ID NO: 14.

[0054] In some embodiments, the present disclosure provides a DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’: (i) a 5’-ITR sequence; (ii) a promoter sequence; (iii) a dimerization domain sequence, said dimerization domain being operably linked to and under control of said promoter; (iv) a splicing acceptor sequence; (v) a C-terminal coding sequence encoding a C-terminal portion of a mid-length dystrophin protein; (vi) a poly(A) signal sequence; and (vii) a 3’-ITR sequence; wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, the splicing acceptor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16. In some embodiments, the dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, the promoter is a muscle tissuespecific promoter. In some embodiments, the muscle tissue-specific promoter is a MHCK7 promoter. In some embodiments, the MHCK7 promoter comprises the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the 5’-ITR is a 5’ AAV2 ITR. In some embodiments, the 5 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the 3’-ITR is a 3’ AAV2 ITR. In some embodiments, the 3’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.

[0055] In some embodiments, the present disclosure provides a DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’: (i) a 5’-ITR sequence of SEQ ID NO: 1; (ii) a promoter sequence of SEQ ID NO: 2; (iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter; (iv) a splicing acceptor sequence of SEQ ID NO: 16; (v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C-terminal portion of a mid-lengthdystrophin protein; (vi) a poly(A) signal sequence of SEQ ID NO: 13; and (vii) a 3’-ITR sequence of SEQ ID NO: 14.

[0056] In some embodiments, the present disclosure provides a packaging cell comprising one or more DNA plasmids described herein. In some embodiments, the DNA plasmid comprising an N-terminal transgene construct and the DNA plasmid comprising a C-terminal transgene construct. In some embodiments, further comprising a helper plasmid containing adenoviral components necessary for recombinant adeno-associated virus production. In some embodiments, further comprising a plasmid comprising AAV replication (rep) and capsid (cap) genes. In some embodiments, the packaging cell is a HEK293 cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The accompanying figures, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and / or features. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[0058] FIG. 1A depicts an exemplary RNA-end joining (REJ) process. A transgene encoding mid-length dystrophin is split into an N-terminal transgene comprising an REJ donor and C-terminal transgene comprising an REJ acceptor (1), packaged into two separate AAV particles (2), co-infected as a 1: 1 formulation into target cells (3), and the split RNA is transcribed from the two AAVs within the cell (4). Structured RNA dimerization domains mediate non-covalent binding (5) and REJ domains facilitate recruitment of the spliceosome (6) resulting in REJ-end joining and generation of mid-length dystrophin mRNA (7). Protein expression from the unjoined fragments is suppressed (8). Desired mid-length dystrophin protein is translated (9).

[0059] FIG. IB depicts exemplary elements designed to inhibit expression of the unjoined N-and C-terminal RNA fragments. The suppressive nucleic acid element may be the absence of a stop codon and inclusion of a degron in the intronic sequence (1); a micro-RNA target site in the splicing donor or acceptor sequence (2); a self-cleaving RNA sequence in the splicing domain (3); and / or an out-of-frame start codon in the splicing acceptor domain (4).

[0060] FIG. 2 is a schematic drawing providing a comparison of mid-length dystrophin (middystrophin) to full-length dystrophin.

[0061] FIG. 3A and FIG. 3B are schematic drawings providing an exemplary dual AAV particle composition. The first AAV particle comprises a first transgene comprising an N-terminal mid-length dystrophin coding sequence (FIG. 3A). The second AAV particle comprises a second transgene comprising a C-terminal mid-length dystrophin coding sequence (FIG. 3B).

[0062] FIG. 4A, FIG. 4B, and FIG. 4C are schematic drawings providing RNA primer probe design for detecting an N-terminal mid-length dystrophin RNA sequence (FIG. 4A), a C-terminal mid-length dystrophin RNA sequence (FIG. 4B), and a joined mid-length dystrophin RNA sequence (FIG. 4C). FIGs. 4A and 4B show the location of half the probe on the exon 5’ of the MsActb-i2-mod intron (rectangle, +) and half the probe on the exon 3’ of the MsActb-i2-mod intron (rectangle, *). FIG. 4D shows transcription of the N-terminal mid-length dystrophin RNA sequence (VL1199), the C-terminal mid-length dystrophin RNA sequence (VL1173), and the joined mid-length dystrophin RNA sequence using various primer-probe mixes (PPMs) by quantitative RT-PCR.

[0063] FIG. 5A shows transcription of the N-terminal mid-length dystrophin RNA sequence (VL1199), the C-terminal mid-length dystrophin RNA sequence (VL1173), and the joined mid-length dystrophin RNA sequence in mouse heart tissue following administration with the dual AAV9 particle composition (encapsidating the VL1199 and VL1173 transgenes, see tables 1 and 2) or an AAV9 control. RT, reverse transcriptase.

[0064] FIG. 5B shows transcription of the N-terminal mid-length dystrophin RNA sequence (VL1199), the C-terminal mid-length dystrophin RNA sequence (VL1173), and the joined mid-length dystrophin RNA sequence in mouse gastrocnemius (GAS), tibialis anterior (TA), and heart tissue following administration with the dual AAV9 particle composition (encapsidating the VL1199 and VL1173 transgenes).

[0065] FIG. 5C shows expression level of the N-terminal mid-length dystrophin RNA sequence (VL1199), the C-terminal mid-length dystrophin RNA sequence (VL1173), and the joined mid-length dystrophin RNA sequence in various mouse tissues. LSC, lumbar spinal cord; DIA, diaphragm; Quad, quadriceps; GAS, gastrocnemius; and TA, tibialis anterior.

[0066] FIG. 6A shows a pseudoblot of mid-length dystrophin protein expression in neuronal progenitor cells following infection with the INSMid composition — comprising two distinct AAV9 particles, one encapsidating the VL1199 transgene and the other encapsidating the VL1173 transgene, formulated at about a 1:1 ratio — administered at either 2.5×105MOI or 5.0×105MOI per transgene, compared to an uninfected control.

[0067] FIG. 6B shows a spectropherogram of mid-length dystrophin protein expression in neuronal progenitor cells following infection with the INSMid composition at either 2.5* 105MOI or 5.0* 105MOI per transgene (VL1199 and VL1173) compared to an uninfected control.

[0068] FIG. 6C shows chemiluminescence of peak height in FIG. 6B measuring mid-length dystrophin protein expression in neuronal progenitor cells following infection with the INSMid composition at either 2.5><105MOI or 5.0><105MOI per transgene (VL1199 and VL1173) compared to an uninfected control. ECM, extracellular matrix.

[0069] FIG. 7A shows tibialis anterior muscle sections obtained from mdx mice 21 days following systemic injection on postnatal day 1 (pl) with the INSMid composition (1.2xlOntotal vg, i.e., 6><1O10vg of each vector, calculated from a 6.0* 1013vg / kg per vector dose) and immunofluorescently stained for dystrophin or laminin.

[0070] FIG. 7B shows gastrocnemius muscle sections obtained from mdx mice 21 days following systemic injection on pl with the INSMid composition (1.2×1011total vg, i.e., 6×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose) and immunofluorescently stained for dystrophin or laminin.

[0071] FIG. 7C shows heart tissue sections obtained from mdx mice 21 days following systemic injection on pl with the INSMid composition (1.2×1011total vg, i.e., 6×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose) and immunofluorescently stained for dystrophin or laminin.

[0072] FIG. 7D shows tibialis anterior and cardiac muscle sections obtained from mdx mice at 21 days following systemic injection on pl with the INSMid composition (1.2×1011total vg, i.e., 6×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), or vehicle control and immunofluorescently stained for dystrophin.

[0073] FIG. 8A shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at 115 days following systemic injection with INSMid D3 (6.0×1010total vg, i.e., 3×1010vg of each vector, calculated from a 3.0×1013vg / kg per vector dose), INSMid D2 (1.2×1011total vg, i.e., 6×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), or INSMid D1 (2.4×1011total vg, i.e., 1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose), or vehicle control at pl. Wild-type (WT) mice are shown as a control.

[0074] FIG. 8B shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at 275 days following systemic injection with INSMid D3 (6.0×1010totalvg, i.e., 3*1010vg of each vector, calculated from a 3.0*1013vg / kg per vector dose), INSMid D2 (1.2* 1011total vg, i.e., 6><1O10vg of each vector, calculated from a 6.0* 1013vg / kg per vector dose), or INSMid DI (2.4* 1011total vg, i.e., 1.2xlOnvg of each vector, calculated from a 1.2* 1014vg / kg per vector dose), or vehicle control at pl. Wild-type (WT) mice are shown as a control.

[0075] FIG. 9A shows hindlimb muscle force (torque) normalized to body weight of mdx mice at about pl 15 (top panel) and p275 (bottom panel) following systemic injection with INSMid D3 (6.0×1010total vg, i.e., 3* IO10vg of each vector, calculated from a 3.0><1013vg / kg per vector dose) or vehicle control at pl. Wild-type (WT) mice are shown as a control.

[0076] FIG. 9B shows hindlimb muscle force (torque) normalized to body weight of mdx mice at about pl 15 (top panel) and p275 (bottom panel) following systemic injection with INSMid D2 (1.2×1011total vg, i.e., 6×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose) or vehicle control at pl. Wild-type (WT) mice are shown as a control.

[0077] FIG. 9C shows hindlimb muscle force (torque) normalized to body weight of mdx mice at about pl 15 (top panel) and p275 (bottom panel) following systemic injection with INSMid D1 (2.4×1011total vg, i.e., 1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose) or vehicle control at pl. Wild-type (WT) mice are shown as a control.

[0078] FIG. 9D summarizes hindlimb muscle force (torque) normalized to body weight of mdx mice at about pl 15 days from FIGS. 9A-9C (top panels) following systemic injection at pl with INSMid D3 (6.0×1010total vg, i.e., 3xl010vg of each vector, calculated from a 3.0xl013vg / kg per vector dose), INSMid D2 (1.2×1011total vg, i.e., 6×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), or INSMid DI (2.4xlOutotal vg, i.e., 1.2x 1011vg of each vector, calculated from a 1.2xl014vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0079] FIG. 9E summarizes hindlimb muscle force (torque) normalized to body weight of mdx mice at about p275 from FIGS. 9A-9C (bottom panels) following systemic injection at pl with INSMid D3 (6.0×1010total vg, i.e., 3xl010vg of each vector, calculated from a 3.0xl013vg / kg per vector dose), INSMid D2 (1.2×1011total vg, i.e., 6×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), or INSMid D1 (2.4×1011total vg, i.e., 1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose) or vehicle control. Wild-type (WT) mice are shown as a control.

[0080] FIG. 10A shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at about pl 15 following intracerebroventricular (ICV) injection at p28 with INSMid D3 (l. OxlO11total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5xl012vg / kg per vector dose), INSMid D2 (4.0×1011total vg, i.e., 2.0×1011vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0xl013vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0081] FIG. 10B shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at about p275 following ICV injection at p28 with INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose), INSMid D2 (4.0xl0ntotal vg, i.e., 2.0xl0uvg of each vector, calculated from a l. OxlO13vg / kg per vector dose), or INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0082] FIG. 10C shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at about p400 following ICV injection at p28 with INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose), INSMid D2 (4.0xl0ntotal vg, i.e., 2.0xl0uvg of each vector, calculated from a l. OxlO13vg / kg per vector dose), or INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0083] FIG. 11A shows hindlimb muscle force (plantar tetanic torque) normalized to body weight of mdx mice at about pl 15 following ICV injection at p28 INSMid D3 (l. OxlO11total vg, i.e., 5. Ox 1010vg of each vector, calculated from a 2.5x 1012vg / kg per vector dose), INSMid D2 (4.0×1011total vg, i.e., 2.0×1011vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0xl013vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0084] FIG. 1 IB shows hindlimb muscle force (plantar tetanic torque) normalized to body weight of mdx mice at about p275 following ICV injection at p28 with INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose), INSMid D2 (4.0xl0ntotal vg, i.e., 2.0xl0uvg of each vector, calculated from a l. OxlO13vg / kg per vector dose), or INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0085] FIG. 11C shows hindlimb muscle force (plantar tetanic torque) normalized to body weight of mdx mice at about p400 following ICV injection at p28 with INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0086] FIG. 12A shows hindlimb muscle force (torque) normalized to body weight of mdx mice at about pl 15 following an ICV injection at p28 with INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0087] FIG. 12B shows hindlimb muscle force (torque) normalized to body weight of mdx mice at about p275 following an ICV injection at p28 with INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0088] FIG. 12C shows hindlimb muscle force (torque) normalized to body weight of mdx mice at about p400 following an ICV injection at p28 with INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose), or vehicle control. Wild-type (WT) mice are shown as a control.

[0089] FIG. 13 shows gastrocnemius muscle sections obtained from mdx mice at 87 days (~pl 15d) following intracerebroventricular (ICV) injection at p28 with INSMid DI (1.6×1012total vg, i.e., 8.0* 1011vg of each vector, calculated from a 4.0* 1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose), or vehicle control, and immunofluorescently stained for laminin (top row) or dystrophin (bottom row). Wild-type (WT) mice are shown as a control. Scale bar = 100 pm.

[0090] FIG. 14A shows DNA levels (vector genomes / diploid genome) in gastrocnemius tissue samples from mdx mice (~pl 15) administered, at p28 via an ICV injection, INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose). DNA levels were assayed using probes specific to each transgene (VL1199 and VL1173) as well as a probe common to both transgenes.

[0091] FIG. 14B shows mRNA levels (copies / pg of total RNA) in gastrocnemius tissue samples from mdx mice (~pl 15) administered, at p28 via an ICV injection, INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose). mRNA levels were assayed using probes specific to each transgene (VL1199 and VL1173) as well as a probe for the joined transgenes.

[0092] FIG. 14C shows DNA levels (vector genomes / diploid genome) in cardiac tissue samples from mdx mice (~pl 15) that were administered, at p28 via an ICV injection, INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose). DNA levels were assayed using probes specific to each transgene as well as a probe common to both transgenes.

[0093] FIG. 14D shows mRNA levels (copies / pg of total RNA) in cardiac tissue samples from mdx mice (~pl 15) that were administered, at p28 via an ICV injection, INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose). mRNA levels were assayed using probes specific to each transgene (VL1199 and VL1173) as well as a probe for the joined transgenes.

[0094] FIG. 14E shows DNA levels (vector genomes / diploid genome) in TA (tibialis anterior) tissue samples from mdx mice (~pl 15) that were administered, at p28 via an ICV injection, INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose). DNA levels were assayed using probes specific to each transgene (VL1199 and VL1173) as well as a probe common to both transgenes.

[0095] FIG. 14F shows mRNA levels (copies / pg of total RNA) in TA tissue samples from mdx mice (~pl 15) that were administered, at p28 via an ICV injection, INSMid Dl (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0×1011total vg, i.e., 2.0×1011vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose). mRNA levels were assayed using probes specific to each transgene (VL1199 and VL1173) as well as a probe for the joined transgenes.

[0096] FIG. 14G shows DNA levels (vector genomes / diploid genome) in diaphragm tissue samples from mdx mice (~pl 15) that were administered, at p28 via an ICV injection, INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4. Ox 1011total vg, i.e., 2. Ox 1011vg of each vector, calculated from a l. OxlO13vg / kg per vector dose), or INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose). DNA levels were assayed using probes specific to each transgene (VL1199 and VL1173) as well as a probe common to both transgenes.

[0097] FIG. 14H shows mRNA levels (copies / pg of total RNA) in diaphragm tissue samples from mdx mice (~pl 15) that were administered, at p28 via an ICV injection, INSMid DI(1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0* 1011total vg, i.e., 2.0* 1011vg of each vector, calculated from a 1.0* 1013vg / kg per vector dose), or INSMid D3 (1.0* 1011total vg, i.e., 5.0* 1010vg of each vector, calculated from a 2.5* 1012vg / kg per vector dose). mRNA levels were assayed using probes specific to each transgene (VL1199 and VL1173) as well as a probe for the joined transgenes.

[0098] FIG. 15A shows membrane dystrophin expression levels in the gastrocnemius muscle of mice (~pl 15). Membrane dystrophin expression levels in the gastrocnemius muscle of mdx mice were assayed at ~pl 15 following administration of the INSMid composition at p28 via an ICV injection of INSMid DI (1.6* 1012total vg, i.e., 8.0* 1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0×1011total vg, i.e., 2.0×1011vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose). Membrane dystrophin levels were assayed in WT mice and vehicle-administered mdx mice at the age of ~pl 15. AU = arbitrary fluorescence unit.

[0099] FIG. 15B shows the percentage of muscle fibers of the gastrocnemius that were positive for dystrophin expression. Dystrophin levels were assayed in the muscle fibers of the gastrocnemius of WT mice and vehicle-administered mdx mice at the age of ~pl 15. Dystrophin levels were assayed in muscle fibers of the gastrocnemius of mdx mice at ~pl 15 following administration of the INSMid composition at p28 via an ICV injection of INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0×1011total vg, i.e., 2.0×1011vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose).

[0100] FIG. 16A shows membrane dystrophin expression levels in the tibialis anterior muscle of mice (~pl 15). Membrane dystrophin expression levels in the tibialis anterior muscle of mdx mice were assayed at ~pl 15 following administration of the INSMid composition at p28 via an ICV injection of INSMid DI (1.6x 1012total vg, i.e., 8. Ox 1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0×1011total vg, i.e., 2.0×1011vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose). Membrane dystrophin levels were assayed in WT mice and vehicle-administered mdx mice at the age of ~pl 15. AU = arbitrary fluorescence unit; TA = tibialis anterior.

[0101] FIG. 16B shows the percentage of muscle fibers of the tibialis anterior that were positive for dystrophin or mid-dystrophin expression. Dystrophin levels were assayed in the muscle fibers of the tibialis anterior of WT mice and vehicle-administered mdx mice at the age of ~pl 15. Mid-dystrophin levels were assayed in muscle fibers of the tibialis anterior of mdx mice at ~pl 15 following administration of INSMid DI (1.6×1012total vg, i.e., 8.0X1011vg of each vector, calculated from a 4. Ox 1013vg / kg per vector dose), INSMid D2 (4. Ox 1011total vg, i.e., 2.0xl0nvg of each vector, calculated from a l. OxlO13vg / kg per vector dose), or INSMid D3 (l. OxlO11total vg, i.e., 5. OxlO10vg of each vector, calculated from a 2.5xl012vg / kg per vector dose). TA = tibialis anterior.

[0102] FIG. 17 shows a tibialis anterior section obtained from mdx mice at approximately 90 days following ICV injection at p28 with INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose). Laminin (left panel) and middystrophin (right panel) were immunofluorescently stained.

[0103] FIG. 18A shows membrane dystrophin and mid-dystrophin expression levels in the diaphragm muscle of mice (~pll5). Membrane mid-dystrophin expression levels in the diaphragm muscle of mdx mice were assayed at ~pl 15 following administration of the INSMid composition at p28 via an ICV injection of INSMid DI (1.6xl012total vg, i.e., 8.0xl0nvg of each vector, calculated from a 4. Ox 1013vg / kg per vector dose), INSMid D2 (4. Ox 1011total vg, i.e., 2.0xl0nvg of each vector, calculated from a l. OxlO13vg / kg per vector dose), or INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose). Membrane dystrophin levels were assayed in WT mice and vehicle-administered mdx mice at the age of ~pl 15. AU = arbitrary fluorescence unit.

[0104] FIG. 18B shows the percentage of muscle fibers of the diaphragm that were positive for dystrophin or mid-dystrophin expression. Dystrophin levels were assayed in the muscle fibers of the diaphragm of WT mice and vehicle-administered mdx mice at the age of ~pl 15. Dystrophin levels were assayed in muscle fibers of the diaphragm of mdx mice at ~pl 15 following administration of the INSMid composition at p28 via an ICV injection of INSMid D1 (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4. Ox 1011total vg, i.e., 2. Ox 1011vg of each vector, calculated from a l. OxlO13vg / kg per vector dose), or INSMid D3 (l. OxlO11total vg, i.e., 5.0xl010vg of each vector, calculated from a 2.5x 1012vg / kg per vector dose).

[0105] FIG. 19 shows a diaphragm section obtained from mdx mice at approximately 90 days following ICV injection at p28 with INSMid DI (1.6×1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose). Laminin (left panel) and middystrophin (right panel) were immunofluorescently stained.

[0106] FIG. 20 shows cardiac muscle sections obtained from mdx mice at approximately 90 days following intracerebroventricular (ICV) injection at p28 with INSMid DI (1.6*1012total vg, i.e., 8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose), INSMid D2 (4.0×1011total vg, i.e., 2.0×1011vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or INSMid D3 (1.0×1011total vg, i.e., 5.0×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose), or vehicle control, and immunofluorescently stained for laminin (top row) or dystrophin (or mid-length dystrophin) (bottom row) demonstrating a robust dose-response of mid-length dystrophin expression in cardiac muscle. Wild-type (WT) mice are shown as a control. Scale bar = 100 pm.

[0107] FIG. 21 A shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at about pl 15 following ICV injection at pl with 6.0×1010total vg (3.0×1010vg of each vector, calculated from a 3.0×1013vg / kg per vector dose), 1.2×1011total vg (6.0×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), 2.4×1011total vg (1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose), or 6.2×1011total vg (3.1×1011vg of each vector, calculated from a 3.1×1014vg / kg per vector dose) of the INSMid composition, or vehicle control. Wild-type (WT) mice are shown as a control.

[0108] FIG. 2 IB shows hindlimb muscle force (plantar tetanic torque) normalized to body weight of mdx mice at about pl 15 following ICV injection at pl with 6.0×1010total vg (3.0×1010vg of each vector, calculated from a 3.0×1013vg / kg per vector dose), 1.2×1011total vg (6.0×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), 2.4×1011total vg (1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose), or 6.2×1011total vg (3.1×1011vg of each vector, calculated from a 3.1×1014vg / kg per vector dose) of the INSMid composition, or vehicle control. Wild-type (WT) mice are shown as a control.

[0109] FIG. 22 shows hindlimb muscle force (torque) normalized to body weight of mdx mice at about pl 15 following an ICV injection at pl with 6.0×1010total vg (3.0×1010vg of each vector, calculated from a 3.0×1013vg / kg per vector dose), 1.2×1011total vg (6.0×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), 2.4×1011total vg (1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose), or 6.2×1011total vg (3.1×1011vg of each vector, calculated from a 3.1*1014vg / kg per vector dose) of the INSMid composition, or vehicle control. Wild-type (WT) mice are shown as a control.

[0110] FIG. 23A shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at about pl 15 following ICV injection at pl with 6.0×1010total vg (3.0×1010vg of each vector, calculated from a 3.0×1013vg / kg per vector dose), 1.2×1011total vg (6.0×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), 2.4×1011total vg (1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose), or 6.2×1011total vg (3.1×1011vg of each vector, calculated from a 3.1×1014vg / kg per vector dose) of the INSMid composition, or vehicle control. Wild-type (WT) mice are shown as a control.[OHl] FIG. 23B shows hindlimb muscle force (plantar twitch torque) normalized to body weight of mdx mice at about pl 15 following ICV injection at p28 with 1.0×1011total vg (3.1×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose), 4.0×1011total vg (5.0×1010vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or 1.6×1012total vg (8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose) of the INSMid composition, or vehicle control. Wild-type (WT) mice are shown as a control.

[0112] FIG. 24A shows hindlimb muscle force (plantar tetanic torque) normalized to body weight of mdx mice at about pl 15 following ICV injection at pl with 6.0×1010total vg (3.0×1010vg of each vector, calculated from a 3.0×1013vg / kg per vector dose), 1.2×1011total vg (6.0×1010vg of each vector, calculated from a 6.0×1013vg / kg per vector dose), 2.4×1011total vg (1.2×1011vg of each vector, calculated from a 1.2×1014vg / kg per vector dose), or 6.2×1011total vg (3.1×1011of each vector, calculated from a 3.1×1014vg / kg per vector dose) of the INSMid composition, or vehicle control. Wild-type (WT) mice are shown as a control.

[0113] FIG. 24B shows hindlimb muscle force (plantar tetanic torque) normalized to body weight of mdx mice at about pl 15 following ICV injection at p28 with 1.0×1011total vg (3.1×1010vg of each vector, calculated from a 2.5×1012vg / kg per vector dose), 4.0×1011total vg (5.0×1010vg of each vector, calculated from a 1.0×1013vg / kg per vector dose), or 1.6×1012total vg (8.0×1011vg of each vector, calculated from a 4.0×1013vg / kg per vector dose) of the INSMid composition, or vehicle control. Wild-type (WT) mice are shown as a control.DETAILED DESCRIPTION

[0114] The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed disclosures, or that any publication specifically orimplicitly referenced is prior art. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter as described.

[0115] Embodiments of the present disclosure relate to dual AAV particle compositions and methods for delivering said compositions to cardiac and / or skeletal muscle to a subject in need of treatment of a dystrophinopathy, such as Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, or DMD-associated dilated cardiomyopathy (DCM).

[0116] Some embodiments, as provided herein, relate to compositions that express a target protein (e.g., mid-length dystrophin) from two or more transgenes which are each encapsidated by a viral or non-viral vector. The two or more vectors can be any viral or non-viral vectors including, but not limited to, adenoviral vectors, retroviral vectors and adeno-associated viral (AAV) vectors. Each transgene drives expression of a split pre-mRNA molecule comprising a portion of the coding sequence of the target gene (e.g., N- or C-terminal fragment of the full coding sequence). The two or more split pre-mRNA molecules (e.g., N-terminal split pre-mRNA and C-terminal split pre-mRNA) are spliced through RNA-end joining (REJ) domains comprising RNA dimerization domains and splicing domains (also referred to as a splicing donor or a splicing acceptor). In some embodiments, the transgenes comprise elements that are designed to inhibit fragment expression and function to block translation of truncated protein from either single vector. Descriptions of REJ technology can be found, for example, in international patent application publication Nos. WO 2020 / 205604 and WO 2021 / 096605, the contents of which are incorporated by reference in their entirety.

[0117] In some embodiments, provided herein are dual AAV particle compositions that express a target protein from two transgenes that are encapsidated by separate AAV vectors (also referred to as “dual AAV-REJ” or “dual AAV vectors”). In some embodiments, provided herein is a dual AAV particle composition that expresses mid-length dystrophin protein from two transgenes that are encapsidated by two AAV vectors. Each transgene drives expression of a split pre-mRNA molecule comprising a portion of the mid-length dystrophin coding sequence (e.g., N- or C-terminal fragment of the mid-length dystrophin coding sequence). The dual AAV vectors, in a preferred embodiment, are dual AAV9 vectors.

[0118] The REJ technology involves in part: (1) splitting the mid-length dystrophin coding sequence into an N-terminal fragment and a C-terminal fragment and constructing two different transgenes, each of which comprises the N- or C-terminal fragment of the coding sequence; (2) packaging each transgene individually into two independent AAV particles; (3) coinfectingtwo independent AAV particles into the same target cell; (4) forming split pre-mRNAs from transcription of the two different transgenes; (5) non-covalent binding between the two RNA dimerization domains; (6) recruiting spliceosome by the splicing domains; (7) spliceosome mediated RNA-end joining; (8) suppressing protein expression from unjoined RNA fragments; and (9) translating a full-length protein. See e.g., FIG. 1 for an overview of the process.

[0119] The present disclosure provides distinct and improved sequences for dimerization domains that promote strong / ra / rs-interactions with a complementary RNA strand, while reducing the likelihood / minimizing cis-binding interactions. These uniquely structured dimerization domains are combined with novel and improved elements including, among others, a splicing donor and acceptor, to enhance RNA splicing processes. By improving the trans-dimerization of the RNA strands in the context of the appropriate elements that mediate efficient splicing, it is demonstrated herein that two different RNA fragments of a coding sequence can be joined precisely and efficiently in the same cell producing physiological or even supraphysiological levels of functional proteins in vivo with no or minimal risk of accumulating unjoined RNAs that encode non-functional and / or deleterious proteins, or truncated protein from either single AAV.

[0120] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0121] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the present application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative methods and materials are herein described.

[0122] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate. Thus, for example, reference to “a carrier” includes mixtures of one or more carriers, two or more carriers, and the like and reference to “the method” includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0123] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims areapproximations that can vary depending upon the desired properties sought to be obtained by the present application. Generally, the term “about”, as used herein in references to a measurable value, such as an amount of weight, time, dose, etc. is meant to encompass values within an acceptable degree of variability in the art. In some embodiments, degree of variability is based on FDA guidelines.

[0124] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). As used herein, “exemplary” means “serving as an example, instance, or illustration,” and shall not be construed as preferred or advantageous over other embodiments disclosed herein.

[0125] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.

[0126] As used herein, the term “wild type” (abbreviated “WT”) refers to a typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms. For example, a wild type protein is the typical form of that protein as it occurs in nature.

[0127] The term “nucleic acid,” “nucleotide,” “polynucleotide” or “oligonucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991). Nucleic Acid Res. 19:5081;Ohtsuka et al. (1985). J. Biol. Chem. 260:2605-2608; and Rossolini et al. (1994). Mol. Cell. Probes 8:91-98).

[0128] In some embodiments, the term “gene” refers to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). In some embodiments, the term “gene” refers to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, guide RNA (gRNA), short-interfering RNA (siRNA), or micro-RNA (miRNA).

[0129] As used herein, the term “transgene” refers to an exogenous gene, for example supplied by a vector, such as AAV. In one embodiment, a transgene comprises a coding region that encodes a portion of a target protein, such as about a third, half, or two-thirds of a target protein, e.g., dystrophin or mid-length dystrophin. Transgenes include regions preceding and following the coding region, regulatory elements, as well as elements necessary for RNA-end joining (e.g., a splicing domain and a dimerization domain). The transgene is flanked on each end by an inverted terminal repeat (ITR), and each ITR is a component of the transgene. The transgene according to embodiments described herein, comprises a fragment of the mid-length dystrophin coding sequence (e.g., N-terminal or C-terminal mid-length dystrophin coding sequence). In the embodiments described herein, a transgene includes, a 5’ ITR; a promoter; an N-terminal mid-length dystrophin coding sequence; a splicing donor; a dimerization domain; a poly(A) signal; and a 3’ ITR. In another embodiment, a transgene includes, a 5’ ITR; a promoter; a dimerization domain; a splicing acceptor; a C-terminal mid-length dystrophin coding sequence; a poly(A) signal; and a 3’ ITR.

[0130] As used herein, a “coding region” is a portion of nucleic acid that consists of codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region.

[0131] As used herein, the term “vector genome” or “vg” refers to a transgene that is encapsidated by a viral capsid, e.g., an AAV capsid. A “vg” dose refers to the number of vgs administered to a subject. A “vg dose” may refer to the dose of a single vector or the dose of multiple vectors (e.g., a first vector and a second vector).

[0132] As used herein, the term “INSMid” or “INSMid composition” refers to the dual AAV serotype 9 (AAV9) composition formulated for gene therapy applications targeting dystrophinopathies, such as Duchenne muscular dystrophy. The INSMid composition comprises two distinct AAV9 particles, each encapsidating a separate recombinant transgene: VL1199 and VL1173 transgenes respectively. When co-administered, these two AAV9 particles enable intracellular RNA recombination through trans-splicing and dimerization interactions, resulting in the expression of a functional mid-length dystrophin protein that restores structural integrity to muscle fibers. The INSMid composition is typically formulated at an approximately 1: 1 ratio of VL 1199 to VL 1173.

[0133] As used herein, the term “endogenous” with reference to a nucleic acid, for example, a gene, or a protein in a cell, is a nucleic acid or protein that occurs in that particular cell as it is found in nature, for example, at its natural genomic location or locus. Moreover, a cell “endogenously expressing” a nucleic acid or protein expresses that nucleic acid or protein as it is found in nature.

[0134] A “promoter” refers to one or more nucleic acid control sequence(s) that direct transcription of a nucleic acid, e.g., a fragment of the mid-length dystrophin coding sequence and REI elements (e.g., a splicing domain and a dimerization domain), and is present within a transgene or when the transgene is encapsidated, a vector genome. As used herein, a promoter includes nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0135] A “regulatory element”, as used herein, refers to a nucleic acid sequence capable of regulating transcription of a gene (e.g., a fragment of the mid-length dystrophin coding sequence and REI elements), and / or regulate the stability or translation of a transcribed mRNA product, and can be present within a vector genome. In some embodiments, regulatory elements can regulate tissue-specific transcription of a gene, for example, skeletal muscle or cardiac tissue. Regulatory elements can comprise at least one transcription factor binding site. Regulatory elements, as used herein, increase or enhance promoter-driven gene expression when compared to the transcription of the gene from the promoter alone in the absence of the regulatory element. The regulatory elements may occur at any distance (i.e., proximal or distal) to the coding sequence. In some embodiments, regulatory elements comprise part of a larger sequence involved in transcriptional control, e.g., part of a promoter sequence. However,regulatory elements alone are typically not sufficient to initiate transcription on its own and require the presence of a promoter.

[0136] A first nucleic acid is “operably linked” to a second nucleic acid when the first nucleic acid is placed into a functional relationship with the second nucleic acid. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation of the coding sequence. A first nucleic acid that is “operably linked” to a second nucleic acid need not be directly linked. In other words, there may be intervening sequences between two operably linked nucleic acids.

[0137] “Polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, and functional fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.

[0138] A nucleic acid sequence that is “flanked” by two nucleic acid elements indicates that one element is located 5’ to the sequence and the other element is located 3’ to the sequence. The term “flanked” is not intended to indicate that the respective sequences are necessarily contiguous. For example, there may be intervening sequences between one nucleotide sequence, e.g., a coding sequence, and an ITR.

[0139] As used herein, a first nucleic acid sequence is “upstream” of a second nucleic acid sequence, if the first nucleic acid sequence is located 5’ of the second nucleic acid. As used herein, a first nucleic acid sequence is “downstream” of a second nucleic acid sequence, if the first nucleic acid sequence is located 3’ of the second nucleic acid.

[0140] As used herein, the term “complementary” refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are, generally, A and T (or A and U), and G and C. The polynucleotides and nucleic acids described herein can comprise sequences that are perfectly complementary or substantially complementary (e.g., having a small fraction of mismatched bases) to a genomic sequence. A single stranded nucleic acid for the purposes described herein, also refers to the complementary nucleic acid and a double stranded nucleic acid comprises both strands.

[0141] The term “binding” refers to an association between two substances or molecules, such as the hybridization of one nucleic acid molecule to another (or itself), such as between two dimerization domains. An oligonucleotide molecule binds or stably binds to another nucleicacid molecule if there are a sufficient number of complementary base pairs between the oligonucleotide molecule and the target nucleic acid to permit detection of that binding. In some examples, binding between nucleic acid molecules may occur directly. In some examples, binding between nucleic acid molecules may occur indirectly, e.g., through an intermediate molecule. Either direct binding or indirect binding may occur by standard base pairing, by non-canonical base pair interactions, by non-base pair interactions, or a combination thereof. Non-canonical base pair interactions may occur by any means of stabilization known to those of skill in the art, including but not limited to Hoogsteen base pairs and wobble base pairs. Nonbase pair interactions can include binding through an intermediate molecule. In some examples, direct binding is between kissing loop dimerization domains.

[0142] The term “C-terminal portion” refers to a region of a protein sequence that includes a contiguous stretch of amino acids that begins at or near the C-terminal residue of the protein. A C-terminal portion of the protein can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues).

[0143] The term “N-terminal portion” refers to a region of a protein sequence that includes a contiguous stretch of amino acids that begins at the N-terminal residue of the protein. An N-terminal portion of the protein can be defined by a contiguous stretch of amino acids (e.g., a number of amino acid residues).

[0144] The term “kissing loop / kissing stem loop” refers to an RNA structure that forms when bases between two hairpin loops form pair interactions. These intermolecular “kissing interactions” occur when the unpaired nucleotides in one hairpin loop, base pair with the unpaired nucleotides in another hairpin loop to form a stable interaction complex.

[0145] Hybridization of a nucleic acid occurs when two nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acids used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993 ). The Tm is the temperature at which 50% of a given strand of nucleic acid is hybridized to its complementary strand.

[0146] As used herein, the term “strand invasion” refers to the displacement of one strand of a first double stranded nucleic acid molecule by a single stranded portion of a second nucleic acid molecule, wherein the single strand has nucleotide sequence that is substantially identical to the displaced strand and can selectively hybridize to the strand complementary to the displaced strand

[0147] The term “substantial identity” or “substantially identical,” as used in the context of polynucleotide or polypeptide sequences, refers to a sequence that has at least 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0148] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0149] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215, pp. 403-410 and Altschul et al. (1977). Nucleic Acids Res. 25, pp.3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977). Nucleic Acids Res. 25, pp.3389-3402). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff (1989). Proc. Natl. Acad. Sci. USA 89, p. 10915).

[0150] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993). Proc. Nat’l. Acad. Sci. USA 90, pp. 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20.

[0151] As used herein, an “adeno-associated virus (AAV) particle”, refers to an AAV virion comprising an AAV capsid encapsidating a transgene or an AAV capsid encapsidating a vector genome. The vector genome comprises a transgene comprising a promoter, a coding sequence, and a REJ domain that are flanked by a 5’ and 3’ AAV ITR sequences. An AAV capsid is typically composed of 60 capsid (cap) protein subunits, VP1, VP2, and VP3, that are arranged in an icosahedral symmetry in a ratio of approximately 1:1:10 to 1:1:20, depending upon the selected AAV capsid proteins. For example, in embodiments described herein, the AAV capsid shell is serotype 9 (AAV9). In a further embodiment, the capsid encapsidates a single-stranded vector genome.

[0152] “Engineered variant” as used herein, means a protein that differs in primary amino acid sequence from a reference (e.g., wild type) protein by one or more amino acids, wherein the one or more amino acid differences (e.g., one or more amino acid substitutions) impart an improved characteristic on the engineered protein, compared to the reference protein. Theimproved characteristic in some embodiments, is improved stability, production yield, improved tissue tropism, or improved immunogenicity profile (i.e., a deimmunized variant).

[0153] As used herein, the terms “introducing” or “delivering” in the context of nucleic acids, for example, AAV vectors, refers to the translocation of the nucleic acid from outside a cell to inside the cell. In some cases, introducing refers to translocation of the nucleic acid from outside the cell to inside the nucleus of the cell. Various methods of such translocation are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome-mediated translocation, and the like.

[0154] As used herein, the terms “packaged” or “encapsidated” refers to the inclusion of a transgene in a capsid comprising viral capsid proteins to form an AAV particle encapsidating a vector genome comprising the transgene. The capsid, in one embodiment of the disclosure, is an AAV9 capsid.

[0155] As used herein, the terms “AAV vector,” “AAV particle,” “recombinant AAV particle,” “AAV,” and “rAAV” are used interchangeably and refer to the AAV capsid with a transgene or vector genome comprising ITRs as produced by a packaging cell.

[0156] The terms “empty capsid,” “empty viral particle,” and “empty AAV” refer to an AAV capsid shell lacking a vector genome packaged within.

[0157] The terms “ITR” or “inverted terminal repeat” refer to the stretch of nucleic acid sequences that exist in AAV and / or rAAV that can form a T-shaped palindromic structure, which is required for completing AAV lytic and latent life cycles. The term “non-resolvable ITR” refers to a modified ITR such that the resolution by the Rep protein is reduced. An ITR can be rendered non-resolvable when the terminal resolution site (trs) and / or D sequence in the ITR is deleted or mutated.

[0158] The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52: 456, Sambrook et al. (1989). Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986). Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981). Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0159] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms include, but are not limited to, mammals, such as humans, simians, murines, equines, bovines, porcines, canines, felines, and the like. In some embodiments, the subject or patient is human. In some embodiments, the subject or patient is a male subject or a male patient.

[0160] As used herein, the term “effective amount” or “effective dose”, refers to the amount of a substance (e.g., an AAV particle of the present disclosure) sufficient to effect beneficial or desired results (e.g., expression of a protein, or a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administration(s), application(s) or dosage(s) and is not intended to be limited to a particular formulation or administration route. Where a dose is provided in “vector genomes”, an “effective dose” may be referred to herein as an “effective vector genome dose”.

[0161] As used herein, the term “treating” includes any effect, e.g., preventing, lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, improving the clinical phenotype of the condition, and the like, or ameliorating a symptom of the condition. “Treating” can thus include one or more of reducing the onset of a dystrophinopathy, reducing the progression of a dystrophinopathy, preventing a dystrophinopathy, reducing the severity of dystrophinopathy symptoms, retarding dystrophinopathy symptom progression, or delaying progression of a dystrophinopathy. In some embodiments, the dystrophinopathy is DMD.Transgene Architecture

[0162] The present disclosure provides dual AAV particle compositions comprising two different AAV particles, each comprising an encapsidated transgene. The transgene of each AAV particle comprises nucleic acid elements required for transcript expression and subsequent mRNA trans-splicing. The transgene encapsidated by an AAV particle can be referred to as a “vector genome”. In the majority of instances discussed herein, nucleic acid elements are discussed as being part of a transgene. However, one of ordinary skill in the art will appreciate that vector genomes of the disclosure will also include such elements, once the transgene is encapsidated by an AAV capsid.

[0163] In some embodiments, the N-terminal transgene comprises one or more of the following nucleic acid sequences: (i) a 5’ ITR; (ii) a promoter; (iii) a Kozak sequence; (iv) an N-terminal mid-length dystrophin coding sequence; (v) a splicing donor (vi) dimerization domain; (vii) apoly(A) signal; and (viii) a 3’ ITR. In some embodiments, the N-terminal transgene comprises one or more of the following nucleic acid sequences from 5’ to 3’: (i) a 5’ ITR; (ii) a promoter; (iii) a Kozak sequence; (iv) an N-terminal mid-length dystrophin coding sequence; (v) a splicing donor (vi) dimerization domain; (vii) a poly(A) signal; and (viii) a 3’ ITR. See, e.g., FIG. 3A.

[0164] In some embodiments, the C-terminal transgene comprises one or more of the following nucleic acid sequences: (i) a 5’ ITR; (ii) a promoter; (iii) a dimerization domain; (iv) a splicing acceptor; (v) a C-terminal mid-length dystrophin coding sequence; (vi) a poly(A) signal; and (vii) a 3’ ITR. In some embodiments, the C-terminal transgene comprises one or more of the following sequences from 5’ to 3’: (i) a 5’ ITR; (ii) a promoter; (iii) a dimerization domain; (iv) a splicing acceptor; (v) a C-terminal mid-length dystrophin coding sequence; (vi) a poly(A) signal; and (vii) a 3’ ITR. See, e.g., FIG. 3B.

[0165] In some embodiments, the one or more additional regulatory elements is upstream of a coding sequence and operably linked thereto. In some embodiments, the one or more additional regulatory elements is downstream of a coding sequence and operably linked thereto. In some embodiments, the one or more additional regulatory elements is an expression control element, such as an enhancer. In some embodiments, the one or more additional regulatory elements is an enhancer, and the enhancer is upstream of the promoter and downstream of the 5’ ITR and operably linked thereto.

[0166] In some embodiments, the one or more additional regulatory elements is upstream of an REJ element (e.g., a dimerization domain) and operably linked thereto. In some embodiments, the one or more additional regulatory elements is downstream of an REJ element (e.g., a dimerization domain) and operably linked thereto. In some embodiments, the one or more additional regulatory elements is an expression control element, such as an enhancer. N-terminal or C-terminal Coding Sequences

[0167] Coding sequences for use in the present disclosure are nucleic acid sequences encoding a target protein, or fragment thereof, and are delivered via the vectors (e.g., AAV particles) described herein. One application of this system is expression of large disease-causing genes using viral vectors with restricted packaging capacity. In some embodiments, the large diseasecausing gene is dystrophin and the disease is Duchenne muscular dystrophy. In some embodiments, the coding sequences for use in the present disclosure are nucleic acid sequences encoding dystrophin, or a fragment thereof, e.g., mid-length dystrophin.

[0168] Dystrophin is the second largest human gene with a genomic region of over 2.2 megabases in length (Chromosome X: 31,117,228-33,344,609 (Genome Reference Consortium — GRCh38 / hg38)). The dystrophin gene contains 79 exons that are processed into an 11,000 base pair mRNA that is translated into a 427 kDa protein. Dystrophin has four domains: (1) the N-terminal region contains binding sites for F-actin; (2) the central rod domain contains 24 spectrin-like repeats interspersed by four hinge regions; (3) the cysteine-rich domain (CRD) binds the dystroglycoprotein complex; and (4) the C-terminal domain. Spectrinlike repeats 16 and 17 in the dystrophin protein comprise a neuronal nitric oxide synthase (nNOS) binding domain. The a2 and a3 helices of spectrin-like repeats 16 and 17 are essential for nNOS binding. A microdomain in the al helix of spectrin-like repeat 17 binds to nNOS in a way that is uniquely defined by the flanking helices. nNOS is an enzyme that produces nitric oxide, which can help increase blood flow in muscle. The binding of dystrophin and nNOS localizes nNOS to the sarcolemma, which is necessary to prevent functional ischemia in skeletal muscle. A further description of dystrophin and its domains is described in Davies and Guiraud, Molecular Therapy, Vol. 27 No. 3m 20, incorporated by reference herein in its entirety.

[0169] Functionally, dystrophin acts as a linker between the actin filaments and the extracellular matrix within muscle fibers. The N-terminus of dystrophin is an actin-binding domain, while the C-terminus interacts with a transmembrane scaffold that anchors the muscle fiber to the extracellular matrix. Upon muscle contraction, dystrophin provides structural support that allows the muscle tissue to withstand mechanical force. DMD is caused by a wide variety of mutations within the dystrophin gene that result in premature stop codons and therefore a truncated dystrophin protein. Truncated dystrophin proteins do not contain the C-terminus, and therefore cannot provide the structural support necessary to withstand the stress of muscle contraction. As a result, the muscle fibers pull themselves apart, which leads to muscle wasting. Other in-frame deletions of the gene result in milder forms of the disease (e.g., Becker muscular dystrophy), since the truncated dystrophin molecules are partially functional. The binding of nNOS involves both the N-terminal and rod domains and has been correlated with the severity of disease, but loss of the nNOS localization is consistent with a mild phenotype in patients.

[0170] In some embodiments, the coding sequences of the present disclosure are nucleic acid sequences encoding dystrophin protein or a fragment thereof. In some embodiments, the coding sequences of the present disclosure are nucleic acid sequences encoding a mid-lengthdystrophin protein. In some embodiments, the mid-length dystrophin protein comprises: (i) an N-terminal region with binding sites for F-actin; (ii) a central rod domain comprising one or more hinge regions and one or more spectrin-like repeats; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the mid-length dystrophin protein comprises: (i) an N-terminal region with binding sites for F-actin; (ii) a central rod domain comprising three hinge regions and ten spectrin-like repeats; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the mid-length dystrophin protein comprises: (i) an N-terminal region with binding sites for F-actin; (ii) a central rod domain comprising: (a) hinge region 1, hinge region 3, and hinge region 4; and (b) spectrin-like repeat 1, spectrin-like repeat 2, spectrin-like repeat 3, spectrin-like repeat 16, spectrin-like repeat 17, spectrin-like repeat 20, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 23, and spectrin-like repeat 24; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the midlength dystrophin protein is approximately 6.9 kilobases. In some embodiments, the mid-length dystrophin protein is approximately 248 kilodaltons.

[0171] In some embodiments, the coding sequence of a target protein is divided into two portions, such as about two equal halves (or other proportions, such as portion A expressing about 1 / 3 and portion B expressing about 2 / 3, or portion A expressing about 1 / 4 and portion B expressing about 3 / 4, etc.). However, it is not required that each portion be the same number of nucleotides (or encode the same number of amino acids). In some embodiments, the target gene is a dystrophin gene. In some embodiments, the coding sequence of the dystrophin protein or fragment thereof, is split into an N-terminal coding sequence encoding an N-terminal portion of the dystrophin protein or fragment thereof, and a C-terminal coding sequence encoding a C-terminal portion of the dystrophin protein or fragment thereof. In some embodiments, the coding sequence of mid-length dystrophin protein, is split into an N-terminal coding sequence encoding an N-terminal portion of the mid-length dystrophin protein, and a C-terminal coding sequence encoding a C-terminal portion of the mid-length dystrophin protein.

[0172] In some embodiments, the N-terminal and / or C-terminal coding sequence of a target protein is a wild type coding sequence. For example, the coding sequence is one that is found in the cell or organism into which the disclosed system is introduced (e.g., a human coding sequence when introduced into a human cell or subject). In some embodiments, the N-terminal and / or C-terminal coding sequence of a target protein is codon-optimized relative to a wild type coding sequence, for example to maximize tRNA availability, or to de-enrich for crypticsplice sites (e.g., to reduce or avoid incorrect splicing and promote the correct junction formation). The coding sequence is the portion of the mRNA sequence that encodes the amino acids for translation. During translation, each of 61 trinucleotide codons are translated to one of 20 amino acids, leading to a degeneracy, or redundancy, in the genetic code. However, different cell types, and different animal species, utilize tRNAs (each bearing an anticodon) coding for the same amino acids at different frequencies. When a gene sequence contains codons that are infrequently represented by the corresponding tRNA, the ribosome translation machinery may slow, impeding efficient translation. Expression can be improved via codon optimization for a particular species, where the coding sequence is altered to encode the same protein sequence, but utilizing codons that are highly represented, and / or utilized by highly expressed human proteins (Cid-Arregui et al., 2003, J. Virol. 77: 4928). In some embodiments, the coding sequence of a target protein (e.g., a dystrophin protein or fragment thereof) is modified to replace codons infrequently expressed in mammal or in primates with codons frequently expressed in primates. In some embodiments, the coding sequence of a target protein (e.g., a dystrophin protein or fragment thereof) is a codon-optimized version of human gene to increase joined mRNA stability upon transcription. For the optimization, GeneArt® software may be used, increasing the GC content and removing cryptic splice sites in order to avoid transcriptional silencing and therefore increase transgene expression. Alternatively, any optimization method known in the art may be used.

[0173] In some embodiments, the N-terminal transgene comprises an N-terminal coding sequence that is a wild-type coding sequence. In some embodiments, the N-terminal transgene comprises an N-terminal coding sequence that is codon-optimized. In some embodiments, the N-terminal transgene comprises an N-terminal coding sequence comprising: (i) an N-terminal region; and (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, and a spectrin-like repeat 21. In some embodiments, the N-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 9. In some embodiments, the N-terminal coding sequence comprises the nucleic acid sequence of SEQ ID NO: 9.

[0174] In some embodiments, the C-terminal transgene comprises a C-terminal coding sequence that is a wild type coding sequence. In some embodiments, the C-terminal transgenecomprises a C-terminal coding sequence that is codon-optimized. In some embodiments, the C-terminal transgene comprises a C-terminal coding sequence comprising: (i) a central rod domain comprising: a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24; and a hinge region 4; (ii) a cysteine rich domain; and (iii) a C-terminal domain. In some embodiments, the C-terminal coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, the C-terminal coding sequence comprises the nucleic acid sequence of SEQ ID NO: 21.

[0175] In some embodiments, the transcript of the N-terminal coding sequence and the transcript of the C-terminal coding sequence can be spliced and joined to form an RNA coding sequence that encodes a full-length target protein (e.g., dystrophin) or fragment thereof (e.g., mid-length dystrophin protein). In some embodiments, the transcript of the N-terminal coding sequence and the transcript of the C-terminal coding sequence can be spliced to form an RNA coding sequence that encodes a mid-length dystrophin protein, wherein the mid-length dystrophin protein comprises: (i) an N-terminal region with binding sites for F-actin; (ii) a central rod domain comprising one or more hinge regions and one or more spectrin-like repeats; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the transcript of the N-terminal coding sequence and the transcript of the C-terminal coding sequence can be spliced to form an RNA coding sequence that encodes a mid-length dystrophin protein, wherein the mid-length dystrophin protein comprises: (i) an N-terminal region with binding sites for F-actin; (ii) a central rod domain comprising three hinge regions and ten spectrin-like repeats; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the transcript of the N-terminal coding sequence and the transcript of the C-terminal coding sequence can be spliced to form an RNA coding sequence that encodes a mid-length dystrophin protein, wherein the mid-length dystrophin protein comprises: (i) an N-terminal region with binding sites for F-actin; (ii) a central rod domain comprising: (a) hinge region 1, hinge region 3, and hinge region 4; and (b) spectrin-like repeat 1, spectrin-like repeat 2, spectrin-like repeat 3, spectrin-like repeat 16, spectrin-like repeat 17, spectrin-like repeat 20, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 23, and spectrin-like repeat 24; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the transcript of the N-terminal coding sequence and the transcript of the C-terminal coding sequence can be spliced to form a coding sequence that encodes a mid-length dystrophin protein, wherein the mid-length dystrophinprotein comprises from 5’ to 3’: (i) an N-terminal region with binding sites for F-actin; (ii) a central rod domain comprising: hinge region 1, spectrin-like repeat 1, spectrin-like repeat 2, spectrin-like repeat 3, spectrin-like repeat 16, spectrin-like repeat 17, hinge region 3, spectrinlike repeat 20, spectrin-like repeat 21, spectrin-like repeat 22, spectrin-like repeat 23, spectrinlike repeat 24, and hinge region 4; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the transcript of the N-terminal RNA coding sequence and the transcript of the C-terminal RNA coding sequence can be spliced and joined to form an RNA coding sequence that encodes a mid-length dystrophin protein, wherein the RNA coding sequence comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the mid-length dystrophin protein comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the mid-length dystrophin protein comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 23. In some embodiments, the mid-length dystrophin protein comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the coding sequence encoding a midlength dystrophin protein is a wild-type coding sequence. In some embodiments, the coding sequence encoding a mid-length dystrophin protein is codon-optimized.

[0176] In some embodiments, the N-terminal coding sequence and / or the C-terminal coding sequence comprises an intervening intron sequence within the coding sequence. The intervening intron sequence is either natural or synthetic in nature. Inclusion of such introns can be used to stimulate splicing machinery attachment to the trans-splicing intron donor and acceptor.

[0177] In some embodiments, the N-terminal coding sequence comprises an intervening intron sequence situated close to the 3’ end of the coding sequence. In some embodiments, the intervening intron sequence is situated about 150 nt, about 155 nt, about 160 nt, about 165 nt, about 170 nt, about 175 nt, about 180 nt, about 185 nt, about 190 nt, about 195 nt, or about 200 nt upstream of the 3’ end of the N-terminal coding sequence. In some embodiments, the intervening intron sequence is situated about 171 nt upstream of the 3’ end of the N-terminal coding sequence. In some embodiments, the intervening intron sequence within the N-terminal coding sequence is a modified mouse beta-actin (Actb) intron 2 sequence. In some embodiments, the intervening intron sequence within the N-terminal coding sequencecomprises a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the intervening intron sequence within the N-terminal coding sequence comprises the nucleotide sequence of SEQ ID NO: 7.

[0178] In some embodiments, the C-terminal coding sequence comprises an intervening intron sequence situated close to the 5’ end of the coding sequence. In some embodiments, the intervening intron sequence is situated about 80 nt, about 85 nt, about 90 nt, about 95 nt, about 100 nt, about 105 nt, about 110 nt, about 115 nt, about 120 nt, about 125 nt, about 130 nt, about 140 nt, about 145 nt, or about 150 nt downstream of the 5’ end of the C-terminal coding sequence. In some embodiments, the intervening intron sequence is situated about 108 nt downstream of the 5’ end of the C-terminal coding sequence. In some embodiments, the intervening intron sequence within the C-terminal coding sequence is a modified mouse betaactin (Actb) intron 2 sequence. In some embodiments, the intervening intron sequence within the C-terminal coding sequence comprises a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the intervening intron sequence within the C-terminal coding sequence comprises the nucleotide sequence of SEQ ID NO: 7.

[0179] In some embodiments, the C-terminal coding sequence comprises an intervening intron sequence situated close to the 3’ end of the coding sequence. In some embodiments, the intervening intron sequence is situated about 850 nt, about 855 nt, about 860 nt, about 865 nt, about 870 nt, about 875 nt, about 880 nt, about 885 nt, about 890 nt, about 895 nt, about 900 nt, about 905 nt, about 910 nt, about 915 nt, about 920 nt, or about 925 nt upstream of the 3’ end of the C-terminal coding sequence. In some embodiments, the intervening intron sequence is situated about 886 nt upstream of the 3’ end of the C-terminal coding sequence. In some embodiments, the intervening intron sequence within the C-terminal coding sequence is a modified mouse Gapdh intron sequence. In some embodiments, the intervening intron sequence within the C-terminal coding sequence is a modified mouse Gapdh intron 5 sequence. In some embodiments, the intervening intron sequence within the C-terminal coding sequence comprises a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the intervening intron sequence within the C-terminal coding sequence comprises the nucleotide sequence of SEQ ID NO: 19.Splicing Domains

[0180] RNA splicing depends on the recruitment of spliceosome components to the splicing domain of each split pre-mRNA. In some embodiments, the N-terminal split pre-mRNA comprises a splicing donor at the 3’ end of the N-terminal coding sequence. See e.g., FIG. 3 A, a synthetic half intron. In some embodiments, the C-terminal split pre-mRNA comprises a splicing acceptor at the 5’ end of the C-terminal coding sequence. See e.g., FIG. 3B, a synthetic half intron. Different ribonucleoproteins are recruited to the synthetic half intron through base pairing of protein associated small nuclear RNA (snRNA) with intronic sequences.

[0181] In some embodiments, the N-terminal transgene comprises a splicing donor that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the C-terminal transgene comprises a splicing acceptor that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 16. In some embodiments, the C-terminal transgene comprises a splicing acceptor comprising the nucleotide sequence of SEQ ID NO: 16.Dimerization Domains

[0182] The association of the two split pre-mRNA molecules (the N-terminal fragment and the C-terminal fragment) is mediated by the RNA dimerization domain, allowing the spliceosome components to recombine the N-terminal RNA coding sequence and C-terminal coding sequence. As shown in FIG. 1, interaction and hybridization (base pairing) between the dimerization domain of the N-terminal split pre-mRNA and the dimerization domain of the C-terminal split pre-mRNA allows the spliceosome components to recombine N-terminal coding sequence and C-terminal coding sequence, which results in the joining of the N-terminal and the C-terminal coding sequences, allowing for expression of the target protein (e.g., mid-length dystrophin).

[0183] In some embodiments, the first and second dimerization domains contain complementary RNA stem loops (also called hairpin loops) that can form strong kissing loop interactions with their counterparts. Each stem loop comprises at least two complementary sequences (e.g., form a stem) separated by a region of non-complementary sequence (e.g., form a loop). Complementary sequences between two stem loops result in base pairing, and generation of a kissing loop / kissing stem loop interaction. In some embodiments, the complementary sequences between the two stem loops comprise at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, or more loop base pairs for intermolecular pairing. In some embodiments, the stems of the kissing loops are chosen to base pair in trans between the two dimerization domains. In such an example, after forming a kissing loop interaction of one stem loop on one dimerization domain with another stem loop on a second dimerization domain, the respective stem (or helix) regions of the initial hairpin loops can base pair in trans between the two dimerization domains through strand replacement / invasion and result in extended duplex formation. In one embodiment, extended duplex formation is favored by inclusion of mismatches in the initial stems that favor pairing in the extended duplex. In some embodiments, the dimerization domains contain helices destabilized by the inclusion of as about 1% to about 30%, about 5% to about 30%, about 10% to about 30%, or about 25% to about 30% mismatches, but match to the other dimerization domain, to favor extended duplex formation after initial kissing / pairing. In some embodiments, these stem loops contain at least 10 nt, such as at least 20 nt, at least 25 nt, at least 50 nt, at least 75 nt, or at least 100 nt in length, such as 10 nt to 50 nt, 20 nt to 25 nt, 10 nt to 100 nt, 10 nt to 20 nt, or 20 nt to 40 nt in length. In some embodiments, each dimerization domain can contain at least 1 individual stem loop, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20, such as 1 to 20, 2 to 5 or 1 to 10 individual stem loops. In one embodiment, each dimerization domain can contain a central stem loop.

[0184] In some embodiments, the first and second dimerization domains are designed to contain sequences that are complementary to each other (e.g., the first dimerization domain comprising a nucleic acid sequence of SEQ ID NO: 11 and the second dimerization domain comprising a nucleic acid sequence of SEQ ID NO: 15). The first and second dimerization domains contain complementary RNA stem loops that can form central kissing loop interaction with four or more loop base pairs for intermolecular pairing. In one embodiment, the positioning of the stem loops is offset in such a way that the respective stem regions of the initial stem loops can base pair in trans through strand invasion, which leads to an extended area of interaction (a wider synapse) between the first and second dimerization domains. In one embodiment, the positioning of the kissing loops is offset by at least 1 nt, at least 2nt, at least 3 nt, at least 4 nt, at least 5 nt, at least 6 nt, at least 7 nt, at least 8 nt, at least 9 nt, or at least 10 nt. In one embodiment, extended strand invasion is followed for the full dimerization of the two dimerization domains. The extended area of interaction facilitates more robust molecule interactions. In addition, mismatches are introduced in the two stem loops to disfavor the intramolecular base pairing and favor intermolecular pairing.

[0185] In some embodiments, the N-terminal transgene comprises a dimerization domain comprising a nucleic acids sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 11. In a further embodiment, the dimerization domain comprises the nucleotide sequence of SEQ ID NO: 11. In some embodiments, the C-terminal transgene comprises a dimerization domain comprising a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 15. In a further embodiment, the dimerization domain comprises the nucleotide sequence of SEQ ID NO: 15. In one embodiment, the N-terminal transgene comprises a dimerization domain comprising the nucleic acid sequence of SEQ ID NO: 11 and the C-terminal transgene comprises a dimerization domain comprising the nucleic acid sequence of SEQ ID NO: 15.Inverted Terminal Repeats (ITRs)

[0186] ITRs are the genetic elements responsible for the replication and packaging of the transgene into an AAV capsid during AAV particle production and are the only viral cis elements required to generate a recombinant AAV particle. The minimal sequences required to package the transgene into an AAV viral particle are the AAV 5’ and 3’ ITRs, which flank the other elements of the transgene, and may be of the same AAV origin as the capsid proteins, or of a different AAV origin. The 5’ and 3’ ITRs of an AAV vector genome are necessary for both the integration of the coding sequence into the host cell genome (e.g., chromosome 19 in humans) and for encapsidation of the transgene into the AAV particle.

[0187] ITRs described herein may be used to facilitate the packaging of either a self-complementary or single stranded vector genome. In some embodiments, one of the ITRs is a non-resolvable ITR that is used to provide a self-complementary vector genome in a host cell. The non-resolvable ITR may be produced by any method known in the art. For example, insertion into the ITR will displace the nicking site and result in a non-resolvable ITR. The designation of the various regions or elements within the ITR are known in the art. In one embodiment, the insertion is made into the sequence of the terminal resolution site (trs). Alternatively, the insertion may be made at a site between the Rep Binding Element (RBE) within the A element and the trs, which is adjacent to the D sequence. In another embodiment, the ITR may be rendered non-resolvable by deletion of the trs site. In addition to the trs, some or all of the D sequence may be deleted. In some embodiments, the 5’ ITR comprises an AAV trs. In some embodiments, the 5’ ITR does not comprise an AAV trs.

[0188] In some embodiments, transgenes of the disclosure comprise ITR sequences from any one AAV serotype, e.g., AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. The ITR sequence, as provided above, can facilitate the formation of either a single stranded or self-complementary vector genome.

[0189] In some embodiments, the transgenes disclosed herein comprise a 5’ AAV2 ITR and a 3’ AAV2 ITR sequence.

[0190] In some embodiments, the N-terminal transgene comprises a 5 ’-ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 1. In a further embodiment, the N-terminal transgene comprises a 3’ AAV ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 14. In a further embodiment, the 3’ AAV ITR comprises SEQ ID NO: 14. In a further embodiment, the 3’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 14.

[0191] In some embodiments, the C-terminal transgene comprises a 5’ ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the 5’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 1. In a further embodiment, the C-terminal transgene comprises a 3’ AAV ITR that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to the nucleic acid sequence of SEQ ID NO: 14. In a further embodiment, the 3’ AAV ITR comprises SEQ ID NO: 14. In a further embodiment, the 3’ AAV ITR consists of the nucleic acid sequence of SEQ ID NO: 14.Promoters

[0192] In some embodiments, a promoter is operably linked to the N-terminal coding sequence of the N-terminal transgene. In some embodiments, the promoter is located upstream (5’) of the N-terminal coding sequence and downstream (3’) of the 5’ ITR.

[0193] In some embodiments, a promoter is operably linked to the REJ dimerization domain. In some embodiments, the promoter is located upstream (5’) of the REJ dimerization domain and downstream (3’) of the 5’ ITR.

[0194] In some embodiments, the N-terminal transgene comprises the same promoter as the C-terminal transgene. In some embodiments, the N-terminal transgene comprises a promoter that is different than the promoter present in the C-terminal transgene.

[0195] In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a muscle tissue-specific promoter.

[0196] In some embodiments, the transgenes of the present disclosure comprise a mammalian promoter, for example, human, non-human primate (e.g., cynomolgus macaque), mouse, horse, cow, pig, cat, or dog promoters. In some embodiments, the transgene comprises a strong, constitutively active promoter to drive high-level expression of the coding sequence. For example, in some embodiments, the promoter is a cytomegalovirus (CMV) promoter / enhancer, an elongation factor 1α (EF1α) promoter, a simian virus 40 (SV40) promoter, a chicken β-actin hybrid promoter, or a CAG promoter. In a further embodiment, the promoter has been engineered to be depleted of CpG (5’-C-phosphate-G-3’) dinucleotides. In even a further embodiment, the promoter has been engineered to be free of CpG (5’-C-phosphate-G-3’) dinucleotides.

[0197] In some embodiments, the transgene of the present disclosure comprises a musclespecific promoter that is operably linked to the transgene to drive high-level and tissue-specific expression in muscle cells (e.g., cardiac and / or skeletal muscle). For example, muscle specific promoters include, but are not limited to, promoters selected from: desmin (DES, also known as CSM1 or CSM2) promoter, the alpha 2 actinin (ACTN2, also known as CMD1AA) promoter, the filamin-C (FLNC, also known as actin-binding-like protein (ABLP), filamin-2 (FLN2), ABP-280, ABP280A, ABPA, ABPL, MFM5 or MPD4) promoter, the sarcoplasmic / endoplasmic reticulum calcium ATPase 1 (ATP2A1, also known as ATP2A or SERCA1) promoter, the troponin I type 1 (TNNI1, also known as SSTNI or 25TTNI) promoter, the myosin-1 (MYH1) promoter, the phosphorylatable, fast skeletal muscle myosin light chain(MYLPF) promoter, myosin 1 (MYH1, also known as MYHSA1, MYHa, MyC-2X / D or MyHC-2x) promoter, the alpha-3 chain tropomyosin (TPM3, also known as CFTD, NEM1, OK / Scl.5, TM-5, TM3, TM30, TM30nm, TM5, TPMsk3, TRK, h TM5 or hscp30) promoter, the ankyrin repeat domain-containing protein 2 (ANKRD2, also known as ARPP) promoter, the myosin heavy-chain (MHC) promoter, the myosin light-chain (MLC) promoter, the muscle creatine kinase (MCK) promoter, synthetic muscle promoters as described in Li et al. (1999. Nat Biotechnol. 17:241-245), such as the SPc5-12 promoter, the muscle creatine kinase (MCK) promoter, the dMCK promoter, the tMCK promoter consisting of respectively, a double or triple tandem of the MCK enhancer to the MCK basal promoter as described in Wang et al. (2008. Gene Ther. 15:1489-1499) and hybrid promoters, such as the hybrid alpha-myosin heavy chain enhancer / MCK enhancer (MHCK7; 770 bp); the MCK-C5-12 promoter as described in Wang et al. (2008. Gene Ther. 15: 1489-1499) and the cardiac and skeletal musclespecific myosin chaperone Unc45b (195 bp) promoter as described in Rudeck S et al. (2016, Genesis. 54(8): 431-8). Non-limiting examples of heart-specific promoters include the calsequestrin 2 (also known as PDIB2, FLJ26321, FLJ93514 or CASQ2 (GenelD 845 for the human gene)) promoter, the ankyrin repeat domain 1 (also known as cardiac ankyrin repeat protein) promoter, the cytokine inducible nuclear protein promoter; the liver ankyrin repeat domain 1 (ANKRD1; GenelD 27063 for the human gene) promoter; the myosin, light chain 2, regulatory, cardiac, slow (MYL2; GenelD 4633 for the human gene) promoter; the myosin, light chain 3, alkali; ventricular, skeletal 10 slow (MYL3; GenelD 4634 for the human gene) promoter; the bromodomain containing 7 (also known as BP75, CELTIX1, NAG4(BRD7; GenelD 29117 for the human gene)) promoter; the alpha myosin heavy chain (αMHC) promoter; the cardiac troponin C promoter and the promoter of the cardiac sodium-calcium exchanger (NCX1), which confers cardiac specificity.

[0198] In some embodiments, the promoter is a MHCK7 promoter. In some embodiments, the MHCK7 promoter comprises the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the N-terminal transgene described herein comprises a promoter comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the C-terminal transgene described herein comprises a promoter comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 2.Poly (A) Signals

[0199] As discussed throughout, the transgenes of the present disclosure comprise a poly(A) signal nucleic acid sequence. In some embodiments, the poly(A) signal sequence is used to assist in RNA export from the nucleus, translation of RNA, and RNA stability.

[0200] In some embodiments, a suitable poly(A) signal sequence is derived from bovine growth hormone (bGH), human growth hormone (hGH), simian virus 40 (SV40), β-globin, rabbit β-globin (RGB), modified RGB (mRGB) or thymidine kinase (TK). The poly(A) signal sequence, in one embodiment, is a SV40-poly(A) signal sequence. The poly(A) signal sequence, in another embodiment, is a bovine growth hormone (bGH)-poly(A) signal sequence. In even another embodiment, the poly(A) signal sequence is a synthetic poly(A) signal sequence or from bovine growth hormone (bGH), human growth hormone (hGH), S V40, rabbit β-globin (RGB), or modified RGB (mRGB).

[0201] In some embodiments, the N-terminal transgene comprises a late SV40 poly(A) signal sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 13. In some embodiments, the late SV40 poly(A) signal sequence comprises the nucleic sequence of SEQ ID NO: 13. In some embodiments, the late SV40 poly(A) signal sequence consists of the nucleic sequence of SEQ ID NO: 13.

[0202] In some embodiments, the C-terminal transgene comprises a late SV40 poly(A) signal sequence. In some embodiments, the C-terminal transgene comprises a late SV40 poly(A) signal sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to SEQ ID NO: 13. In some embodiments, the late SV40 poly(A) signal sequence comprises the nucleic sequence of SEQ ID NO: 13. In some embodiments, the late SV40 poly(A) signal sequence consists of the nucleic sequence of SEQ ID NO: 13.Nucleic Acid Elements Designed to Inhibit Unjoined N- and C-terminal Fragment Expression

[0203] In some embodiments, the transgenes described herein comprise at least one nucleic acid element that suppresses expression of the unjoined or unrecombined RNA transcript. In such an example, if the N-terminal and C-terminal transcripts encoding a portion of the midlength dystrophin protein do not recombine, the at least one nucleic acid element suppresses expression of the unjoined N-terminal transcript and / or the C-terminal transcript. For example, such a suppressive nucleic acid element can destabilize the RNA once outside the nucleus, prevent translation, stimulate translation from a shifted start codon, contain microRNA target sites, or contain protein degron or destabilization domains that when translated suppress the protein activity or flag it for degradation. See e.g., FIG. IB for exemplary suppressive nucleic acid elements.

[0204] In some embodiments, destabilization of the un-recombined protein product from an open reading frame can be achieved by depleting stop codon occurrence in the intronic sequence and an additional inclusion of an RNA sequence coding for an in frame protein signal that can flag a protein for degradation (e.g., a degron sequence) that is placed at any position within the intronic sequence and which is in frame with the open reading frame that is extended out from the coding sequence (shown as 1 in FIG. 4). In one embodiment, a degron sequence can be a ssrA tag sequence, a PEST sequence, or a CL 1 degron sequence. Degron sequences used can employ proteasome-dependent, proteasome-independent, ubiquitin-dependent, or ubiquitin-independent pathways. In one embodiment, un-recombined protein destabilization is enhanced by inclusion of several of the same or different degron sequences. In this example, the degron sequence would be C -terminally joined to the un-recombined protein fragment that will be suppressed by being flagged for degradation.

[0205] In some embodiments, a suppressive nucleic acid element comprises one or more micro-RNA target sites (miR-targets) at any position within the intronic sequence of a splicing donor or a splicing acceptor (shown as 2 in FIG. IB). If an unjoined RNA is exported from the nucleus, it becomes subject to micro-RNA / small hairpin RNA-dependent degradation which can suppress unintended unjoined fragment expression by degrading / suppressing unjoined RNA that is exported from the nucleus. In some embodiments, such a micro-RNA target sequence is complementary to a micro-RNA known to be expressed in the cell, or tissue, or animal into which dual AAV vectors are introduced. In some embodiments, a micro-RNA target sequence is complementary to a sequence that is introduced into the cell, or tissue, or animal. In some embodiments, a microRNA is expressed from an RNA-polymerase III dependent promoter in the form of a small hairpin RNA. In some embodiments, a microRNAis expressed from an RNA polymerase II dependent promoter and embedded in a micro RNA processing loop ( e.g., mir30 scaffold).

[0206] In some embodiments, destabilization of the unjoined RNA molecule is achieved by including a self-cleaving RNA sequence (e.g., Glucosamine-6-phosphate synthase (glmS) ribozyme, Hatchet ribozyme, hairpin ribozyme, Pistol ribozyme, Twister ribozyme, Twister sister ribozyme, Neurospora Varkud satellite (VS) ribozyme, Hovlinc ribozyme, Hammerhead ribozyme or HDV ribozyme) in the splicing domain (i.e., a synthetic half-intron portion) (shown as 3 in FIG. IB). In one embodiment, cleaving the RNA molecule leads to a loss of the RNA stabilizing poly(A) tail, which can suppress expression of an un-recombined protein from open reading frame. In one embodiment, a self-cleaving RNA sequence is included at any position within an intronic sequence to cleave off the 5’ terminal CAP which in one example can lead to reduced expression of an open reading frame that includes parts or the whole of coding sequence. In one embodiment, self-cleaving RNA sequences are substituted with an RNA cleaving enzyme target site, such as a Csy4 target site.

[0207] In some embodiments, a suppressive nucleic acid molecule comprises a weak poly(A) signal sequence or an alternative termination signal sequence, such as e.g. an RNA triplex at the 3’ end of the N-terminal transgene, to suppress nuclear export and translation of the N-terminal fragment.

[0208] In some embodiments, a suppressive nucleic acid element comprises one or more out-of-frame start codon(s) (OOF-AUGs) in the splicing acceptor domain to suppress translation of C-terminal fragment (shown as 4 in FIG. IB).

[0209] In some embodiments, the suppressive nucleic acid element comprises a start codon (ATG) and a Kozak sequence at any position in the intronic sequence that directs translation of an open reading frame that is shifted -1, -2, +1, or +2 nucleotides relative to the open reading frame sequence of the coding sequence. In one example, the unjoined RNA fragment is suppressed by using this decoy start codon strategy to direct translation away from the to be suppressed open reading frame of the coding sequence.

[0210] In some embodiments, destabilization of the un-recombined protein product from open reading frame sequence is achieved by introduction of a start codon (ATG) followed by a degron sequence at any position within the intronic sequence of the splicing acceptor which is in frame with an open reading frame within the coding sequence. In this example, the degronsequence will N-terminally joined to the un -recombined protein fragment that will be suppressed by being flagged for degradation.

[0211] In some embodiments, the N-terminal transgene comprises at least one nucleic acid element to suppress expression of an unjoined transcript. In some embodiments, the at least one nucleic acid element is the absence of a stop codon in the N-terminal coding sequence. In some embodiments, the at least one nucleic acid element is a micro-RNA target site 3’ to the splicing donor. In some embodiments, the at least one nucleic acid element is two micro-RNA target sites 3’ to the splicing donor. In some embodiments, the at least one micro-RNA target site is a micro-RNA- 16 target site. In some embodiments, the micro-RNA- 16 target site comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence of SEQ ID NO: 12. In some embodiments, the micro-RNA-16 target site comprises the nucleic acid of SEQ ID NO: 12.

[0212] In some embodiments, the C-terminal transgene comprises at least one nucleic acid element to suppress expression of an unjoined transcript. In some embodiments, the at least one nucleic acid element is an out-of-frame start codon 5’ of the splicing acceptor. In some embodiments, the at least one nucleic acid element is a modified mouse Gapdh intron sequence in the C-terminal coding sequence. In some embodiments, the modified Gapdh intron sequence is improperly spliced leading to retention of the intron, which triggers nonsense-mediated decay and degradation of the C-terminal fragment. In some embodiments, the at least one nucleic acid element is a modified mouse Gapdh intron 5 sequence in the C-terminal coding sequence. In some embodiments, the modified mouse Gapdh intron 5 sequence site comprises a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence of SEQ ID NO: 19. In some embodiments, the modified mouse Gapdh intron 5 sequence site comprises the nucleic acid sequence of SEQ ID NO: 19.Enhancers

[0213] In some embodiments, the transgenes provided herein comprise one or more enhancer sequences. In one embodiment, the one or more enhancers are operably linked to the coding sequence in the N-terminal transgene. In one embodiment, the one or more enhancers areoperably linked to the dimerization domain sequence in the C-terminal transgene. An enhancer sequence, in one embodiment, can increase the level of transcription of the coding sequence, for example, by serving as a binding site for transcription factors and co-regulators that assist in DNA looping and recruitment of the transcriptional machinery to promoters.

[0214] In some embodiments, the enhancer is downstream (i.e., 3’) of the 5’ ITR and upstream (i.e., 5’) of the promoter in the N-terminal transgene. In some embodiments, the enhancer is downstream (i.e., 3’) of the promoter and upstream (i.e., 5’) of the coding sequence in the N-terminal transgene. In some embodiments, the enhancer is downstream (i.e., 3’) of the coding sequence and upstream (i.e., 5’) of the 3’ ITR in the N-terminal transgene.

[0215] In some embodiments, the enhancer is downstream (i.e., 3’) of the 5’ ITR and upstream (i.e., 5’) of the promoter in the C-terminal transgene. In some embodiments, the enhancer is downstream (i.e., 3’) of the promoter and upstream (i.e., 5’) of the dimerization domain in the C-terminal transgene. In some embodiments, the enhancer is downstream (i.e., 3’) of the coding sequence and upstream (i.e., 5’) of the 3’ ITR in the C-terminal transgene.

[0216] In some embodiments, a transgene of the present disclosure comprises an enhancer that significantly promotes the transcription of a coding sequence in skeletal and / or cardiac muscle cells. In some embodiments, the enhancer is a myosin light chain (MLC) enhancer. In some embodiments, the enhancer is a muscle creatine kinase (MCK) enhancer. In some embodiments, the enhancer is a myogenic differentiation 1 (MYODI) enhancer. In some embodiments, the enhancer is a glucocorticoid receptor (GR) enhancer. In some embodiments, the enhancer is a myogenin (MYOG) enhancer. In some embodiments, the enhancer is a smooth muscle-myosin heavy chain (SM-MHC) enhancer. In some embodiments, the enhancer is a skeletal-cis-regulatory module 4 (SK-CRM4) enhancer. In some embodiments, the enhancer is upstream of a promoter sequence.

[0217] In some embodiments, a transgene of the present disclosure comprises a cytomegalovirus (CMV) enhancer nucleic acid sequence. In some embodiments, the CMV enhancer is upstream of a promoter sequence.

[0218] In some embodiments, two or more enhancers are present in a transgene of the disclosure. Such combination may include more than one copy of any of the enhancers described herein, and / or more than one type of enhancer.Other Regulatory Sequences

[0219] In addition to a promoter and optionally an enhancer, a transgene of the present disclosure may contain other appropriate transcription initiation, termination and / or RNA processing signals (e.g., a Kozak sequence). In some embodiments, a Kozak sequence is present in the transgene provided herein. The Kozak sequence refers to a translation initiation enhancer element to enhance expression of a gene or open reading frame. The Kozak sequence directs the pre-initiation complex (PIC) and ribosome to the translation initiation site (start codon) and mediates ribosome assembly ensuring the correct protein sequence is translated. In one embodiment, the N-terminal transgene comprises a Kozak sequence immediately upstream of the ATG start codon in the coding sequence. In some embodiments, the Kozak sequence comprises a nucleic acid sequence selected from any one of: GCCGCCACC (SEQ ID NO: 24), GCCACC (SEQ ID NO: 3), CTGCCACC (SEQ ID NO: 25), GCTGCCACC (SEQ ID NO: 26). In some embodiments, the N-terminal transgene comprises a Kozak sequence of GCCACC (SEQ ID NO: 3).AAV Vector Backbones

[0220] In some embodiments, a transgene of the disclosure, and ultimately, an AAV vector genome of the present disclosure, is assembled by inserting the transgene nucleic acid sequence, or a portion thereof, as described herein, into an appropriate adenovirus plasmid backbone using standard molecular biology techniques (see, for example, Sambrook et al. (1989). “Molecular Cloning: A Laboratory Manual, 2nd Ed.”; Ausubel et al. (1987). “Current Protocols in Molecular Biology”). The adenovirus plasmid backbone, in one embodiment, comprises the 5’ ITR and 3’ ITR sequences described herein. As such, in one embodiment, the transgene components other than the ITR sequences can be inserted into the adenovirus plasmid backbone between the ITR sequences, i.e., downstream of the 5’ ITR sequence and upstream of the 3’ ITR sequence. In another embodiment, a DNA plasmid comprising one of the transgenes provided herein can be assembled by inserting the ITR sequences into the DNA plasmid backbone, along with the remaining nucleic acid sequences that make up the transgene.

[0221] In some embodiments, provided herein are methods to express a mid-length dystrophin protein from two or more AAV vectors, such as at least two, at least three, at least four, or at least five different AAV vectors (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different AAV vectors). Each AAV vector drives expression of a split pre-mRNA molecule comprising a portion of the mid-length dystrophin coding sequence. Each split pre-mRNA molecule can be spliced sequentially through RNA-end joining domains to generate the complete mid-lengthdystrophin RNA sequence encoding the mid-length dystrophin protein. In some embodiments, the methods described herein express a mid-length dystrophin protein from two AAV vectors.

[0222] In some embodiments, a DNA plasmid is provided comprising the transgenes of the present disclosure. In some embodiments, the DNA plasmid comprises components of the N-terminal transgene with a nucleic acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence provided in Table 1. In some embodiments, the DNA plasmid comprises components of the C-terminal transgene with a nucleic acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identical to a sequence provided in Table 2. In some embodiments, the plasmid backbone comprises one or more components comprising a nucleic acid sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence provided in Table 1 or Table 2.AAV Particles

[0223] Some embodiments, as provided herein, relate to AAV particles, methods for producing the same, and methods for delivering the AAV particles to a subject in need of treatment. The AAV particle for example, an AAV9 particle, comprises a capsid comprising one or more AAV9 capsid proteins; and a transgene encapsidated by the AAV9 capsid (i.e., a vector genome). The vector genome comprises a transgene (e.g., an N-terminal or C-terminal transgene encoding a portion of the mid-length dystrophin protein) and regulatory elements that promote gene expression of the transgene when delivered into muscle cells, for example skeletal and / or cardiac muscle cells.

[0224] One aspect of the present disclosure relates to a dual AAV particle composition comprising: a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises: i) a first 5 ’-inverted terminal repeat (5’-ITR) sequence; ii) a first promoter sequence; iii) a Kozak sequence; iv) an N-terminal coding sequence encoding an N-terminal portion of a target protein, said N-terminal coding sequence being operably linked to and under control of said first promoter; v) a splicing donor sequence; vi) a first dimerization domain sequence; vii) a first poly(A) signal sequence; and viii) a first 3’-ITR sequence; and b) a second AAV particle comprising an AAV capsid encapsidating a secondtransgene, wherein the second transgene comprises: i) a second 5’-ITR sequence; ii) a second promoter sequence; iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter; iv) a splicing acceptor sequence; v) a C-terminal coding sequence encoding a C-terminal portion of the target protein; vi) a second poly(A) signal sequence; and vii) a second 3’-ITR sequence; wherein the N-terminal coding sequence and the C-terminal coding sequence can be spliced and joined to form a recombined nucleic acid that encodes a full-length target protein. In some embodiments, the target protein is a mid-length dystrophin protein.

[0225] The vector genomes comprising the transgenes provided herein may be single stranded or self-complementary. In one embodiment, the vector genome is single stranded. It should be noted that reference to “single stranded” or “self-complementary” is not intended to limit the structure of the vector genome when encapsidated by an AAV capsid. Without wishing to be bound by theory, it is believed that DNA packaging in AAV likely precludes a double-stranded structure of the vector genome while encapsidated. Rather, “single stranded” or “self-complementary” is intended to refer to the transgene structure once present within a target cell, subsequent to viral uncoating.

[0226] The vector genomes and transgenes provided herein are described as including a coding sequence (i.e., the “plus” or “sense” sequence). However, one of ordinary skill will appreciate that the vector genomes and transgenes of the disclosure are described in their broadest sense to indicate the strand of DNA corresponding to the mRNA transcript which is translatable into a polypeptide sequence. As such, it is understood that the vector genomes and transgenes describe herein also encompass non-translated sequences, including the complementary sequence to the transgene, i.e., the “minus” or “antisense” nucleic acid sequences.

[0227] Single stranded (ss) vector genomes provided herein can encompass both the coding (plus or sense) nucleic acid sequence and the minus (antisense) nucleic acid sequence of the transgene. For the ss vector genomes provided herein, without wishing to be bound by theory, packaging of the vector genome into assembled capsids occurs from the 3’ end of both the plus (coding) and minus (antisense) strand of the transgene. Without wishing to be bound by theory, it is thought that the resulting AAV particle population comprises about an equal mix of capsids containing the plus strand of the transgene (coding strand) and the minus strand (antisense) of the transgene. When encapsidated into particles, such transgenes are referred to as vector genomes. Each AAV particle can then deliver a single stranded version of the transgene to the target cell, which needs to become double-stranded before it can express the desired therapeuticprotein. This can occur either by annealing of the plus strand of a transgene to a minus strand delivered to the same cell or from native nuclear mechanisms initiating second-strand synthesis from a single-stranded transgene.

[0228] The assembly of self-complementary AAV vector genomes is known in the art, and is described, for example, in U. S. Patent No. 8,361,457, the contents of which are incorporated by reference in their entirety. One of ordinary skill in the art will understand, for example, that by employing a non-resolvable ITR in a transgene, together with a resolvable ITR, that the transgene will ultimately comprise resolvable ITRs at both ends, with a centrally-located non-resolvable ITR. Additionally, one of skill in the art will appreciate that each half of the transgene on either side of the non-resolvable ITR is approximately the same length and substantially complementary to the other half. One portion will include a coding sequence, a REJ domain, a poly(A) signal and one or more regulatory elements, and the other portion will be complementary thereto.

[0229] Various AAVs and combinations thereof may be selected as sources for capsids and capsid proteins. See, e.g., U. S. Patent Application Publication No. 2007 / 0036760; U. S. Patent Application Publication No. 2009 / 0197338; EP 1,310,571; WO 2003 / 042397 (AAV7 and other simian AAV), U. S. Patent Nos. 7,790,449 and 7,282,199 (AAV8); WO 2005 / 033321 and US 7,906,111 (AAV9); WO 2019 / 207132 (AAVMYO); WO 2021 / 1077000 (MyoAAV), and W02006 / 110689, and WO 2003 / 042397 (rh.10), each of which is incorporated by reference in its entirety for all purposes. Unless otherwise specified, the AAV capsid, ITRs, and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, the AAVs commonly identified as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh.74, AAV9, AAV8bp, AAV7M8 and AAVAnc80, engineered variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. In one embodiment, the AAV capsid is an AAV8 capsid or variant thereof, an AAV9 capsid or variant thereof, an AAVrh.10 capsid or variant thereof, an AAVrh64Rl capsid or variant thereof, an AAVhu.37 capsid or variant thereof, or an AAV3B or variant thereof, or an AAVrh.74 capsid or variant thereof. In one embodiment, the capsid is an AAV9 capsid. In even another embodiment, the capsid is an engineered variant of an AAV9 capsid. In even another embodiment, the engineered variant of the AAV9 capsid includes an AAVMYO capsid. In even another embodiment, the engineered variant of the AAV9 capsid includes an MyoAAV capsid. In some embodiments, the capsid is an AAVrh.74capsid. In even another embodiment, the capsid is an engineered variant of the AAVrh.74 capsid.

[0230] The AAV capsid proteins can be from the same or different AAV serotypes and can be wild-type or engineered. Vector genomes described herein can be replicated, and packaged into AAV capsids when introduced into a host cell also comprising one or more plasmids encoding the respective rep and cap gene products. In one embodiment, a helper plasmid is also transfected into the host cell to aid in AAV particle production by the host cell. In one embodiment, the AAV capsid is an AAV9 capsid.

[0231] In some embodiments, the AAV particle is an AAV9 particle comprising an AAV9 capsid. In some embodiments, the AAV particle is an AAV9 particle and the AAV capsid comprises one or more AAV9 capsid proteins. In some embodiments, the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP1. A non-limiting example of an AAV9 capsid protein VP1 is shown in Table 3. In some embodiments, the AAV9 capsid protein VP1 comprises an amino acid sequence of SEQ ID NO:33. In some embodiments, the AAV9 capsid protein VP1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO:33. In some embodiments, the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP2. In some embodiments, the AAV9 capsid protein VP2 comprises amino acids 138-736 of SEQ ID NO:33. In some embodiments, the AAV9 capsid protein VP2 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to amino acids 138-736 of SEQ ID NO:33. In some embodiments, the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP3. In some embodiments, the AAV9 capsid protein VP3 comprises amino acids 203-736 of SEQ ID NO:33. In some embodiments, the AAV9 capsid protein VP3 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to amino acids 203-736 of SEQ ID NO:33. In some embodiments, the AAV9 capsid encapsidates the N-terminal transgene encoding mid-length dystrophin. In some embodiments, the AAV9 capsid encapsidates the C-terminal transgene encoding mid-length dystrophin.

[0232] In some embodiments, the AAV capsid comprises one or more engineered variants of an AAV9 capsid protein. In some embodiments, the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP1. In some embodiments, the AAV9 capsidprotein comprises an engineered variant of AAV9 capsid protein VP2. In some embodiments, the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP3. In some embodiments, the one or more engineered variants of an AAV9 capsid protein are one or more deimmunized variants of an AAV9 capsid protein. In some embodiments, the engineered AAV9 capsid encapsidates the N-terminal transgene encoding mid-length dystrophin. In some embodiments, the engineered AAV9 capsid encapsidates the C-terminal transgene encoding mid-length dystrophin.

[0233] In some embodiments, the capsid of one or both of the dual AAV vectors comprises one or more AAVMYO capsid proteins. In some embodiments, the one or more AAVMYO capsid proteins comprise AAVMYO capsid protein VP1. AAVMYO capsid protein VP1 is characterized by a 7-mer amino acid insertion at amino acid 588 or amino acid 589 of the AAV9 capsid protein VP1. Non-limiting examples of AAVMYO capsid protein VP1 are shown in Table 3. In some embodiments, the AAVMYO capsid protein VP1 comprises an amino acid sequence of any one of SEQ ID NOS:35-38. In some embodiments, the AAVMYO capsid protein VP1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO:35, 36, 37 or 38. In some embodiment, the AAVMYO capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:35. In some embodiment, the AAVMYO capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:36. In some embodiment, the AAVMYO capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:37. In some embodiment, the AAVMYO capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the one or more AAVMYO capsid proteins comprise AAVMYO VP2. In some embodiments, the one or more AAVMYO capsid proteins comprise AAVMYO VP3.

[0234] In some embodiments, the capsid of one or both of the dual AAV vectors comprises one or more MyoAAV capsid proteins. MyoAAV is an engineered variant of AAV9. In some embodiments, the one or more MyoAAV capsid proteins comprise MyoAAV capsid protein VP1. MyoAAV capsid protein VP1 is characterized by a 10-mer amino acid insertion at amino acid 588 or amino acid 589 of the AAV9 capsid protein VP1. For example, in some embodiments, the MyoAAV capsid protein VP1 comprises a the amino acid sequence of SEQ ID NO: 39 or 40 (see table 3). In some embodiments, the MyoAAV capsid protein VP1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 39 or 40. In some embodiment, the MyoAAV capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:39. In some embodiment, the MyoAAV capsid comprises a VP1 protein comprising the amino acid sequence of SEQ ID NO:40. In some embodiments, the one or more MyoAAV capsid proteins comprises MyoAAV VP2. In some embodiments, the one or more MyoAAV capsid proteins comprise MyoAAV VP3.

[0235] In some embodiments, one or both of the dual AAV particles is an AAVrh.74 particle comprising an AAVrh.74 capsid. In some embodiments, the AAV particle is an AAVrh.74 particle and the AAV capsid comprises one or more AAVrh.74 capsid proteins. In some embodiments, the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP1. In some embodiments, the AAVrh.74 capsid protein VP1 comprises an amino acid sequence of SEQ ID NO:34 (see table 3). In some embodiments, the AAVrh.74 capsid protein VP1 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO:34. In some embodiments, the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP2. In some embodiments, the AAVrh.74 capsid protein VP2 comprises amino acids 138-738 of SEQ ID NO:34. In some embodiments, the AAV9 capsid protein VP2 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to amino acids 138-738 of SEQ ID NO: 34. In some embodiments, the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP3. In some embodiments, the AAVrh.74 capsid protein VP3 comprises amino acids 204-738 of SEQ ID NO:34. In some embodiments, the AAVrh.74 capsid protein VP3 comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to amino acids 204-738 of SEQ ID NO:34.

[0236] In some embodiments, the AAV capsid comprises one or more engineered variants of an AAVrh.74 capsid protein. In some embodiments, the AAVrh.74 capsid protein comprises an engineered variant of AAVrh.74 capsid protein VP1. In some embodiments, the AAVrh.74 capsid protein comprises an engineered variant of AAVrh.74 capsid protein VP2. In some embodiments, the AAVrh.74 capsid protein comprises an engineered variant of AAVrh.74 capsid protein VP3. In some embodiments, the one or more engineered variants ofan AAVrh.74 capsid protein are one or more deimmunized variants of an AAVrh.74 capsid protein.AAV Particle Production

[0237] AAV particles can be produced by any standard method (see, for example, WO 2001 / 083692; Masic etal. 2014. Molecular Therapy, 22(11): 1900-1909; Carter, 1992, Current Opinions in Biotechnology, 1533-539; Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129); Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al, J. Virol, 62: 1963 (1988); and Lebkowski et al, Mol. Cell. Biol, 7:349 (1988). Samulski etal, J. Virol., 63:3822-3828 (1989); U. S. Patent No. 5,173,414; WO 95 / 13365; U. S. Patent No. 5,658.776; WO95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US 96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin etal. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4:609-615 (1993); Clark et al. Gene Therapy 3: 1124- 1132 (1996); U. S. Patent. No. 5,786,211; U. S. Patent No. 5,871,982; and U. S. Patent. No. 6,258,595, herein incorporated by reference in their entireties).

[0238] In some embodiments, DNA plasmids comprising the transgenes described herein can be transformed into Escherichia coli to scale-up DNA production, purified using any standard method (for example, a Maxi-Prep K, Thermo Scientific), and verified by restriction digest or sequencing. Purified transgene plasmids can then be transfected using a standard method (e.g., calcium phosphate transfection, polyethyleneimine, electroporation, and the like) into an appropriate packaging cell line (e.g., HEK293, HeLa, or PerC.6, MRC-5, WI-38, Vera, and FRhL-2 cells) in combination with a plasmid comprising AAV rep and AAV cap genes, and an AAV helper plasmid.

[0239] As such, in one aspect of the disclosure, a packaging cell is provided comprising a DNA plasmid comprising one of the transgenes provided herein. In a further embodiment, the packaging cell comprises AAV replication (rep) and capsid (cap) genes, and a nucleic acid encoding helper virus protein sequences. The packaging cell, in a further embodiment, is a HEK293 packaging cell.

[0240] The AAV rep and cap genes may be from any AAV serotype and may be the same or different from that of the recombinant AAV vector ITRs including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74,AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In some embodiments, the AAV particles described herein comprise AAV rep and cap genes derived from AAV2 and AAV9, respectively.

[0241] AAV particles can be produced, in one embodiment, by one of the methods described in U. S. Patent Application Publication No. 2021 / 0317474, the contents of which are incorporated by reference in their entirety. In various methods of recombinant AAV particles disclosed herein, AAV particles may be produced in packaging cell lines used to produce viral vectors, such as, immortalized human embryonic kidney 293 (HEK293) cell line and Sf9 insect cell lines.

[0242] In one embodiment, the present disclosure provides a method for manufacturing an AAV particle described herein, comprising, (i) culturing adherent cells, (ii) transfecting the adherent cells with plasmids for less than 60 minutes to enable production of the AAV particle, and optionally applying further processing, e.g., purification, formulation and filling steps to produce a finished product.

[0243] Production of AAV particles typically requires the presence of three elements in the packaging cell: (1) a vector genome comprising 5’ and 3’ AAV inverted terminal repeat (ITR) sequences, (2) AAV replication (rep) and capsid (cap) genes, and (3) nucleic acid encoding helper virus protein sequences. In one embodiment, a host cell, e.g., a HEK293 cell, is transfected with three plasmids encoding the aforementioned three components to produce one of the AAV particles described herein.

[0244] As such, in one aspect of the disclosure, a packaging cell is provided comprising a plasmid comprising one of the vector genomes described herein. The packaging cell, in one embodiment, comprises a vector genome comprising a nucleic acid sequence in Table 1 or Table 2.

[0245] In one embodiment, the packaging cell comprises a plasmid comprising AAV replication (rep) and capsid (cap) gene sequences. In even a further embodiment, the packaging cell comprises a plasmid encoding helper AAV protein sequences. The packaging cell in one embodiment, is aHEK293 cell.

[0246] The plasmid comprising the vector genome is a plasmid in the packaging cell that encodes the information for a recombinant AAV DNA vector genome that transcribe a fragment of the mid-length dystrophin coding sequence under the control of the chosen promoter and in some embodiments, an enhancer element. The plasmid contains the vectorgenome components described herein. In embodiments described herein, the 5’ ITR is modified to preferentially package self-complementary AAV genomes into AAV capsids. Together, the regions between and including the ITRs are packaged into recombinant AAV capsids during the manufacture of the AAV particle. Plasmid components that are not intended for packaging into encapsidated vector genomes, in one embodiment, include an open reading frame encoding resistance to kanamycin (KanR) and an origin of replication (ori).

[0247] The second plasmid includes the rep and cap open reading frames. Rep and cap encode viral replication and capsid proteins, respectively. In the production of recombinant adeno-associated viral particles; the viral ITRs are the only elements used in cis while the viral rep and cap open reading frames are supplied in trans. Using the transfection of packaging cells to make AAV particles addresses the cis / trans roles for the different genetic elements by dividing them to separate plasmids.

[0248] The helper plasmid (also referred to as “pHELP,” component (3), above) contains the Trans acting adenoviral components necessary for recombinant adeno-associated virus production. The pHELP plasmid contains the regions of the adenovirus genome that provide factors that are important for AAV replication, namely E2A, E4, and VA RNA. The adenovirus El functions involved in AVV replication are provided by the transfection host cells. The pHELP plasmid does not, however, contain other adenovirus replication or structural genes. The adenovirus sequences do not contain the cis elements critical for replication, such as the inverted terminal repeats. Therefore, no infectious adenovirus is expected to be generated from such a production system.

[0249] In some embodiments, the step of producing the AAV particle further comprises a step of collecting the AAV particle from the cell. The AAV particle can be collected from the medium and / or by lysing the cells. The vector genome can be provided to the cell using any method known in the art, for example, a non-viral (e.g., plasmid) or viral vector. In some embodiments, the vector genome is supplied by a herpesvirus, an adenovirus vector, a baculovirus vector, or an Epstein-Barr virus (EBV)-derived vector.

[0250] The AAV rep and cap genes, and / or the helper virus protein sequences are provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell). For example, the AAV rep and cap genes and / or the helper virus protein sequences may be provided by a hybrid adenovirus, a hybrid herpesvirus vector (e.g., hybrid herpes simplex virus type I (HSV-1) vector), an Epstein-Barr virus (EBV)-derived vector, abaculovirus vector, or a non-infectious adenovirus mini-plasmid. In some embodiments, helper virus functions may be provided by the packaging cell, which has helper sequences embedded in the chromosome, or maintained as a stable extrachromosomal element.

[0251] Transfection can be performed using techniques known in the art, including but not limited to electroporation, lipofection, e.g., with lipofectamine, cationic polymers and cationic lipids. Any suitable transfection media may be used. In one embodiment, adherent human embryonic kidney (HEK293) cells are transfected with a triple DNA plasmid polyethylenimine (PEI) co-precipitation. In one embodiment, an AAV particle described herein is produced using triple DNA plasmid transfection into adherent cells using a PEI coprecipitation. In one embodiment, the DMEM growth medium used for cell expansion is replaced with a modified DMEM transfection media. This media is formulated without calcium and L-glutamine. In one embodiment, the transfection media is DMEM with no FBS, no calcium, no L-glutamine and 4.5 g / L glucose. In some embodiments, transfection media without serum (e.g., without FBS) improves transfection efficacy. In an embodiment, the transfection media is OptiMEM (Invitrogen / Thermo Fisher). In one embodiment, the three plasmids described herein are mixed together with PEI in transfection media and allowed to react. In some embodiments, the three plasmids are mixed together in about 1:1:1 molar ratio. In one embodiment, the plasmids and PEI are mixed in a ratio of 1:1 by weight of DNA: PEI. In one embodiment, the plasmids and PEI are mixed in a ratio of less than 1: 1 by weight of DNA: PEI.

[0252] In some embodiments, AAV particles described herein can be harvested from packaging cells and purified by methods standard in the art (e.g., Clark et al, Hum. Gene Ther., 10(6): 1031-1039 (1999); Schnepp and Clark, Methods Mol. Med., 69 427-443 (2002); U. S. Patent No. 6,566,118 and WO 98 / 09657, incorporated herein in their entirety by reference) such as by cesium chloride ultracentrifugation gradient or column chromatography. In one embodiment, a harvesting or purification step provided in U. S. Patent Application Publication No. 2021 / 0317474 is used to harvest or purify one of the AAV particles described herein. The contents of U. S. Patent Application Publication No. 2021 / 0317474 are incorporated herein in their entirety.Pharmaceutical Compositions

[0253] One aspect of the disclosure is directed to a pharmaceutical composition comprising the dual AAV particles according to the disclosure, and a pharmaceutically acceptable carrier, excipient, diluent or buffer. By “pharmaceutically acceptable” it is meant a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject withoutcausing any undesirable biological effects. In some embodiments, the pharmaceutical composition further comprises an adjuvant or stabilizing agent.

[0254] In some embodiments, the pharmaceutical composition comprising the dual AAV particle composition disclosed herein is formulated for intrathecal administration. In some embodiments, the pharmaceutical composition comprising the dual AAV particle composition disclosed herein is formulated for intravenous administration. In some embodiments, the pharmaceutical composition comprising the dual AAV particle composition disclosed herein is formulated for intramuscular administration. In some embodiments, the pharmaceutical composition comprising the dual AAV particle composition disclosed herein is formulated for intracerebroventricular (ICV) administration.

[0255] In some embodiments, the pharmaceutical compositions provided herein comprise sterile aqueous and non-aqueous injection solutions, which are optionally isotonic with the blood of the subject to whom the pharmaceutical composition is to be delivered. Pharmaceutical compositions can contain antioxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended subject to be administered. Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol; polyethylene glycol; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In some embodiments pharmaceutical compositions comprise pharmaceutically acceptable vehicles and can include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0256] In some embodiments, pharmaceutical compositions can be presented in unit / dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.REJ Efficiency

[0257] The dual AAV particle composition described herein allows for the efficient RNA recombination between individual fragments. Achieving efficient recombination between multiple RNA molecules allows for packaging and delivery of transgenes into AAVs, which exceed the packaging limit of a single AAV. AAV packaging limits represent a major hurdlefor gene therapy approaches for diseases caused by the absence / defect of large genes. One application of the AAV-REJ system described herein is expression of large disease-causing genes (e.g., dystrophin or mid-length dystrophin) using viral vectors with restricted packaging capacity.

[0258] In some embodiments, reconstitution (i.e., splicing or recombination) efficiency achieved using the compositions of the disclosure is determined using any suitable method known to one of skill in the art. In some embodiments, reconstitution efficiency is represented by a measure of correctly joined RNA relative to a control RNA, or a measure of mid-length dystrophin protein or protein activity relative to that of a control protein. In some embodiments, the control RNA is the unjoined RNA, wherein reconstitution efficiency is represented by a measure of joined RNA relative to unjoined RNA. This measurement can be made by detecting and comparing junction RNA and the unjoined C-terminal RNA species (e.g., junction RNA: unjoined C-terminal RNA). In some embodiments, reconstitution efficiency is represented by a measure of mid-length dystrophin or protein activity relative to a protein fragment or inactive protein.

[0259] The measurements of RNA or protein used to determine recombination efficiency or production level can be made by any suitable method known to those of skill in the art. In some embodiments, recombination efficiency or production level is determined by measuring an amount of functional mid-length dystrophin protein expressed, for example by Western blotting. In some embodiments, recombination efficiency or production level is determined by measuring the RNA transcript, for example using two probe based quantitative real-time PCR. For example, the first assay spans a sequence fully contained in the 3’ exonic coding sequence (labelled 3’ probe). The second assay spans the junction between the 5’ and the 3’ exonic coding sequence (labelled junction probe). Reconstitution efficiency can be calculated as the ratio of (junction probe count) / (3’ probe count). “Reconstitution efficiency,” “recombination efficiency,” and “splicing efficiency” are used interchangeably herein.

[0260] In some embodiments, the reconstitution, recombination or splicing efficiency (a measure of the correct joining of the two different coding sequences present on different vector genome relative to a control RNA, and / or the production of the desired mid-length dystrophin protein relative to a control protein) is about 10% to about 200%. In some embodiments, the reconstitution efficiency is about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about90%, about 10% to about 100%, about 10% to about 110%, about 10% to about 120%, about 10% to about 130%, about 10% to about 140%, about 10% to about 150%, about 10% to about 160%, about 10% to about 170%, about 10% to about 180%, about 10% to about 190%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 15% to about 110%, about 15% to about 120%, about 15% to about 130%, about 15% to about 140%, about 15% to about 150%, about 15% to about 160%, about 15% to about 170%, about 15% to about 180%, about 15% to about 190%, about 15% to about 200%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 20% to about 110%, about 20% to about 120%, about 20% to about 130%, about 20% to about 140%, about 20% to about 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 200%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 100%, about 25% to about 110%, about 25% to about 120%, about 25% to about 130%, about 25% to about 140%, about 25% to about 150%, about 25% to about 160%, about 25% to about 170%, about 25% to about 180%, about 25% to about 190%, about 25% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 110%, about 30% to about 120%, about 30% to about 130%, about 30% to about 140%, about 30% to about 150%, about 30% to about 160%, about 30% to about 170%, about 30% to about 180%, about 30% to about 190%, about 30% to about 200%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%. In some embodiments, the reconstitution efficiency is about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments,the reconstitution efficiency is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the reconstitution efficiency is at most about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%.

[0261] In some embodiments, the dual AAV particle compositions described herein are evaluated by determining an RNA production level using any suitable method known to one of skill in the art. In some embodiments, the RNA production level is represented by a measure of correctly joined RNA relative to a control RNA. In some embodiments, the control RNA is a corresponding mutant RNA or an endogenous RNA. For example, the ratio of the amount of joined RNA to the amount of mutant or endogenous RNA produced in the transfected cell is compared with same ratio in non-transfected cells, to determine the production level of the correctly joined RNA. In some embodiments, the control RNA is the corresponding endogenous RNA produced in a non-transfected cell from a normal subject that expresses the corresponding full-length protein, and the RNA production level is determined by measuring the amount of the joined RNA in the transfected cell and comparing it to that of the control RNA in a non-transfected cell from a normal subject.

[0262] In some embodiments, the RNA production level (i.e., a measure of the amount of correctly joined RNA relative to that of a control RNA) achieved is about 5% to about 200%. In some embodiments, the RNA production level achieved is about 5% to about 10%, about 5% to about 20%, about 5% to about 25%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 110%, about 10% to about 120%, about 10% to about 130%, about 10% to about 140%, about 10% to about 150%, about 10% to about 160%, about 10% to about 170%, about 10% to about 180%, about 10% to about 190%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 15% to about 110%, about 15% to about 120%, about 15% to about 130%, about 15% to about 140%, about 15% to about 150%, about 15% to about 160%, about 15% to about 170%, about 15% to about 180%, about 15% to about 190%, about 15% to about 200%, about20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 20% to about 110%, about 20% to about 120%, about 20% to about 130%, about 20% to about 140%, about 20% to about 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 200%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 100%, about 25% to about 110%, about 25% to about 120%, about 25% to about 130%, about 25% to about 140%, about 25% to about 150%, about 25% to about 160%, about 25% to about 170%, about 25% to about 180%, about 25% to about 190%, about 25% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 110%, about 30% to about 120%, about 30% to about 130%, about 30% to about 140%, about 30% to about 150%, about 30% to about 160%, about 30% to about 170%, about 30% to about 180%, about 30% to about 190%, about 30% to about 200%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%. In some embodiments, the RNA production level achieved is about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the RNA production level achieved is at least about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the RNA production level achieved is at most about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%.

[0263] In some embodiments, the protein expression level is represented by a measure of the amount of mid-length dystrophin protein or protein activity relative to that of a control protein. In some embodiments, the control protein is a corresponding mutant protein or an endogenous protein (e.g., dystrophin). For example, the ratio of the amount of mid-length dystrophin protein or protein activity to the amount of mutant or endogenous protein produced in the transfected cell is compared with same ratio in non-transfected cells. In some embodiments, the control protein is the full-length dystrophin protein produced in, e.g., a cell that is engineered to express a control full-length dystrophin protein (wherein the cell is not transfected with the inventive constructs) or a non-transfected cell from a normal subject that expresses a control full-length dystrophin protein, and the protein expression level is determined by measuring the amount or activity of the mid-length dystrophin protein in the transfected cell and comparing it to that of the control protein. In some embodiments, the control protein is a mutant form of the protein, produced in a cell that is transfected or non-transfected with the vector, and the amount of mid-length dystrophin protein or protein activity is compared with that of the control protein to determine the protein expression level. In some embodiments, the amount of mid-length dystrophin protein or protein activity is compared with that of an endogenous, or housekeeping, protein to determine the protein production level.

[0264] In some embodiments, the protein expression level (i.e., a measure of the amount of mid-length dystrophin protein or protein activity relative to that of a control protein) achieved is about 1% to about 200%. In some embodiments, the mid-length dystrophin protein expression level achieved is about 10% to about 200%. In some embodiments, the mid-length dystrophin protein expression level achieved is about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 100%, about 10% to about 110%, about 10% to about 120%, about 10% to about 130%, about 10% to about 140%, about 10% to about 150%, about 10% to about 160%, about 10% to about 170%, about 10% to about 180%, about 10% to about 190%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 15% to about 110%, about 15% to about 120%, about 15% to about 130%, about 15% to about 140%, about 15% to about 150%, about 15% to about 160%, about 15% to about 170%, about 15% to about 180%, about 15% to about 190%, about 15% to about 200%, about20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 20% to about 110%, about 20% to about 120%, about 20% to about 130%, about 20% to about 140%, about 20% to about 150%, about 20% to about 160%, about 20% to about 170%, about 20% to about 180%, about 20% to about 190%, about 20% to about 200%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 100%, about 25% to about 110%, about 25% to about 120%, about 25% to about 130%, about 25% to about 140%, about 25% to about 150%, about 25% to about 160%, about 25% to about 170%, about 25% to about 180%, about 25% to about 190%, about 25% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 110%, about 30% to about 120%, about 30% to about 130%, about 30% to about 140%, about 30% to about 150%, about 30% to about 160%, about 30% to about 170%, about 30% to about 180%, about 30% to about 190%, about 30% to about 200%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%.. In some embodiments, the mid-length dystrophin protein expression level achieved is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the mid-length dystrophin protein expression level achieved is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%. In some embodiments, the mid-length dystrophin protein expression level achieved is at most about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200%.

[0265] In some embodiments, the mid-length dystrophin protein activity level (i.e. a measure of protein activity of mid-length dystrophin protein relative to that of a control protein) achieved is about 50% to about 200%. In some embodiments, the mid-length dystrophin protein activity level achieved is about 50% to about 200%. In some embodiments, the mid-length dystrophin protein activity level achieved is about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about 130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 55% to about 60%, about 55% to about 65%, about 55% to about 70%, about 55% to about 75%, about 55% to about 80%, about 55% to about 85%, about 55% to about 90%, about 55% to about 95%, about 55% to about 100%, about 55% to about 110%, about 55% to about 120%, about 55% to about 130%, about 55% to about 140%, about 55% to about 150%, about 55% to about 160%, about 55% to about 170%, about 55% to about 180%, about 55% to about 190%, about 55% to about 200%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 100%, about 65% to about 110%, about 65% to about 120%, about 65% to about 130%, about 65% to about 140%, about 65% to about 150%, about 65% to about 160%, about 65% to about 170%, about 65% to about 180%, about 65% to about 190%, about 65% to about 200%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 100%, about 75% to about 110%, about 75% to about 120%, about 75% to about 130%, about 75% to about 140%, about 75% to about 150%, about 75% to about 160%, about 75% to about 170%, about 75% to about 180%, about 75% to about190%, about 75% to about 200%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 85% to about 90%, about 85% to about 95%, about 85% to about 100%, about 85% to about 110%, about 85% to about 120%, about 85% to about 130%, about 85% to about 140%, about 85% to about 150%, about 85% to about 160%, about 85% to about 170%, about 85% to about 180%, about 85% to about 190%, about 85% to about 200%, about 90% to about 95%, about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90% to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 95% to about 100%, about 95% to about 110%, about 95% to about 120%, about 95% to about 130%, about 95% to about 140%, about 95% to about 150%, about 95% to about 160%, about 95% to about 170%, about 95% to about 180%, about 95% to about 190%, about 95% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200%. In some embodiments, the mid-length dystrophin protein activity level achieved is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%. In some embodiments, the midlength dystrophin protein activity level achieved is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the mid-length dystrophin protein activity level achieved is at most about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%.

[0266] In some embodiments, the amount of correctly joined RNA or mid-length dystrophin protein produced in a cell is sufficient to ameliorate or cure a condition or disease in a subject, as understood by one of skill in the art for the particular condition or disease. In some embodiments, the amount of correctly joined RNA or mid-length dystrophin protein produced in a cell is an effective amount. In some embodiments, this amount is equivalent to about 50% to 200% the amount of the RNA or dystrophin protein produced in a normal cell. In some embodiments, this amount is equivalent to about 40% to about 200% the amount of the RNA or dystrophin protein produced in a normal cell. In some embodiments, this amount is equivalent to about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 100%, about 40% to about 110%, about 40% to about 120%, about 40% to about 130%, about 40% to about 140%, about 40% to about 150%, about 40% to about 160%, about 40% to about 170%, about 40% to about 180%, about 40% to about 190%, about 40% to about 200%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 45% to about 65%, about 45% to about 70%, about 45% to about 75%, about 45% to about 80%, about 45% to about 85%, about 45% to about 90%, about 45% to about 100%, about 45% to about 110%, about 45% to about 120%, about 45% to about 130%, about 45% to about 140%, about 45% to about 150%, about 45% to about 160%, about 45% to about 170%, about 45% to about 180%, about 45% to about 190%, about 45% to about 200%, about 50% to about 55%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 100%, about 50% to about 110%, about 50% to about 120%, about 50% to about130%, about 50% to about 140%, about 50% to about 150%, about 50% to about 160%, about 50% to about 170%, about 50% to about 180%, about 50% to about 190%, about 50% to about 200%, about 55% to about 60%, about 55% to about 65%, about 55% to about 70%, about 55% to about 75%, about 55% to about 80%, about 55% to about 85%, about 55% to about 90%, about 55% to about 100%, about 55% to about 110%, about 55% to about 120%, about 55% to about 130%, about 55% to about 140%, about 55% to about 150%, about 55% to about 160%, about 55% to about 170%, about 55% to about 180%, about 55% to about 190%, about 55% to about 200%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 100%, about 60% to about 110%, about 60% to about 120%, about 60% to about 130%, about 60% to about 140%, about 60% to about 150%, about 60% to about 160%, about 60% to about 170%, about 60% to about 180%, about 60% to about 190%, about 60% to about 200%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 100%, about 65% to about 110%, about 65% to about 120%, about 65% to about 130%, about 65% to about 140%, about 65% to about 150%, about 65% to about 160%, about 65% to about 170%, about 65% to about 180%, about 65% to about 190%, about 65% to about 200%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 100%, about 70% to about 110%, about 70% to about 120%, about 70% to about 130%, about 70% to about 140%, about 70% to about 150%, about 70% to about 160%, about 70% to about 170%, about 70% to about 180%, about 70% to about 190%, about 70% to about 200%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 100%, about 75% to about 110%, about 75% to about 120%, about 75% to about 130%, about 75% to about 140%, about 75% to about 150%, about 75% to about 160%, about 75% to about 170%, about 75% to about 180%, about 75% to about 190%, about 75% to about 200%, about 80% to about 85%, about 80% to about 90%, about 80% to about 100%, about 80% to about 110%, about 80% to about 120%, about 80% to about 130%, about 80% to about 140%, about 80% to about 150%, about 80% to about 160%, about 80% to about 170%, about 80% to about 180%, about 80% to about 190%, about 80% to about 200%, about 85% to about 90%, about 85% to about 100%, about 85% to about 110%, about 85% to about 120%, about 85% to about 130%, about 85% to about 140%, about 85% to about 150%, about 85% to about 160%, about 85% to about 170%, about 85% to about 180%, about 85% to about 190%, about 85% to about 200%, or about 90% to about 100%, about 90% to about 110%, about 90% to about 120%, about 90% to about 130%, about 90% to about 140%, about 90% to about 150%, about 90%to about 160%, about 90% to about 170%, about 90% to about 180%, about 90% to about 190%, about 90% to about 200%, about 95% to about 100%, about 95% to about 110%, about 95% to about 120%, about 95% to about 130%, about 95% to about 140%, about 95% to about 150%, about 95% to about 160%, about 95% to about 170%, about 95% to about 180%, about 95% to about 190%, about 95% to about 200%, about 100% to about 110%, about 100% to about 120%, about 100% to about 130%, about 100% to about 140%, about 100% to about 150%, about 100% to about 160%, about 100% to about 170%, about 100% to about 180%, about 100% to about 190%, about 100% to about 200%, about 110% to about 120%, about 110% to about 130%, about 110% to about 140%, about 110% to about 150%, about 110% to about 160%, about 110% to about 170%, about 110% to about 180%, about 110% to about 190%, about 110% to about 200%, about 120% to about 130%, about 120% to about 140%, about 120% to about 150%, about 120% to about 160%, about 120% to about 170%, about 120% to about 180%, about 120% to about 190%, about 120% to about 200%, about 130% to about 140%, about 130% to about 150%, about 130% to about 160%, about 130% to about 170%, about 130% to about 180%, about 130% to about 190%, about 130% to about 200%, about 140% to about 150%, about 140% to about 160%, about 140% to about 170%, about 140% to about 180%, about 140% to about 190%, about 140% to about 200%, about 150% to about 160%, about 150% to about 170%, about 150% to about 180%, about 150% to about 190%, about 150% to about 200%, about 160% to about 170%, about 160% to about 180%, about 160% to about 190%, about 160% to about 200%, about 170% to about 180%, about 170% to about 190%, about 170% to about 200%, about 180% to about 190%, about 180% to about 200%, or about 190% to about 200% the amount of the RNA or dystrophin protein produced in a normal cell. In some embodiments, this amount is equivalent to about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200% the amount of the RNA or dystrophin protein produced in a normal cell. In some embodiments, this amount is equivalent to about at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% the amount of the RNA or dystrophin protein produced in a normal cell. In some embodiments, this amount is equivalent to about at most about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 100%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200% the amount of the RNA or dystrophin protein produced in a normal cell.

[0267] The present disclosure relates in part to a method of treating a dystrophinopathy in a subject in need thereof, comprising administering to the subject an effective amount of a dual AAV particle composition described herein or a pharmaceutical composition comprising the same. In some embodiments, the dual AAV particle composition is used to treat a dystrophinopathy including, but not limited to, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy (DCM). In one preferred embodiment, the dual AAV particle composition encodes a mid-length dystrophin protein for the treatment of a dystrophinopathy, such as DMD.

[0268] In some embodiments, the methods of the present disclosure deliver an effective amount of a dual AAV particle composition encoding mid-length dystrophin protein to cardiac and / or skeletal muscle of the subject, for example, to treat dystrophinopathy, such as DMD, Becker muscular dystrophy, or DCM. In some embodiments, subsequent to administration with an effective amount of the dual AAV composition to the subject, the mid-length dystrophin protein is expressed at higher levels in skeletal and / or cardiac muscle compared to the expression level of mid-length dystrophin protein in liver tissue.

[0269] In some embodiments, the subject in need of treatment, e.g., a subject with DMD, is administered an effective amount of the dual AAV particle composition comprising a first AAV particle comprising an AAV capsid encapsidating a first transgene and a second AAV particle encapsidating a second transgene. In some embodiments, a first transcript of the first transgene and a second transcript of the second transgene are spliced and joined to form an RNA coding sequence that encodes a mid-length dystrophin protein. In some embodiments, the mid-length dystrophin protein comprises (i) an N-terminal region; (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, a spectrin-like repeat 21, a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24, and a hinge region 4; (iii) a cysteine-rich domain; and (iv) a C-terminal domain. In some embodiments, the RNA sequence encoding the mid-length dystrophin protein comprises a nucleic acid sequence of SEQ ID NO: 22. In another embodiment, the mid-length dystrophin comprises the amino acid sequence set forth in SEQ ID NO: 23. In someembodiments, the subject is intrathecally administered the dual AAV composition. In some embodiments, the subject is administered the dual AAV composition in a single dose.

[0270] According to the embodiments described herein, coding sequence expression may refer to gene expression (i.e., by measuring mRNA levels) or expression of the corresponding protein. It will be understood by those of ordinary skill in the art that in order to determine levels of coding sequence expression in different tissue types, substantially the same amount of tissue, or substantially the same number of cells should be compared for gene expression levels. In some embodiments, the level of mid-length dystrophin expression is measured about 1 week, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months or about 24 months following administration of the dual AAV particle composition. In some embodiments, the level of mid-length dystrophin expression in the skeletal and / or cardiac muscle is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% or at least about 80% higher compared to the amount of mid-length dystrophin expression in the liver tissue. In a further embodiment, the mid-length dystrophin comprises the RNA coding sequence set for in SEQ ID NO: 22. In another embodiment, the mid-length dystrophin comprises the protein coding sequence set forth in SEQ ID NO: 23.

[0271] In some embodiments, the subject is a human. In some embodiments, the subject is a neonate. In some embodiments, the subject is a human and the subject is an infant. In some embodiments, the subject is a human and the subject is an adolescent. In some embodiments, the subject is a human and the subject is an adult.

[0272] In some embodiments, the subject is human. In some embodiments, the human subject is from about 4 years old to about 7 years old, a newborn, about 1 year to about 7 years old, about 2 years to about 7 years old, about 2 years to about 6 years old, about 2 years to about 5 years old, about 2 years to about 4 years old, about 3 years to about 7 years old, about 3 years to about 6 years old, about 1 month to about 6 years old, about 1 month to about 5 years old, about 1 month to about 4 years old, about 1 month to about 3 years old, about 1 month to about 2 years old, or about 1 month to about 12 months old. In some embodiments, the subject is a male human subject.

[0273] In some embodiments, the subject is a male human subject from about 4 years old to about 7 years old. In some embodiments, the subject is a male human subject from about 3years old to about 7 years old. In some embodiments, the subject is a male human subject from about 2 years old to about 7 years old.

[0274] In some embodiments, treating a subject with the dual AAV particle compositions described herein decreases the number of symptoms, or reduces the severity of one or more symptoms in the subject being treated, compared to the symptoms exhibited prior to treatment. In one embodiment, treating comprises reducing the severity of one or more dystrophinopathy symptoms in a subject compared to the severity of one or more symptoms in the subject prior to treatment. Such symptoms may include but are not limited to: increasing mobility (e.g., walking or climbing), reducing skeletal muscle weakness, reducing muscle pain, reducing muscle stiffness, improving cognitive ability, reducing calf muscle size, reducing cardiomyopathy, improving vision, improving hearing, improving blood clotting, or improving respiratory function.

[0275] In some embodiments, treating a subject with the dual AAV particle compositions described herein comprises decreasing serum creatine kinase (CK) levels in a subject compared to the serum CK levels prior to treatment. In some embodiments, the serum CK levels are decreased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more compared to serum CK levels prior to treatment with the dual AAV particle composition described herein. In a further embodiment, serum CK levels are assessed prior to treatment with a dual AAV particle composition and at about 12 months, about 18 months, about 24 months, or about 30 months subsequent to administration of the dual AAV particle composition. In some embodiments, the dual AAV particle composition encodes a mid-length dystrophin protein.

[0276] In some embodiments, the dual AAV particle composition is administered once or multiple times to a subject in need of treatment, e.g., a subject with DMD. In some embodiments, the dual AAV particle composition is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more to a subject in need of treatment, e.g., a subject with DMD. In some embodiments, the dual AAV particle composition is administered once to a subject in need of treatment, e.g., a subject with DMD.

[0277] In some embodiments, the dual AAV particle composition is administered intravenously to a subject with a dystrophinopathy. In some embodiments, the dual AAV particle composition is administered intramuscularly to a subject with a dystrophinopathy. In some embodiments, the dual AAV particle composition is administered subcutaneously to asubject with a dystrophinopathy. In some embodiments, the dual AAV particle composition is administered intraperitoneally to a subject with a dystrophinopathy. Other administration routes may also be selected (e.g., intranasal, intratracheal, intraarterial, or oral). In some embodiments, the dystrophinopathy is DMD.

[0278] In some embodiments, the dual AAV particle composition is administered intrathecally to a subject with a dystrophinopathy. In a further embodiment, the subject is positioned in the Trendelenburg position for intrathecal administration of the dual AAV particle composition. In some embodiments, the dual AAV particle composition is administered intracerebroventricularly to a subject with a dystrophinopathy.

[0279] In some embodiments, treatment with the dual AAV particle compositions described herein results in an increase in the subject’s score from baseline (i.e., prior to treatment) in the NorthStar Ambulatory Assessment (NSAA). The NSAA is a 17-item rating scale that is used to measure functional motor abilities in ambulatory DMD subjects. The scale is ordinal with 34 as the maximum score indicating fully independent function. Each activity is graded as either a 0 (unable to achieve independently), 1 (modified method but achieves goal independent of physical assistance from another), or 2 (normal - no obvious modification of activity). See, e.g., Mazonne et al. (2009). Neuromuscular Disorders 19, pp. 458-461 and researchrom.com / masterlist / view / 18#form2, the disclosure of each of which is incorporated by reference in its entirety for all purposes. The change from baseline, in one embodiment, is measured 12 months subsequent to administration of the dual AAV particle composition. In another embodiment, the change from baseline is measured 18 months subsequent to administration of the dual AAV particle composition. In another embodiment, the change from baseline is measure 24 months subsequent to administration of the dual AAV particle composition. In even another embodiment, the subject’s increased score from baseline in the NSAA as measured 12 months subsequent to administration is substantially unchanged or increased at 18 months subsequent to administration. In yet even another embodiment, the subject’s increased score from baseline in the NSAA as measured 12 months subsequent to administration is substantially unchanged or increased at 24 months subsequent to administration of the dual AAV particle composition. In yet even another embodiment, the subject’s increased score from baseline in the NSAA as measured 12 months subsequent to administration is substantially unchanged or increased at 60 months subsequent to administration of the dual AAV particle composition.

[0280] Increasing the NSAA score, in one embodiment, comprises increasing the NSAA score by from about 5 to about 25, from about 5 to about 20, from about 5 to about 15 or from about 5 to about 10. In another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 2 to about 12 points. In another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 2 to about 10 points. In yet another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 3 to about 10 points. In even another embodiment, increasing the NSAA score comprises increasing the score by from about 4 to about 10 points. In yet even another embodiment, increasing the NSAA score comprises increasing the score by from about 2 to about 8 points. In another embodiment, increasing the NSAA score, comprises increasing the NSAA score by from about 2 to about 6 points.

[0281] In some embodiments, treating with the dual AAV particle compositions described herein comprises increasing the number of meters walked in a 6-minute walk test (6MWT) compared to the number of meters walked prior to treatment. In some embodiments, the baseline number of meters walked by the subject in the 6MWT is measured prior to the subject undergoing treatment with the dual AAV particle composition described herein. In some embodiments, the 6MWT is assessed 6 months after administration of the dual AAV particle composition. In some embodiments, the 6MWT is assessed 12 months after administration of the dual AAV particle composition. In some embodiments, the 6MWT is assessed 18 months after administration of the dual AAV particle composition. In some embodiments, the 6MWT is measured 24 months after administration of the dual AAV particle composition. In some embodiments, the 6MWT is measured 36 months after administration of the dual AAV particle composition. In some embodiments, the 6MWT is measured 48 months after administration of the dual AAV particle composition. In some embodiments, the 6MWT is measured 60 months after administration of the dual AAV particle composition.

[0282] In some embodiments, increasing the number of meters walked by the subject in the 6MWT compared to the number of meters walked by the subject prior to treatment comprises increasing by from about 5 meters to about 50 meters, about 5 meters to about 45 meters, about 5 meters to about 40 meters, about 5 meters to about 35 meters, about 5 meters to about 30 meters, about 5 meters to about 25 meters, about 5 meters to about 20 meters, about 5 to about 15 meters, or about 5 meters to about 10 meters.

[0283] In some embodiments, treating comprises decreasing the time to complete a 10-meter walk / run test (10MWR) for the subject, compared to the time to complete a 10-meter walk / runfor the subject prior to administration of the composition (i.e., a baseline time). The 10MWR covers a flat, 10-meter walkway marked with a start and finish. On “go” or “start,” the subject is timed as the subject walks or runs as fast and safely as possible from the start to the finish line. The time to complete the 10 meters is then measured. In some embodiments, the 10MWR is assessed 6 months after administration of the dual AAV particle composition to assess whether treating was successful. In some embodiments, the 10MWR is assessed 12 months after administration of the dual AAV particle composition. In some embodiments, the 10MWR is assessed 18 months after administration of the dual AAV particle composition. In some embodiments, the 10MWR is measured 24 months after administration of the dual AAV particle composition. In some embodiments, the 10MWR is measured 36 months after administration of the dual AAV particle composition. In some embodiments, the 10MWR is measured 48 months after administration of the dual AAV particle composition. In some embodiments, the 10MWR is measured 60 months after administration of the dual AAV particle composition.

[0284] In some embodiments, treating comprises decreasing the time for the subject to finish the 10MWR compared to the time for the subject to finish the 10MWR prior to treatment by about 0.5 seconds to about 4 seconds, by about 0.5 seconds to about 3.5 seconds, by about 0.5 seconds to about 3 seconds, by about 0.5 seconds to about 2.5 seconds, by about 0.5 seconds to about 2 seconds, by about 0.5 seconds to about 1.5 seconds, or by about 0.5 seconds to about 1 second.

[0285] In some embodiments of the methods described herein, treating comprises decreasing the time to complete a 100-meter walk / run test (100MWR) for the subject, compared to the time to complete a 100MWR prior to administration of the dual particle composition. The 100MWR covers a flat, 100-meter walkway marked with a start and finish. On “go” or “start,” the subject is timed as the subject walks or runs as fast and safely as possible from the start to the finish line. The time to complete the 100 meters is then measured. In some embodiments, the 100MWR is measured 6 months subsequent to the administration of the dual AAV particle composition. In some embodiments, the 100MWR is assessed 12 months after administration of the dual AAV particle composition. In some embodiments, the 100MWR is assessed 18 months after administration of the dual AAV particle composition. In some embodiments, the 100MWR is measured 24 months after administration of the dual AAV particle composition. In some embodiments, the 100MWR is measured 36 months after administration of the dual AAV particle composition. In some embodiments, the 100MWR is measured 48 months afteradministration of the dual AAV particle composition. In some embodiments, the 100MWR is measured 60 months after administration of the dual AAV particle composition.

[0286] In some embodiments, treating comprises decreasing the time to finish the 100MWR for the subject by about 1 second to about 20 seconds, by about 1 second to about 15 seconds, by about 1 second to about 10 seconds, by about 1 second to about 8 seconds, by about 1 second to about 6 seconds, by about 1 second to about 4 seconds, or by about 1 second to about 2 seconds.

[0287] In some embodiments, treating comprises decreasing the time to complete the 4-stair climb test (4SC) for the subject, compared to a baseline time of the subject to complete the 4SC. In some embodiments, the baseline time for the subject to complete the 4SC is measured prior to the administration of the dual vector composition. The 4SC measures the time required for the subject to ascend 4 standard steps (typically each step is 6 inches in height) and is considered a reliable non DMD-specific measure of motor function in assessing dynamic balance, functional abilities, and falling risk in patients, particularly children, with DMD. The 4SC test also measures the velocity of the stair climb in the 4SC test in stairs / second (s / s). In some embodiments, the time to complete the 4SC or the velocity of the stair climb is measured about 6 months after the administration of the dual AAV particle composition about 12 months after the administration of the dual AAV particle composition, about 18 months after the administration of the dual AAV particle composition, about 24 months after the administration of the dual AAV particle composition, about 36 months after the administration of the dual AAV particle composition, about 48 months after the administration of the dual AAV particle composition, or about 60 months after the administration of the dual AAV particle composition.

[0288] In some embodiments, treating comprises decreasing the subject’s 4SC time compared to the subject’s 4SC time measured prior to the administration of the dual vector composition. In some embodiments, treating comprises decreasing the subject’s 4SC time subsequent to treatment by about 1 second to about 8 seconds, by about 1 second to about 7 seconds, by about 1 second to about 6 seconds, by about 1 second to about 5 seconds, by about 1 second to about 4 seconds, by about 1 second to about 3 seconds, or by about 1 second to about 2 seconds. In some embodiments, treating comprises increasing a velocity of the subject’s climb in the 4SC subsequent to treatment compared to the velocity of the subject’s climb in the 4SC prior to the administration of the dual vector composition. In some embodiments, treating comprises increasing the subject’s 4SC velocity subsequent to treatment by about 0.5 s / s to about 3.5 s / s, by about 0.5 s / s to by about 3 s / s, by about 0.5 s / s to about 2.5 s / s, by about 0.5 s / s to about 2s / s, by about 0.5 s / s to about 1.5 s / s, by about 0.5 s / s to about 1 s / s, as compared to the subject’s 4SC velocity prior to administration of the dual vector composition.

[0289] In some embodiments, treating comprises decreasing a time to stand (TTSTAND) for the subject, compared to a baseline TTSAND measured for the subject. In some embodiments, the baseline TTSTAND for the subject is measured prior to administration of the composition. In some embodiments, the TTSTAND is measured about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, or about 60 months after administration of the composition to assess whether the treating was successful. In some embodiments, treating comprises decreasing the TTSTAND for the subject by about 1 second to about 10 seconds, by 1 to about 9 seconds, by about 1 second to about 8 seconds, by about 1 second to about 7 seconds, by about 1 second to about 6 seconds, by about 1 second to about 5 seconds, by about 1 second to about 4 seconds, by about 1 second to about 3 seconds, or by about 1 second to about 2 seconds as compared to the TTSTAND for the subject prior to administration of the composition.

[0290] In some embodiments, treating comprises decreasing the TTSTAND for the subject by about 1 second to about 10 seconds, by 1 to about 9 seconds, by about 1 second to about 8 seconds, by about 1 second to about 7 seconds, by about 1 second to about 6 seconds, by about 1 second to about 5 seconds, by about 1 second to about 4 seconds, by about 1 second to about 3 seconds, or by about 1 second to about 2 seconds, as compared to a TTSTAND for the subject measured prior to administration of the dual vector composition.

[0291] The Neuromuscular Gross Motor Outcome (GRO) is a tool to quantify and assess whole body strength, motor development, and function for all levels of ability across the lifespan in those diagnosed with neuromuscular diseases across the span of abilities. GRO includes various physical assessments including, but not limited to, distal finger or hand movement, seated head control, sliding arm, anti-gravity elbow strength, anti-gravity, shoulder strength, grip strength, distal leg movement, supine reaching, hip strength, midline head control, head rotation, rolls to sidelying, independent rolling, turns head in prone, lifts head in prone, props on forearms, props on extended arms, supine to sitting, brief sitting, independent sitting, hands to head, rotates in sitting, bench sitting, bench sitting - lean forward, sitting to lying or quadruped, independent mobility, crawling, TTSTAND, TTSTAND (mature), kneeling to standing, standing balance, walks with support, independent walking, stands up from a chair, runs, 10MWR, single limb stance, tiptoes, tandem stance, tandem walk, touches floor, squat, jump, turning jump, long jump, hops, ascends 1 step (6-in or 15 cm step), descends 1 step (height ofstep: 6-in or 15 cm), 4SC, and 100MWR. Each of these assessments are scored on a 0-2 scale, with 0 indicating the inability to perform the physical task required by the assessment and 2 for successful completion of the physical task. By way of example, if there are 50 physical assessments under the GRO protocol, then the GRO cumulative score is out of 100 (cumulative 2 scores for each of the 50 assessments). In some embodiments, treatment with the dual AAV particle composition increases the GRO score of a subject, as compared to a GRO score for the subject measured prior to the administration of the dual vector composition.

[0292] In a preferred embodiment, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 1:1 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 2:1 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 1:2 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 3:1 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 1:3 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 4:1 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 1:4 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 5:1 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 1:5 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 10:1 ratio of N-terminal AAV particle to C-terminal AAV particle. In some embodiments, the pharmaceutical composition comprises an effective amount of dual AAV particles at a 1:10 ratio of N-terminal AAV particle to C-terminal AAV particle.

[0293] In some embodiments, the dose of a first or second particle of a dual AAV particle composition, perparticle, is about 1×109to about 1×1016vector genomes (vg), about 1×1010vg to about 1×1016vg, about 1×1011vg to about 1×1016vg, about 1×1012vg to about 1×1016vg, about 1×1013vg to about 1×1016vg, about 1×1014vg to about 1×1016vg, about 1×1015vg toabout 1×1016vg, about 1×109vg to about 1×1015vg, about 1×109vg to about 1×1014vg, about 1×109vg to about 1×1013vg, about 1×109vg to about 1×1012vg, about 1×109vg to about 1×1011vg, about 1×109vg to about 1×1010vg, about 1×1010vg to about 1×1013vg, about 1×1010vg to about 1×1012vg, about 2.5×1010vg to about 1×1013vg, about 3.0×1010vg to about 1×1013vg, about 6.0×1010vg to about 1×1013vg, about 7.5×1010vg to about 1×1013vg, about 1×1011vg to about 1×1013vg, about 1.2×1011vg to about 1×1013vg, about 2.5×1011to about 1×1013vg, about 5×1011to about 1×1013vg, about 7.5×1011vg to about 1×1013vg, about 1×1011vg to about 7.5×1012vg, about 2.5×1011vg to about 7.5×1012vg, about 3.0×1011vg to about 7.5×1012vg, about 5×1011vg to about 7.5×1012vg, about 7.5×1011vg to about 7.5×1012vg, about 1×1011vg to about 1×1012vg, about 2.5×1011vg to about 1×1012vg, about 3.0×1011vg to about 1×1012vg, about 5×1011vg to about 1×1012vg, about 7.5×1011vg to about 1×1012vg. The dual particle composition can be delivered as either a single or dual administrations, e.g., as a single injection or multiple injections. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about lxl012vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 5×1012vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 1×1013vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 5×1013vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 1×1014vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 5×1014vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 1×1015vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 5×1015vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 1×1016vg. In some embodiments, the dose of a first or second AAV particle of the dual AAV vector composition is about 5×1016vg. In some embodiments, the dual AAV vector composition is administered intravenously. In a preferred embodiment, the dual AAV vector composition is administered intrathecally. In some embodiments, the AAV particles are administered intracerebroventricularly.

[0294] In some embodiments, a dose is provided as vg per AAV vector. A “total dose” or “total vg dose”, as used herein, refers to the total number of vgs administered to a subject. The total number of vgs administered to a subject in embodiments described herein, comprises the sum of (i) the dose of a first AAV particle (a first AAV vector) and a (ii) the dose of a secondAAV particle (a second AAV vector). In preferred embodiments of the disclosure, the first AAV particle and the second AAV particle are administered at equal vg doses. As described in embodiments herein, the first AAV particle comprises a transgene comprising an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein and the second AAV particle comprises a C-terminal coding sequence encoding a C-terminal portion of a mid-length dystrophin protein (or vice versa). In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×109to about 1×1016vg per AAV vector, about 1×1010to about 1×1016vg per AAV vector, about 1×1011to about 1×1016vg per AAV vector, about 1 x 1012to about 1 x 1016vg per AAV vector, about 1 x 1013to about lxl016vg per AAV vector, about 1×1014to about 1×1016vg per AAV vector, about 1 x 1015to about IxlO16vg per AAV vector, about 1 x 109to about IxlO15vg per AAV vector, about 1×109to about 1×1014vg per AAV vector, about 1×109to about 1×1013vg per AAV vector, about 1×109to about 1×1012vg per AAV vector, about 1×109to about 1×1011vg per AAV vector, about 1×109to about 1×1010vg per AAV vector, about 1×1010to about 1×1015vg per AAV vector, about 1×1010to about 1×1014vg per AAV vector, about 1×1010to about 1×1013vg per AAV vector, about 1×1010to about 1×1012vg per AAV vector, about 1×1010to about 1×1011vg per AAV vector, about 1×1011to about 1×1015vg per AAV vector, 1×1011to about 1×1014vg per AAV vector, 1×1011to about 1×1013vg per AAV vector, 1×1011to about 1×1012vg per AAV vector, 1×1012to about 1×1015vg per AAV vector, 1×1012to about 1×1014vg per AAV vector, 1×1012to about 1×1013vg per AAV vector, 1×1013to about 1×1015vg per AAV vector, 1×1013to about 1×1014vg per AAV vector, about 2.5×1010to about 1×1013vg per AAV vector, about 3.0×1010to about 1×1013vg per AAV vector, about 6.0×1010to about 1×1013vg per AAV vector, about 7.5×1010to about 1×1013vg per AAV vector, about 1×1011to about 1×1013vg per AAV vector, about 1.2×1011to about 1×1013vg per AAV vector, about 2.5×1011to about 1×1013vg per AAV vector, about 5×1011to about 1×1013vg per AAV vector, about 7.5 x 1011to about 1 x 1013vg per AAV vector, about 1×1011to about 7.5×1012vg per AAV vector, about 2.5×1011to about 7.5×1012vg per AAV vector, about 3.0×1011to about 7.5×1012vg per AAV vector, about 5x 1011to about 7.5x 1012vg per AAV vector, about 7.5 x 1011to about 7.5xl012vg per AAV vector, about 1×1011to about 1×1012vg per AAV vector, about 2.5×1011to about 1×1012vg per AAV vector, about 3.0×1011to about 1×1012vg per AAV vector, about 5x 1011to about 1 x 1012vg per AAV vector, about 7.5x 1011to about 1 x 1012vg per AAV vector, delivered as a single or as divided doses. In a further embodiment, the first and second vector (the first or second particle) are administered at equal doses.

[0295] In some embodiments, the dual AAV particle composition is administered intravenously. In one preferred embodiment, the dual AAV particle composition is administered intrathecally. In some embodiments, the dual AAV particle composition is administered intracerebroventricularly.

[0296] In a preferred embodiment, the first AAV particle (first AAV vector) and the second AAV particle (second AAV vector) are administered at equal vg doses. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1013to about 5×1016vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1013to about 1×1016vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1013to about 5×1015vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1013to about 1×1015vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1013to about 5×1014vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1 x 1013to about 1 x 1014vg.

[0297] In a preferred embodiment, the first AAV particle (first AAV vector) and the second AAV particle (second AAV vector) are administered at equal vg doses. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1013to about 5×1016vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 5×1013to about 5×1016vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1014to about 5×1016vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 5×1014to about 5×1016vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1015to about 5×1016vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 5×1016to about 5×1016vg.

[0298] In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1013vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 5×1013vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1014vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 5×1014vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1015vg. In someembodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 5×1015vg. In some embodiments, the dose per AAV vector of the dual AAV particle compositions described herein is about 1×1016vg.

[0299] In a preferred embodiment, the first AAV particle and the second AAV particle are administered at equal vg doses. In some embodiments, the dose per vector of the dual AAV particle compositions described herein is about 1×1014to about 5×1015vg per vector. In some embodiments, the dose per vector of the dual AAV particle compositions described herein is about 5x 1014to about 1 x 1015vg per vector. In some embodiments, the dose per vector of the dual AAV particle compositions described herein is about 1×1014to about 1×1015vg per vector.

[0300] In some embodiments, the dose per vector of the dual AAV particle compositions described herein is about 1×1014vg per vector. In some embodiments, the dose per vector of the dual AAV particle compositions described herein is about 5×1014vg per vector. In some embodiments, the dose per vector of the dual AAV particle compositions described herein is about 1 x 1015vg per vector. In some embodiments, the dose per vector of the dual AAV particle compositions described herein is about 5x 1015vg per vector.

[0301] In some embodiments, the AAV particles are administered intravenously.

[0302] In a preferred embodiment, the dual AAV particle composition is administered intrathecally. In some embodiments, the AAV particles are administered intracerebroventri cul arly.Numbered Embodiments

[0303] Additional non-limiting embodiments of the present disclosure are provided in the following numbered embodiments.

[0304] 1. A composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a deoxyribonucleic acid (DNA) from 5’ to 3’:(i) a first 5’-ITR sequence;(ii) a first promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;(v) a splicing donor sequence;(vi) a first dimerization domain sequence;(vii) a first poly(A) signal sequence; and(viii) a first 3’-ITR sequence; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a second 5’-ITR sequence;(ii) a second promoter sequence;(iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter;(iv) a splicing acceptor sequence;(v) a C-terminal coding sequence encoding a C-terminal portion of the midlength dystrophin protein;(vi) a second poly(A) signal sequence; and(vii) a second 3’ -ITR sequence;wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form a ribonucleic acid (RNA) coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence that encodes the mid-length dystrophin protein; andwherein the mid-length dystrophin protein comprises: (i) an N-terminal region; (ii) a central rod domain comprising: one or more hinge regions and ten spectrin-like repeats; (iii) a cysteine-rich domain; and (iv) a C-terminal domain.

[0305] 2. A composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a DNA from 5’ to 3’:(i) a first 5 ’-ITR sequence;(ii) a first promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;(v) a splicing donor sequence;(vi) a first dimerization domain sequence;(vii) a first poly(A) signal sequence; and(viii) a first 3’-ITR sequence; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a second 5’-ITR sequence;(ii) a second promoter sequence;(iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter;(iv) a splicing acceptor sequence;(v) a C-terminal coding sequence encoding a C-terminal portion of the midlength dystrophin protein;(vi) a second poly(A) signal sequence; and(vii) a second 3’ -ITR sequence;wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form an RNA coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence that encodes the mid-length dystrophin protein; andwherein the mid-length dystrophin protein comprises: (i) an N-terminal region; (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, a spectrin-like repeat 21, a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24, and a hinge region 4; (iii) a cysteine-rich domain; and (iv) a C-terminal domain.

[0306] 3. A composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a DNA from 5’ to 3’:(i) a first 5 ’-ITR sequence;(ii) a first promoter sequence;(iii) a Kozak sequence;(iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;(v) a splicing donor sequence;(vi) a first dimerization domain sequence;(vii) a first poly(A) signal sequence; and(viii) a first 3’-ITR sequence; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a second 5’-ITR sequence;(ii) a second promoter sequence;(iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter;(iv) a splicing acceptor sequence;(v) a C-terminal coding sequence encoding a C-terminal portion of the midlength dystrophin protein;(vi) a second poly(A) signal sequence; and(vii) a second 3’ -ITR sequence;wherein the N-terminal coding sequence encoding the N-terminal portion of the midlength dystrophin protein comprises: (i) an N-terminal region; and (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, and a spectrin-like repeat 21; andwherein the C-terminal coding sequence encoding the C-terminal portion of the midlength dystrophin protein comprises: (i) a central rod domain comprising: a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24; and a hinge region 4; (ii) a cysteine rich domain; and (iii) a C-terminal domain.

[0307] 4. The composition of embodiment 1, wherein the N-terminal coding sequence encoding the N-terminal portion of the mid-length dystrophin protein comprises: (i) the N-terminal region; and (ii) the central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrinlike repeat 17, a hinge region 3, a spectrin-like repeat 20, and a spectrin-like repeat 21.

[0308] 5. The composition of embodiment 2, wherein the N-terminal coding sequence encoding the N-terminal portion of the mid-length dystrophin protein comprises: (i) the N-terminal region; and (ii) the central rod domain comprising: the hinge region 1, the spectrinlike repeat 1, the spectrin-like repeat 2, the spectrin-like repeat 3, the spectrin-like repeat 16, the spectrin-like repeat 17, the hinge region 3, the spectrin-like repeat 20, and the spectrin-like repeat 21.

[0309] 6. The composition of any one of embodiments 1-5, wherein the N-terminal coding sequence is codon-optimized.

[0310] 7. The composition of any one of embodiments 1-6, wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9.

[0311] 8. The composition of any one of embodiments 1-6, wherein the N-terminal coding sequence comprises a nucleic acid sequence of SEQ ID NO: 9.

[0312] 9. The composition of any one of embodiments 1, 2, and 5-8, wherein the C-terminal coding sequence encoding the C-terminal portion of the mid-length dystrophin protein comprises: (i) a central rod domain comprising: a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24; and a hinge region 4; (ii) a cysteine rich domain; and (iii) a C-terminal domain.

[0313] 10. The composition of any one of embodiments 1-9, wherein the C-terminal coding sequence is codon-optimized.

[0314] 11. The composition of any one of embodiments 1-10, wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21.

[0315] 12. The composition of any one of embodiments 1-10, wherein the C-terminal coding sequence comprises a nucleic acid sequence of SEQ ID NO: 21.

[0316] 13. The composition of any one of embodiments 3 and 6-12, wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form an RNA coding sequence encoding the mid-length dystrophin protein, the RNA coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence.

[0317] 14. The composition of embodiment 13, wherein the mid-length dystrophin protein comprises: (i) the N-terminal region; (ii) the central rod domain comprising: the hinge region 1, the spectrin-like repeat 1, the spectrin-like repeat 2, the spectrin-like repeat 3, the spectrinlike repeat 16, the spectrin-like repeat 17, the hinge region 3, the spectrin-like repeat 20, thespectrin-like repeat 21, the spectrin-like repeat 22, the spectrin-like repeat 23, the spectrin-like repeat 24, and the hinge region 4; (iii) the cysteine-rich domain; and (iv) the C-terminal domain.

[0318] 15. The composition of embodiment 2 or 14, wherein the spectrin-like repeat 16 and / or the spectrin-like repeat 17 bind to neuronal nitric oxide synthase.

[0319] 16. The composition of any one of embodiments 13-15, wherein the RNA coding sequence encoding the mid-length dystrophin protein is codon-optimized.

[0320] 17. The composition of any one of embodiments 13-15, wherein the RNA coding sequence encoding the mid-length dystrophin protein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 22.

[0321] 18. The composition of any one of embodiments 13-15, wherein the RNA coding sequence encoding the mid-length dystrophin protein comprises a nucleic acid sequence of SEQ ID NO: 22.

[0322] 19. The composition of any one of embodiments 1-18, wherein the mid-length dystrophin protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23.

[0323] 20. The composition of any one of embodiments 1-18, wherein the mid-length dystrophin protein comprises an amino acid sequence of SEQ ID NO: 23.

[0324] 21. The composition of any one of embodiments 1-20, wherein the N-terminal coding sequence comprises an intervening intron sequence.

[0325] 22. The composition of embodiment 21, wherein the intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence.

[0326] 23. The composition of embodiment 22, wherein the intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7.

[0327] 24. The composition of any one of embodiments 1-23, wherein the C-terminal coding sequence comprises a first intervening intron sequence and a second intervening intron sequence.

[0328] 25. The composition of embodiment 24, wherein the first intervening intron sequence is a modified mouse beta-actin intron 2 sequence and the second intervening intron sequence is a modified mouse Gapdh intron 5 sequence.

[0329] 26. The composition of embodiment 25, wherein the first intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7.

[0330] 27. The composition of embodiment 25 or 26, wherein the second intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19.

[0331] 28. The composition of any one of embodiments 1-27, wherein the splicing donor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.

[0332] 29. The composition of any one of embodiments 1-28, wherein the splicing acceptor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

[0333] 30. The composition of any one of embodiments 1-29, wherein the first dimerization domain and the second dimerization domain encode RNA sequences that are complementary to each other.

[0334] 31. The composition of embodiment 30, wherein the first dimerization domain and the second dimerization domain encode RNA sequences with complementary RNA stem loops.

[0335] 32. The composition of embodiment 31, wherein the complementary RNA stem loops form a central kissing loop interaction with four or more loop base pairs for intermolecular pairing.

[0336] 33. The composition of embodiment 32, wherein the positioning of the complementary RNA stem loops is offset by at least 1 nt, at least 2 nt, at least 3 nt, at least 4 nt, at least 5 nt, at least 6 nt, at least 7 nt, at least 8 nt, at least 9 nt, or at least 10 nt so that the respective stem regions of the complementary RNA stem loops base pair in trans through strand invasion.

[0337] 34. The composition of embodiment 33, wherein the stem regions of the complementary RNA stem loops contain about 1% to about 30% mismatches, but match to the other dimerization domain.

[0338] 35. The composition of embodiment 33 or 34, wherein the RNA sequences encoding the first dimerization domain and the second dimerization domain form an extended duplex through strand invasion.

[0339] 36. The composition of any one of embodiments 1-35, wherein the first dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11.

[0340] 37. The composition of any one of embodiments 1-36, wherein the first dimerization domain comprises a nucleic acid sequence of SEQ ID NO: 11.

[0341] 38. The composition of any one of embodiments 1-37, wherein the second dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15.

[0342] 39. The composition of any one of embodiments 1-38, wherein the second dimerization domain comprises a nucleic acid sequence of SEQ ID NO: 15.

[0343] 40. The composition of any one of embodiments 1-39, wherein the first promoter is a muscle tissue-specific promoter.

[0344] 41. The composition of embodiment 40, wherein the muscle tissue-specific promoter is a muscle- and heart-specific enhancer 7 (MHCK7) promoter.

[0345] 42. The composition of embodiment 41, wherein the MHCK7 promoter comprises a nucleic acid sequence of SEQ ID NO: 2.

[0346] 43. The composition of any one of embodiments 1-42, wherein the second promoter is a muscle tissue-specific promoter.

[0347] 44. The composition of embodiment 43, wherein the muscle tissue-specific promoter is a MHCK7 promoter.

[0348] 45. The composition of embodiment 44, wherein the MHCK7 promoter comprises a nucleic acid sequence of SEQ ID NO: 2.

[0349] 46. The composition of any one of embodiments 1-45, wherein the first 5’-ITR is a 5’ AAV2 ITR.

[0350] 47. The composition of embodiment 46, wherein the first 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 1.

[0351] 48. The composition of any one of embodiments 1-47, wherein the first 3’-ITR is a 3’ AAV2 ITR.

[0352] 49. The composition of embodiment 48, wherein the first 3 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 14.

[0353] 50. The composition of any one of embodiments 1-49, wherein the second 5 ’-ITR is a 5’ AAV2 ITR.

[0354] 51. The composition of embodiment 50, wherein the second 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 1.

[0355] 52. The composition of any one of embodiments 1-51, wherein the second 3 ’-ITR is a 3’ AAV2 ITR.

[0356] 53. The composition of embodiment 52, wherein the second 3 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 14.

[0357] 54. The composition of any one of embodiments 1-53, wherein the first poly(A) signal sequence comprises a late SV40 poly(A) signal sequence.

[0358] 55. The composition of embodiment 54, wherein the first poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 13.

[0359] 56. The composition of any one of embodiments 1-55, wherein the second poly(A) signal sequence comprises a late SV40 poly(A) signal sequence.

[0360] 57. The composition of embodiment 56, wherein the second poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 13.

[0361] 58. The composition of any one of embodiments 1-57, wherein the Kozak sequence comprises a nucleic acid sequence of any one of SEQ ID NOs: 3 and 24-26.

[0362] 59. The composition of any one of embodiments 1-57, wherein the Kozak sequence comprises a nucleic acid sequence of SEQ ID NO: 3.

[0363] 60. The composition of any one of embodiments 1-59, wherein the first transgene comprises at least one nucleic acid element to suppress expression of a first unjoined transcript.

[0364] 61. The composition of embodiment 60, wherein the at least one nucleic acid element is the absence of a stop codon in the N-terminal coding sequence.

[0365] 62. The composition of embodiment 60 or 61, wherein the at least one nucleic acid element is a micro-RNA target site 3’ to the splicing donor.

[0366] 63. The composition of any one of embodiments 60-62, wherein the at least one nucleic acid element is two micro-RNA target sites 3’ to the splicing donor.

[0367] 64. The composition of embodiment 62 or 63, wherein the at least one micro-RNA target site is a micro-RNA- 16 target site.

[0368] 65. The composition of embodiment 64, wherein the micro-RNA- 16 target site comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to a sequence of SEQ ID NO: 12.

[0369] 66. The composition of any one of embodiments 1-65, wherein the second transgene comprises at least one nucleic acid element to suppress expression of a second unjoined transcript.

[0370] 67. The composition of embodiment 66, wherein the at least one nucleic acid element is an out-of-frame start codon 5’ of the splicing acceptor.

[0371] 68. The composition of embodiment 66 or 67, wherein the at least one nucleic acid element is a modified mouse Gapdh intron 5 sequence in the C-terminal coding sequence.

[0372] 69. A composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’:(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a Kozak sequence of SEQ ID NO: 3;(iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N- terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a splicing donor sequence of SEQ ID NO: 10;(vi) a dimerization domain sequence of SEQ ID NO: 11;(vii) a poly(A) signal sequence of SEQ ID NO: 13; and(viii) a 3 ’-ITR sequence of SEQ ID NO: 14; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter;(iv) a splicing acceptor sequence of SEQ ID NO: 16;(v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C-terminal portion of mid-length dystrophin protein;(vi) a poly(A) signal sequence of SEQ ID NO: 13; and(vii) a 3 ’-ITR sequence of SEQ ID NO: 14.

[0373] 70. The composition of any one of embodiments 1-69, wherein the AAV capsid of the first AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins, or an engineered variant of one of the foregoing.

[0374] 71. The composition of embodiment 70, wherein the first AAV particle is an AAV9 particle and the AAV capsid of the first AAV particle comprises one or more AAV9 capsid proteins.

[0375] 72. The composition of embodiment 71, wherein the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP1.

[0376] 73. The composition of embodiment 72, wherein the AAV9 capsid protein VP1 comprises the amino acid sequence of SEQ ID NO: 33.

[0377] 74. The composition of any one of embodiments 71-73, wherein the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP2.

[0378] 75. The composition of embodiment 74, wherein the AAV9 capsid protein VP2 comprises amino acids 138-736 of SEQ ID NO: 33.

[0379] 76. The composition of any one of embodiments 71-75, wherein the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP3.

[0380] 77. The composition of embodiment 76, wherein the AAV9 capsid protein VP3 comprises amino acids 203-736 of SEQ ID NO: 33.

[0381] 78. The composition of embodiment 70, wherein the AAV capsid of the first AAV particle comprises one or more engineered variants of an AAV9 capsid protein.

[0382] 79. The composition of embodiment 78, wherein the one or more engineered variants of the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP1.

[0383] 80. The composition of embodiment 78 or 79, wherein the one or more engineered variants of the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP2.

[0384] 81. The composition of any one of embodiments 78-80, wherein the one or more engineered variants of the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP3.

[0385] 82. The composition of any one of embodiments 78-81, wherein the one or more engineered variants of the AAV9 capsid protein of the first AAV particle are one or more deimmunized variants of the AAV9 capsid protein.

[0386] 83. The composition of any one of embodiments 1-82, wherein the AAV capsid of the second AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins, or an engineered variant of one of the foregoing.

[0387] 84. The composition of embodiment 83, wherein the second AAV particle is an AAV9 particle and the AAV capsid of the second AAV particle comprises one or more AAV9 capsid proteins.

[0388] 85. The composition of embodiment 84, wherein the one or more AAV9 capsid proteins of the AAV capsid of the second AAV particle comprise AAV9 capsid protein VP1.

[0389] 86. The composition of embodiment 85, wherein the AAV9 capsid protein VP1 comprises the amino acid sequence of SEQ ID NO: 33.

[0390] 87. The composition of any one of embodiments 84-86, wherein the one or more AAV9 capsid proteins of the AAV capsid of the second AAV particle comprise AAV9 capsid protein VP2.

[0391] 88. The composition of embodiment 87, wherein the AAV9 capsid protein VP2 comprises amino acids 138-736 of SEQ ID NO: 33.

[0392] 89. The composition of any one of embodiments 84-88, wherein the one or more AAV9 capsid proteins of the AAV capsid of the second AAV particle comprise AAV9 capsid protein VP3.

[0393] 90. The composition of embodiment 89, wherein the AAV9 capsid protein VP3 comprises amino acids 203-736 of SEQ ID NO: 33.

[0394] 91. The composition of embodiment 83, wherein the AAV capsid of the second AAV particle comprises one or more engineered variants of the AAV9 capsid protein.

[0395] 92. The composition of embodiment 91, wherein the AAV9 capsid of the second AAV particle comprises an engineered variant of AAV9 capsid protein VP1.

[0396] 93. The composition of embodiment 91 or embodiment 92, wherein the AAV9 capsid of the second AAV particle comprises an engineered variant of AAV9 capsid protein VP2.

[0397] 94. The composition of any one of embodiments 91-93, wherein the AAV9 capsid protein of the second AAV particle comprises an engineered variant of AAV9 capsid protein VP3.

[0398] 95. The composition of any one of embodiments 91-94, wherein the one or more engineered variants of the AAV9 capsid protein of the second AAV particle are one or more deimmunized variants of the AAV9 capsid protein.

[0399] 96. The composition of any one of embodiments 1-69, wherein the AAV capsid of the first AAV particle comprises one or more AAVMYO capsid proteins.

[0400] 97. The composition of embodiment 96, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP1.

[0401] 98. The composition of embodiment 97, wherein the AAVMYO capsid protein VP1 comprises an amino acid sequence of any one of SEQ ID NOS: 35-38.

[0402] 99. The composition of any one of embodiments 96-98, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP2.

[0403] 100. The composition of any one of embodiments 96-99, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP3.

[0404] 101. The composition of any one of embodiments 96-100, wherein the one or more AAVMYO capsid proteins comprises one or more deimmunized variants of AAVMYO capsid proteins.

[0405] 102. The composition of any one of embodiments 96-101, wherein the AAV capsid of the second AAV particle comprises one or more AAVMYO capsid proteins.

[0406] 103. The composition of embodiment 102, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP1.

[0407] 104. The composition of embodiment 103, wherein the AAVMYO capsid protein VP1 comprises an amino acid sequence of any one of SEQ ID NOS: 35-38.

[0408] 105. The composition of any one of embodiments 102-104, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP2.

[0409] 106. The composition of any one of embodiments 102-105, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP3.

[0410] 107. The composition of any one of embodiments 102-106, the one or more AAVMYO capsid proteins are one or more deimmunized variants of an AAVMYO capsid protein.

[0411] 108. The composition of any one of embodiments 1-69, wherein the AAV capsid of the first AAV particle comprises one or more MyoAAV capsid proteins.

[0412] 109. The composition of embodiment 108, wherein the one or more MyoAAV capsid proteins comprises an MyoAAV capsid protein VP1.

[0413] 110. The composition of embodiment 109, wherein the MyoAAV capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 39 or 40.

[0414] 111. The composition of any one of embodiments 108-110, wherein the one or more MyoAAV capsid protein of comprises an MyoAAV capsid protein VP2.

[0415] 112. The composition of any one of embodiments 108-111, wherein the one or more MyoAAV capsid protein comprises an MyoAAV capsid protein VP3.

[0416] 113. The composition of any one of embodiments 108-112, the one or more MyoAAV capsid proteins are one or more deimmunized variants of the MyoAAV capsid protein.

[0417] 114. The composition of any one of embodiments 108-113, wherein the AAV capsid of the second AAV particle comprises one or more MyoAAV capsid proteins.

[0418] 115. The composition of embodiment 114, wherein the one or more MyoAAV capsid proteins comprises an MyoAAV capsid protein VP1.

[0419] 116. The composition of embodiment 115, wherein the MyoAAV capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 39 or 40.

[0420] 117. The composition of any one of embodiments 114-116, wherein the one or more MyoAAV capsid proteins of the second AAV particle comprises an MyoAAV capsid protein VP2.

[0421] 118. The composition of any one of embodiments 114-117, wherein the one or more MyoAAV capsid protein of the second AAV particle comprises an MyoAAV capsid protein VP3.

[0422] 119. The composition of any one of embodiments 114-118, the one or more MyoAAV capsid protein of the second AAV particle are one or more deimmunized variants of a MyoAAV capsid protein.

[0423] 120. The composition of embodiment 70, wherein the first AAV particle is an AAVrh.74 particle and the AAV capsid of the first AAV particle comprises one or more AAVrh.74 capsid proteins.

[0424] 121. The composition of embodiment 120, wherein the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP1.

[0425] 122. The composition of embodiment 121, wherein the AAVrh.74 capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 34.

[0426] 123. The composition of any one of embodiments 120-122, wherein the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP2.

[0427] 124. The composition of embodiment 123, wherein the AAVrh.74 capsid protein VP2 comprises amino acids 138-738 of SEQ ID NO: 34.

[0428] 125. The composition of any one of embodiments 120-124, wherein the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP3.

[0429] 126. The composition of embodiment 125, wherein the AAVrh.74 capsid protein VP3 comprises amino acids 204-738 of SEQ ID NO:34.

[0430] 127. The composition of any one of embodiments 120-126, wherein the second AAV particle is an AAVrh.74 particle and the AAV capsid of the second AAV particle comprises one or more AAVrh.74 capsid proteins.

[0431] 128. The composition of embodiment 127, wherein the one or more AAVrh.74 capsid proteins of the AAV capsid of the second AAV particle comprise AAVrh.74 capsid protein VP1.

[0432] 129. The composition of embodiment 128, wherein the AAVrh.74 capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 34.

[0433] 130. The composition of any one of embodiments 127-129, wherein the one or more AAVrh.74 capsid proteins of the AAV capsid of the second AAV particle comprise AAVrh.74 capsid protein VP2.

[0434] 131. The composition of embodiment 130, wherein the AAVrh.74 capsid protein VP2 comprises amino acids 138-738 of SEQ ID NO: 34.

[0435] 132. The composition of any one of embodiments 127-131, wherein the one or more AAVrh.74 capsid proteins of the AAV capsid of the second AAV particle comprise AAVrh.74 capsid protein VP3.

[0436] 133. The composition of embodiment 132, wherein the AAVrh.74 capsid protein VP3 comprises amino acids 204-738 of SEQ ID NO:34.

[0437] 134. The composition of any one of embodiments 1-69, wherein the AAV capsid of the first AAV particle comprises one or more AAV9 capsid proteins; and wherein the AAV capsid of the second AAV particle comprises one or more AAV9 capsid proteins.

[0438] 135. The composition of embodiment 134, wherein the one or more AAV9 capsid proteins of the first AAV particle comprise AAV9 capsid protein VP1, AAV9 capsid protein VP2, and / or AAV9 capsid protein VP3; and wherein the one or more AAV9 capsid proteins of the second AAV particle comprise AAV9 capsid protein VP1, AAV9 capsid protein VP2, and / or AAV9 capsid protein VP3.Ill

[0439] 136. A composition comprising:(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’:(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a Kozak sequence of SEQ ID NO: 3;(iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N- terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter;(v) a splicing donor sequence of SEQ ID NO: 10;(vi) a dimerization domain sequence of SEQ ID NO: 11;(vii) a poly(A) signal sequence of SEQ ID NO: 13; and(viii) a 3 ’-ITR sequence of SEQ ID NO: 14; and(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;(ii) a promoter sequence of SEQ ID NO: 2;(iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter;(iv) a splicing acceptor sequence of SEQ ID NO: 16;(v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C-terminal portion of mid-length dystrophin protein;(vi) a poly(A) signal sequence of SEQ ID NO: 13; and(vii) a 3 ’-ITR sequence of SEQ ID NO: 14;wherein the first AAV particle comprises AAV9 capsid proteins; and wherein the second AAV particle comprises AAV9 capsid proteins.

[0440] 137. A pharmaceutical composition comprising an effective amount of the composition of any one of embodiments 1-136 and a pharmaceutically acceptable carrier, excipient, diluent, or buffer.

[0441] 138. The pharmaceutical composition of embodiment 137, wherein the effective amount of the composition is a composition comprising about the same number of first AAV particles and second AAV particles.

[0442] 139. The pharmaceutical composition of embodiment 137, wherein the composition comprises about the same number of vector genomes of the first AAV particle and the second AAV particle.

[0443] 140. The pharmaceutical composition of embodiment 137, wherein the composition comprises the same number of vector genomes of the first AAV particle and the second AAV particle.

[0444] 141. The pharmaceutical composition of embodiment 137, wherein the composition comprises the first AAV particle and the second AAV particle combined at a 1: 1 ratio.

[0445] 142. The pharmaceutical composition of embodiment 141, wherein the 1: 1 ratio is a 1: 1 ratio of encapsidated transgenes.

[0446] 143. The pharmaceutical composition of embodiment 141, wherein the 1:1 ratio is a 1:1 ratio of first AAV particles to second AAV particles.

[0447] 144. A method of treating a dystrophinopathy in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of embodiments 138-143.

[0448] 145. The method of embodiment 144, wherein the subject is a male subject from about 6 months to about 7 years old....

Claims

1. CLAIMS1. A composition comprising:3.(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a deoxyribonucleic acid (DNA) from4.5’ to 3’:5.(i) a first 5’ -ITR sequence;6.(ii) a first promoter sequence;7.(iii) a Kozak sequence;8.(iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;9.(v) a splicing donor sequence;10.(vi) a first dimerization domain sequence;11.(vii) a first poly(A) signal sequence; and12.(viii) a first 3 ’-ITR sequence; and13.(b) a second AAV particle comprising an AAV capsid encapsidating14.a second transgene, wherein the second transgene comprises from 5’ to 3’:15.(i) a second 5 ’-ITR sequence;16.(ii) a second promoter sequence;17.(iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said18.second promoter;19.(iv) a splicing acceptor sequence;20.(v) a C-terminal coding sequence encoding a C-terminal portion of the mid-length dystrophin protein;21.(vi) a second poly(A) signal sequence; and22.(vii) a second 3 ’-ITR sequence;23.wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form a ribonucleic acid (RNA) coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence that encodes the mid-length dystrophin protein; and24.wherein the mid-length dystrophin protein comprises: (i) an N-terminal region;25.(ii) a central rod domain comprising: one or more hinge regions and ten spectrin-like repeats; (iii) a cysteine-rich domain; and (iv) a C-terminal domain.

2. A composition comprising:27.(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a DNA from 5’ to 3’:28.(i) a first 5’ -ITR sequence;29.(ii) a first promoter sequence;30.(iii) a Kozak sequence;31.(iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;32.(v) a splicing donor sequence;33.(vi) a first dimerization domain sequence;34.(vii) a first poly(A) signal sequence; and35.(viii) a first 3 ’-ITR sequence; and36.(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:37.(i) a second 5 ’-ITR sequence;38.(ii) a second promoter sequence;39.(iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter;40.(iv) a splicing acceptor sequence;41.(v) a C-terminal coding sequence encoding a C-terminal portion of the mid-length dystrophin protein;42.(vi) a second poly(A) signal sequence; and43.(vii) a second 3 ’-ITR sequence;44.wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form an RNA coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence that encodes the mid-length dystrophin protein; and45.wherein the mid-length dystrophin protein comprises: (i) an N-terminal region; (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, a spectrin-like repeat 21, a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24, and a hinge region 4; (iii) a cysteine-rich domain; and (iv) a C-terminal domain.

3. A composition comprising:47.(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises a DNA from 5’ to 3’:48.(i) a first 5’ -ITR sequence;49.(ii) a first promoter sequence;50.(iii) a Kozak sequence;51.(iv) an N-terminal coding sequence encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said first promoter;52.(v) a splicing donor sequence;53.(vi) a first dimerization domain sequence;54.(vii) a first poly(A) signal sequence; and55.(viii) a first 3 ’-ITR sequence; and56.(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:57.(i) a second 5 ’-ITR sequence;58.(ii) a second promoter sequence;59.(iii) a second dimerization domain sequence, said second dimerization domain being operably linked to and under control of said second promoter;60.(iv) a splicing acceptor sequence;61.(v) a C-terminal coding sequence encoding a C-terminal portion of the mid-length dystrophin protein;62.(vi) a second poly(A) signal sequence; and63.(vii) a second 3 ’-ITR sequence;64.wherein the N-terminal coding sequence encoding the N-terminal portion of the mid-length dystrophin protein comprises: (i) an N-terminal region; and (ii) a central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, and a spectrin-like repeat 21; and wherein the C-terminal coding sequence encoding the C-terminal portion of the mid-length dystrophin protein comprises: (i) a central rod domain comprising: a spectrinlike repeat 22, a spectrin-like repeat 23, a spectrin-like repeat 24; and a hinge region 4;65.(ii) a cysteine rich domain; and (iii) a C-terminal domain.

4. The composition of claim 1, wherein the N-terminal coding sequence encoding the N-terminal portion of the mid-length dystrophin protein comprises: (i) the N-terminal region; and (ii) the central rod domain comprising: a hinge region 1, a spectrin-like repeat 1, a spectrin-like repeat 2, a spectrin-like repeat 3, a spectrin-like repeat 16, a spectrin-like repeat 17, a hinge region 3, a spectrin-like repeat 20, and a spectrin-like repeat 21.

5. The composition of claim 2, wherein the N-terminal coding sequence encoding the N-terminal portion of the mid-length dystrophin protein comprises: (i) the N-terminal region; and (ii) the central rod domain comprising: the hinge region 1, the spectrin-like repeat 1, the spectrin-like repeat 2, the spectrin-like repeat 3, the spectrin-like repeat 16, the spectrin-like repeat 17, the hinge region 3, the spectrin-like repeat 20, and the spectrin-like repeat 21.

6. The composition of any one of claims 1-5, wherein the N-terminal coding sequence is codon-optimized.

7. The composition of any one of claims 1-6, wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9.

8. The composition of any one of claims 1-6, wherein the N-terminal coding sequence comprises a nucleic acid sequence of SEQ ID NO: 9.

9. The composition of any one of claims 1, 2, and 5-8, wherein the C-terminal coding sequence encoding the C-terminal portion of the mid-length dystrophin protein comprises: (i) a central rod domain comprising: a spectrin-like repeat 22, a spectrin-like repeat 23, a spectrinlike repeat 24; and a hinge region 4; (ii) a cysteine rich domain; and (iii) a C-terminal domain.

10. The composition of any one of claims 1-9, wherein the C-terminal coding sequence is codon-optimized.

11. The composition of any one of claims 1-10, wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21.

12. The composition of any one of claims 1-10, wherein the C-terminal coding sequence comprises a nucleic acid sequence of SEQ ID NO: 21.

13. The composition of any one of claims 3 and 6-12, wherein a first transcript of the first transgene and a second transcript of the second transgene can be spliced and joined to form an RNA coding sequence encoding the mid-length dystrophin protein, the RNA coding sequence comprising the N-terminal coding sequence operably linked to the C-terminal coding sequence.

14. The composition of claim 13, wherein the mid-length dystrophin protein comprises: (i) the N-terminal region; (ii) the central rod domain comprising: the hinge region 1, the spectrinlike repeat 1, the spectrin-like repeat 2, the spectrin-like repeat 3, the spectrin-like repeat 16, the spectrin-like repeat 17, the hinge region 3, the spectrin-like repeat 20, the spectrin-like repeat 21, the spectrin-like repeat 22, the spectrin-like repeat 23, the spectrin-like repeat 24, and the hinge region 4; (iii) the cysteine-rich domain; and (iv) the C-terminal domain.

15. The composition of claim 2 or 14, wherein the spectrin-like repeat 16 and / or the spectrin-like repeat 17 bind to neuronal nitric oxide synthase.

16. The composition of any one of claims 13-15, wherein the RNA coding sequence encoding the mid-length dystrophin protein is codon-optimized.

17. The composition of any one of claims 13-15, wherein the RNA coding sequence encoding the mid-length dystrophin protein comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 22.

18. The composition of any one of claims 13-15, wherein the RNA coding sequence encoding the mid-length dystrophin protein comprises a nucleic acid sequence of SEQ ID NO: 22.

19. The composition of any one of claims 1-18, wherein the mid-length dystrophin protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23.

20. The composition of any one of claims 1-18, wherein the mid-length dystrophin protein comprises an amino acid sequence of SEQ ID NO: 23.

21. The composition of any one of claims 1-20, wherein the N-terminal coding sequence comprises an intervening intron sequence.

22. The composition of claim 21, wherein the intervening intron sequence is a modified mouse beta-actin (Actb) intron 2 sequence.

23. The composition of claim 22, wherein the intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7.

24. The composition of any one of claims 1-23, wherein the C-terminal coding sequence comprises a first intervening intron sequence and a second intervening intron sequence.

25. The composition of claim 24, wherein the first intervening intron sequence is a modified mouse beta-actin intron 2 sequence and the second intervening intron sequence is a modified mouse Gapdh intron 5 sequence.

26. The composition of claim 25, wherein the first intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7.

27. The composition of claim 25 or 26, wherein the second intervening intron sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19.

28. The composition of any one of claims 1-27, wherein the splicing donor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.

29. The composition of any one of claims 1-28, wherein the splicing acceptor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

30. The composition of any one of claims 1-29, wherein the first dimerization domain and the second dimerization domain encode RNA sequences that are complementary to each other.

31. The composition of claim 30, wherein the first dimerization domain and the second dimerization domain encode RNA sequences with complementary RNA stem loops.

32. The composition of claim 31, wherein the complementary RNA stem loops form a central kissing loop interaction with four or more loop base pairs for intermolecular pairing.

33. The composition of claim 32, wherein the positioning of the complementary RNA stem loops is offset by at least 1 nt, at least 2 nt, at least 3 nt, at least 4 nt, at least 5 nt, at least 6 nt, at least 7 nt, at least 8 nt, at least 9 nt, or at least 10 nt so that the respective stem regions of the complementary RNA stem loops base pair in trans through strand invasion.

34. The composition of claim 33, wherein the stem regions of the complementary RNA stem loops contain about 1% to about 30% mismatches, but match to the other dimerization domain.

35. The composition of claim 33 or 34, wherein the RNA sequences encoding the first dimerization domain and the second dimerization domain form an extended duplex through strand invasion.

36. The composition of any one of claims 1-35, wherein the first dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11.

37. The composition of any one of claims 1-36, wherein the first dimerization domain comprises a nucleic acid sequence of SEQ ID NO: 11.

38. The composition of any one of claims 1-37, wherein the second dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15.

39. The composition of any one of claims 1-38, wherein the second dimerization domain comprises a nucleic acid sequence of SEQ ID NO: 15.

40. The composition of any one of claims 1-39, wherein the first promoter is a muscle tissue-specific promoter.

41. The composition of claim 40, wherein the muscle tissue-specific promoter is a muscle-and heart-specific enhancer 7 (MHCK7) promoter.

42. The composition of claim 41, wherein the MHCK7 promoter comprises a nucleic acid sequence of SEQ ID NO: 2.

43. The composition of any one of claims 1-42, wherein the second promoter is a muscle tissue-specific promoter.

44. The composition of claim 43, wherein the muscle tissue-specific promoter is a MHCK7 promoter.

45. The composition of claim 44, wherein the MHCK7 promoter comprises a nucleic acid sequence of SEQ ID NO: 2.

46. The composition of any one of claims 1-45, wherein the first 5’-ITR is a 5’ AAV2 ITR.

47. The composition of claim 46, wherein the first 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 1.

48. The composition of any one of claims 1-47, wherein the first 3’-ITR is a 3’ AAV2 ITR.

49. The composition of claim 48, wherein the first 3 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 14.

50. The composition of any one of claims 1-49, wherein the second 5 ’-ITR is a 5’ AAV2 ITR.

51. The composition of claim 50, wherein the second 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 1.

52. The composition of any one of claims 1-51, wherein the second 3 ’-ITR is a 3’ AAV2 ITR.

53. The composition of claim 52, wherein the second 3’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 14.

54. The composition of any one of claims 1-53, wherein the first poly(A) signal sequence comprises a late SV40 poly(A) signal sequence.

55. The composition of claim 54, wherein the first poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 13.

56. The composition of any one of claims 1-55, wherein the second poly(A) signal sequence comprises a late SV40 poly(A) signal sequence.

57. The composition of claim 56, wherein the second poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 13.

58. The composition of any one of claims 1-57, wherein the Kozak sequence comprises a nucleic acid sequence of any one of SEQ ID NOs: 3 and 24-26.

59. The composition of any one of claims 1-57, wherein the Kozak sequence comprises a nucleic acid sequence of SEQ ID NO: 3.

60. The composition of any one of claims 1-59, wherein the first transgene comprises at least one nucleic acid element to suppress expression of a first unjoined transcript.

61. The composition of claim 60, wherein the at least one nucleic acid element is the absence of a stop codon in the N-terminal coding sequence.

62. The composition of claim 60 or 61, wherein the at least one nucleic acid element is a micro-RNA target site 3’ to the splicing donor.

63. The composition of any one of claims 60-62, wherein the at least one nucleic acid element is two micro-RNA target sites 3’ to the splicing donor.

64. The composition of claim 62 or 63, wherein the at least one micro-RNA target site is a micro-RNA-16 target site.

65. The composition of claim 64, wherein the micro-RNA-16 target site comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to a sequence of SEQ ID NO: 12.

66. The composition of any one of claims 1-65, wherein the second transgene comprises at least one nucleic acid element to suppress expression of a second unjoined transcript.

67. The composition of claim 66, wherein the at least one nucleic acid element is an out-of-frame start codon 5’ of the splicing acceptor.

68. The composition of claim 66 or 67, wherein the at least one nucleic acid element is a modified mouse Gapdh intron 5 sequence in the C-terminal coding sequence.

69. A composition comprising:126.(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’:127.(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;128.(ii) a promoter sequence of SEQ ID NO: 2;129.(iii) a Kozak sequence of SEQ ID NO: 3;130.(iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N- terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter;131.(v) a splicing donor sequence of SEQ ID NO: 10;132.(vi) a dimerization domain sequence of SEQ ID NO: 11;133.(vii) a poly(A) signal sequence of SEQ ID NO: 13; and134.(viii) a 3 ’-ITR sequence of SEQ ID NO: 14; and135.(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:136.(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;137.(ii) a promoter sequence of SEQ ID NO: 2;138.(iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter; (iv) a splicing acceptor sequence of SEQ ID NO: 16;139.(v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C- terminal portion of mid-length dystrophin protein;140.(vi) a poly(A) signal sequence of SEQ ID NO: 13; and141.(vii) a 3 ’-ITR sequence of SEQ ID NO: 14.

70. The composition of any one of claims 1-69, wherein the AAV capsid of the first AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins, or an engineered variant of one of the foregoing.

71. The composition of claim 70, wherein the first AAV particle is an AAV9 particle and the AAV capsid of the first AAV particle comprises one or more AAV9 capsid proteins.

72. The composition of claim 71, wherein the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP1.

73. The composition of claim 72, wherein the AAV9 capsid protein VP1 comprises the amino acid sequence of SEQ ID NO: 33.

74. The composition of any one of claims 71-73, wherein the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP2.

75. The composition of claim 74, wherein the AAV9 capsid protein VP2 comprises amino acids 138-736 of SEQ ID NO: 33.

76. The composition of any one of claims 71-75, wherein the one or more AAV9 capsid proteins comprise AAV9 capsid protein VP3.

77. The composition of claim 76, wherein the AAV9 capsid protein VP3 comprises amino acids 203-736 of SEQ ID NO: 33.

78. The composition of claim 70, wherein the AAV capsid of the first AAV particle comprises one or more engineered variants of an AAV9 capsid protein.

79. The composition of claim 78, wherein the one or more engineered variants of the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP 1.

80. The composition of claim 78 or 79, wherein the one or more engineered variants of the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP2.

81. The composition of any one of claims 78-80, wherein the one or more engineered variants of the AAV9 capsid protein comprises an engineered variant of AAV9 capsid protein VP3.

82. The composition of any one of claims 78-81, wherein the one or more engineered variants of the AAV9 capsid protein of the first AAV particle are one or more deimmunized variants of the AAV9 capsid protein.

83. The composition of any one of claims 1-82, wherein the AAV capsid of the second AAV particle comprises one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVrh.8, AAVrh.10, AAVrh.39, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13 capsid proteins, or an engineered variant of one of the foregoing.

84. The composition of claim 83, wherein the second AAV particle is an AAV9 particle and the AAV capsid of the second AAV particle comprises one or more AAV9 capsid proteins.

85. The composition of claim 84, wherein the one or more AAV9 capsid proteins of the AAV capsid of the second AAV particle comprise AAV9 capsid protein VP1.

86. The composition of claim 85, wherein the AAV9 capsid protein VP1 comprises the amino acid sequence of SEQ ID NO: 33.

87. The composition of any one of claims 84-86, wherein the one or more AAV9 capsid proteins of the AAV capsid of the second AAV particle comprise AAV9 capsid protein VP2.

88. The composition of claim 87, wherein the AAV9 capsid protein VP2 comprises amino acids 138-736 of SEQ ID NO: 33.

89. The composition of any one of claims 84-88, wherein the one or more AAV9 capsid proteins of the AAV capsid of the second AAV particle comprise AAV9 capsid protein VP3.

90. The composition of claim 89, wherein the AAV9 capsid protein VP3 comprises amino acids 203-736 of SEQ ID NO: 33.

91. The composition of claim 83, wherein the AAV capsid of the second AAV particle comprises one or more engineered variants of the AAV9 capsid protein.

92. The composition of claim 91, wherein the AAV9 capsid of the second AAV particle comprises an engineered variant of AAV9 capsid protein VP1.

93. The composition of claim 91 or claim 92, wherein the AAV9 capsid of the second AAV particle comprises an engineered variant of AAV9 capsid protein VP2.

94. The composition of any one of claims 91-93, wherein the AAV9 capsid protein of the second AAV particle comprises an engineered variant of AAV9 capsid protein VP3.

95. The composition of any one of claims 91-94, wherein the one or more engineered variants of the AAV9 capsid protein of the second AAV particle are one or more deimmunized variants of the AAV9 capsid protein.

96. The composition of any one of claims 1-69, wherein the AAV capsid of the first AAV particle comprises one or more AAVMYO capsid proteins.

97. The composition of claim 96, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP1.

98. The composition of claim 97, wherein the AAVMYO capsid protein VP1 comprises an amino acid sequence of any one of SEQ ID NOS: 35-38.

99. The composition of any one of claims 96-98, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP2.

100. The composition of any one of claims 96-99, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP3.

101. The composition of any one of claims 96-100, wherein the one or more AAVMYO capsid proteins comprises one or more deimmunized variants of AAVMYO capsid proteins.

102. The composition of any one of claims 96-101, wherein the AAV capsid of the second AAV particle comprises one or more AAVMYO capsid proteins.

103. The composition of claim 102, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP1.

104. The composition of claim 103, wherein the AAVMYO capsid protein VP1 comprises an amino acid sequence of any one of SEQ ID NOS: 35-38.

105. The composition of any one of claims 102-104, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP2.

106. The composition of any one of claims 102-105, wherein the one or more AAVMYO capsid proteins comprises an AAVMYO capsid protein VP3.

107. The composition of any one of claims 102-106, the one or more AAVMYO capsid proteins are one or more deimmunized variants of an AAVMYO capsid protein.

108. The composition of any one of claims 1-69, wherein the AAV capsid of the first AAV particle comprises one or more MyoAAV capsid proteins.

109. The composition of claim 108, wherein the one or more MyoAAV capsid proteins comprises an MyoAAV capsid protein VP1.

110. The composition of claim 109, wherein the MyoAAV capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 39 or 40.

111. The composition of any one of claims 108-110, wherein the one or more MyoAAV capsid protein of comprises an MyoAAV capsid protein VP2.

112. The composition of any one of claims 108-111, wherein the one or more MyoAAV capsid protein comprises an MyoAAV capsid protein VP3.

113. The composition of any one of claims 108-112, the one or more MyoAAV capsid proteins are one or more deimmunized variants of the MyoAAV capsid protein.

114. The composition of any one of claims 108-113, wherein the AAV capsid of the second AAV particle comprises one or more MyoAAV capsid proteins.

115. The composition of claim 114, wherein the one or more MyoAAV capsid proteins comprises an MyoAAV capsid protein VP1.

116. The composition of claim 115, wherein the MyoAAV capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 39 or 40.

117. The composition of any one of claims 114-116, wherein the one or more MyoAAV capsid proteins of the second AAV particle comprises an MyoAAV capsid protein VP2.

118. The composition of any one of claims 114-117, wherein the one or more MyoAAV capsid protein of the second AAV particle comprises an MyoAAV capsid protein VP3.

119. The composition of any one of claims 114-118, the one or more MyoAAV capsid protein of the second AAV particle are one or more deimmunized variants of a MyoAAV capsid protein.

120. The composition of claim 70, wherein the first AAV particle is an AAVrh.74 particle and the AAV capsid of the first AAV particle comprises one or more AAVrh.74 capsid proteins.

121. The composition of claim 120, wherein the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP1.

122. The composition of claim 121, wherein the AAVrh.74 capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 34.

123. The composition of any one of claims 120-122, wherein the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP2.

124. The composition of claim 123, wherein the AAVrh.74 capsid protein VP2 comprises amino acids 138-738 of SEQ ID NO: 34.

125. The composition of any one of claims 120-124, wherein the one or more AAVrh.74 capsid proteins comprise AAVrh.74 capsid protein VP3.

126. The composition of claim 125, wherein the AAVrh.74 capsid protein VP3 comprises amino acids 204-738 of SEQ ID NO:34.

127. The composition of any one of claims 120-126, wherein the second AAV particle is an AAVrh.74 particle and the AAV capsid of the second AAV particle comprises one or more AAVrh.74 capsid proteins.

128. The composition of claim 127, wherein the one or more AAVrh.74 capsid proteins of the AAV capsid of the second AAV particle comprise AAVrh.74 capsid protein VP1.

129. The composition of claim 128, wherein the AAVrh.74 capsid protein VP1 comprises an amino acid sequence of SEQ ID NO: 34.

130. The composition of any one of claims 127-129, wherein the one or more AAVrh.74 capsid proteins of the AAV capsid of the second AAV particle comprise AAVrh.74 capsid protein VP2.

131. The composition of claim 130, wherein the AAVrh.74 capsid protein VP2 comprises amino acids 138-738 of SEQ ID NO: 34.

132. The composition of any one of claims 127-131, wherein the one or more AAVrh.74 capsid proteins of the AAV capsid of the second AAV particle comprise AAVrh.74 capsid protein VP3.

133. The composition of claim 132, wherein the AAVrh.74 capsid protein VP3 comprises amino acids 204-738 of SEQ ID NO:34.

134. The composition of any one of claims 1-69, wherein the AAV capsid of the first AAV particle comprises one or more AAV9 capsid proteins; and wherein the AAV capsid of the second AAV particle comprises one or more AAV9 capsid proteins.

135. The composition of claim 134, wherein the one or more AAV9 capsid proteins of the first AAV particle comprise AAV9 capsid protein VP1, AAV9 capsid protein VP2, and / or AAV9 capsid protein VP3; and wherein the one or more AAV9 capsid proteins of the second AAV particle comprise AAV9 capsid protein VP1, AAV9 capsid protein VP2, and / or AAV9 capsid protein VP3.

136. A composition comprising:204.(a) a first AAV particle comprising an AAV capsid encapsidating a first transgene, wherein the first transgene comprises from 5’ to 3’:205.(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;206.(ii) a promoter sequence of SEQ ID NO: 2;207.(iii) a Kozak sequence of SEQ ID NO: 3;208.(iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N- terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter; (v) a splicing donor sequence of SEQ ID NO: 10;209.(vi) a dimerization domain sequence of SEQ ID NO: 11;210.(vii) a poly(A) signal sequence of SEQ ID NO: 13; and211.(viii) a 3’-ITR sequence of SEQ ID NO: 14; and212.(b) a second AAV particle comprising an AAV capsid encapsidating a second transgene, wherein the second transgene comprises from 5’ to 3’:213.(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;214.(ii) a promoter sequence of SEQ ID NO: 2;215.(iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter;216.(iv) a splicing acceptor sequence of SEQ ID NO: 16;217.(v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C- terminal portion of mid-length dystrophin protein;218.(vi) a poly(A) signal sequence of SEQ ID NO: 13; and219.(vii) a 3 ’-ITR sequence of SEQ ID NO: 14;220.wherein the first AAV particle comprises AAV9 capsid proteins; and wherein the second AAV particle comprises AAV9 capsid proteins.

137. A pharmaceutical composition comprising an effective amount of the composition of any one of claims 1-136 and a pharmaceutically acceptable carrier, excipient, diluent, or buffer.

138. The pharmaceutical composition of claim 137, wherein the effective amount of the composition is a composition comprising about the same number of first AAV particles and second AAV particles.

139. The pharmaceutical composition of claim 137, wherein the composition comprises about the same number of vector genomes of the first AAV particle and the second AAV particle.

140. The pharmaceutical composition of claim 137, wherein the composition comprises the same number of vector genomes of the first AAV particle and the second AAV particle.

141. The pharmaceutical composition of claim 137, wherein the composition comprises the first AAV particle and the second AAV particle combined at a 1:1 ratio.

142. The pharmaceutical composition of claim 141, wherein the 1:1 ratio is a 1:1 ratio of encapsidated transgenes.

143. The pharmaceutical composition of claim 141, wherein the 1:1 ratio is a 1:1 ratio of first AAV particles to second AAV particles.

144. A method of treating a dystrophinopathy in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 138-143.

145. The method of claim 144, wherein the subject is a male subject from about 6 months to about 7 years old.

146. The method of claim 144, wherein the subject is a male subject from about 1 year to about 7 years old.

147. The method of claim 144, wherein the subject is a male subject from about 2 years to about 7 years old.

148. The method of claim 144, wherein the subject is a male subject from about 2 years to about 5 years old.

149. The method of any one of claims 144-148, wherein the dystrophinopathy is Duchenne muscular dystrophy (DMD).

150. The method of any one of claims 144-148, wherein the dystrophinopathy is Becker muscular dystrophy.

151. The method of any one of claims 144-148, wherein the dystrophinopathy is DMD-associated dilated cardiomyopathy (DCM).

152. The method of any one of claims 144-151, wherein the subject is placed in the Trendelenburg position during the treating.

153. The method of any one of claims 144-152, wherein the composition is administered intrathecally to the subject.

154. The method of any one of claims 144-152, wherein the composition is administered intravenously to the subject.

155. The method of any one of claims 144-154, wherein the composition increases midlength dystrophin expression in skeletal and / or cardiac muscle compared to the pre-treatment level.

156. The method of claim 155, wherein the treating comprises increasing the mid-length dystrophin protein level in the subject to at least 200%, about 195%, about 190%, about 185%, about 180%, about 175%, about 170%, about 165%, about 160%, about 155%, about 150%, about 145%, about 140%, about 135%, about 130% about 125%, about 120%, about 115%, about 110%, about 105%, about 100%, about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30% about 25%, about 20%, about 15%, about 10%, about 5%, about 2%, or about 1% of the pre-treatment level.

157. The method of any one of claims 144-156, wherein the effective amount of the administered AAV particles reduces the severity of one or more dystrophinopathy symptoms in the subject.

158. The method of claim 157, wherein the one or more dystrophinopathy symptoms comprise muscle weakness, muscle stiffness, muscle pain, reduced motor skills, difficulty swallowing, difficulty breathing, or irregular heartbeat.

159. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about l><109to about l><1016vector genomes (vg) of the first AAV particle and 1 x 109to about 1 x 1016vg of the second AAV particle.

160. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×109to about 1×1015vg of the first AAV particle and 1×109to about 1×1015vg of the second AAV particle.

161. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×109to about 1×1014vg of the first AAV particle and about 1×109to about 1×1014vg of the second AAV particle.

162. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×109to about 1×1013vg of the first AAV particle and from about 1×109to about 1×1013vg of the second AAV particle.

163. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about l><109to about l><1012vg of the first AAV particle and.

164. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×109to about 1×1011vg of the first AAV particle and from about 1×109to about 1×1011vg of the second AAV particle.

165. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1010to about 1×1016total vg of the first AAV particle and from about 1×1010to about 1×1016vg of the second AAV particle.

166. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1010to about 1×1015total vg of the first AAV particle and from about 1×1010to about 1×1015vg of the second AAV particle.

167. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1010to about 1×1014total vg of the first AAV particle and from about 1×1010to about 1×1014vg of the second AAV particle.

168. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1010to about 1×1013total vg of the first AAV particle and from about 1×1010to about 1×1013vg of the second AAV particle.

169. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1010to about 1×1012total vg of the first AAV particle and from about 1×1010to about 1×1012vg of the second AAV particle.

170. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1011to about 1×1016total vg of the first AAV particle and from about 1×1011to about 1×1016vg of the second AAV particle.

171. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1011to about 1×1015total vg of the first AAV particle and from about 1×1011to about 1×1015vg of the second AAV particle.

172. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about IxlO11to about l><1014total vg of the first AAV particle and from about 1 x 1011to about 1 x 1014vg of the second AAV particle.

173. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1011to about 1×1013total vg of the first AAV particle and from about 1×1011to about 1×1013vg of the second AAV particle.

174. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1012to about 1×1016total vg of the first AAV particle and from about 1×1012to about 1×1016vg of the second AAV particle.

175. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1012to about 1×1015total vg of the first AAV particle and from about 1×1012to about 1×1015vg of the second AAV particle.

176. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1012to about 1×1014total vg of the first AAV particle and from about 1×1012to about 1×1014vg of the second AAV particle.

177. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1.0×1013to about 1×1016total vg of the first AAV particle and from about 1×1013to about 1×1016vg of the second AAV particle.

178. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1.0×1013to about 1×1015total vg of the first AAV particle and from about IxlO13to about IxlO15vg of the second AAV particle.

179. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 1×1012vg of the first AAV particle and about 1×1012vg of the second AAV particle.

180. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 5×1012vg of the first AAV particle and about 5×1012vg of the second AAV particle.

181. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 1×1013vg of the first AAV particle and about 1×1013vg of the second AAV particle.

182. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 5×1013vg of the first AAV particle and about 5×1013vg of the second AAV particle.

183. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 1×1014vg of the first AAV particle and about 1×1014vg of the second AAV particle.

184. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 5×1014vg of the first AAV particle and about 5×1014vg of the second AAV particle.

185. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 1×1015vg of the first AAV particle and about 1×1015vg of the second AAV particle.

186. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 5×1015vg of the first AAV particle and about 5×1015vg of the second AAV particle.

187. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 1×1016vg of the first AAV particle and about 1×1016vg of the second AAV particle.

188. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is about 5×1016vg of the first AAV particle and about 5×1016vg of the second AAV particle.

189. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1014to about 5×1015vg of the first AAV particle and from about 1×1014to about 5×1015vg of the second AAV particle.

190. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 5×1014to about 1×1015vg of the first AAV particle and from about 5×1014to about 1×1015vg of the second AAV particle.

191. The method of any one of claims 144-158, wherein the effective amount of the AAV particles in the composition is from about 1×1014to about 1×1015vg of the first AAV particle and from about 1×1014to about 1×1015vg of the second AAV particle.

192. The method of any one of claims 144-191, wherein the effective amount of the AAV particles is administered as a single dose.

193. The method of any one of claims 144-191, wherein the effective amount of the AAV particles is administered as two doses.

194. The method of any one of claims 144-191, wherein treating comprises increasing the subject’s NorthStar Ambulatory Assessment (NSAA) score subsequent to treatment, compared to a baseline NSAA score of the subject, wherein the baseline NSAA score of the subject is measured prior to administration of the composition.

195. The method of claim 194, wherein increasing the score comprises increasing the score from about 5 points to about 25 points, about 5 points to about 20 points, about 5 points to about 15 points, or about 5 points to about 10 points.

196. The method of claim 194 or claim 195, wherein the NSAA score subsequent to treatment is measured about 6 months, about 12 months, about 18 months, or about 24 months after administration of the composition.

197. The method of any one of claims 144-196, wherein treating comprises increasing the number of meters walked by the subject in the six-minute walk test (6MWT) subsequent to treatment, compared to a baseline number of meters walked by the subject in the 6MWT, wherein the baseline number of meters walked is measured prior to administration of the composition..

198. The method of claim 197, wherein the number of meters walked by the subject in the 6MWT subsequent to treatment is measured about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, or about 60 months after administration of the composition.

199. The method of claim 197 or claim 198, wherein increasing the number of meters walked by the subject in the 6MWT comprises increasing by about 5 meters to about 50 meters, about 5 meters to about 45 meters, about 5 meters to about 40 meters, about 5 meters to about 35 meters, about 5 meters to about 30 meters, about 5 meters to about 25 meters, about 5 meters to about 20 meters, about 5 to about 15 meters, or about 5 meters to about 10 meters.

200. The method of any one of claims 144-199, wherein treating comprises decreasing the time to complete the 4-stair climb test (4SC) for the subject subsequent to treatment, as compared to a baseline time of the subject to complete the 4SC, wherein the baseline time is measured prior to the administration of the composition.

201. The method of claim 200, wherein the time to complete the 4SC subsequent to treatment is measured about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, or about 60 months after administration of the composition.

202. The method of claim 200 or claim 201, wherein treating comprises decreasing the time to complete the 4SC for the subject subsequent to the treatment by about 1 second to about 8 seconds, by about 1 second to about 7 seconds, by about 1 second to about 6 seconds, by about 1 second to about 5 seconds, by about 1 second to about 4 seconds, by about 1 second to about 3 seconds, or by about 1 second to about 2 seconds.

203. The method of any one of claims 144-202, wherein treating comprises decreasing a time to stand (TTSTAND) for the subject subsequent to treatment, compared to a baseline TTSAND for the subject, wherein the baseline TTSAND is measured prior to administration of the composition.

204. The method of claim 203, wherein the TTSTAND subsequent to treatment is measured about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, or about 60 months after administration of the composition.

205. Th method of claim 203 or claim 204, wherein treating comprises decreasing the TTSTAND by about 1 second to about 10 seconds, by 1 to about 9 seconds, by about 1 second to about 8 seconds, by about 1 second to about 7 seconds, by about 1 second to about 6 seconds, by about 1 second to about 5 seconds, by about 1 second to about 4 seconds, by about 1 second to about 3 seconds, or by about 1 second to about 2 seconds.

206. The method of any one of claims 144-205, wherein treating comprises increasing a Gross Motor Outcome (GRO) score of the subject subsequent to treatment, compared to a baseline GRO score, wherein the baseline GRO score is measured in the subject prior to administration of the composition.

207. A method for delivering a mid-dystrophin transgene to skeletal and / or cardiac muscle of a subject, comprising: intrathecally or intravenously administering to a subject an effective amount of the pharmaceutical composition of claim 142 or claim 143.

208. A DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’:285.(i) a 5’-ITR sequence;286.(ii) a promoter sequence;287.(iii) a Kozak sequence;288.(iv) an N-terminal coding sequence encoding an N-terminal portion of a midlength dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter;289.(v) a splicing donor sequence;290.(vi) a dimerization domain sequence;291.(vii) a poly(A) signal sequence; and292.(viii) a 3 ’ -ITR sequence;293.wherein the N-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9.

209. The DNA plasmid of claim 208, wherein the splicing donor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.

210. The DNA plasmid of claim 208 or claim 209, wherein the dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11.

211. The DNA plasmid of any one of claims 208-210, wherein the promoter is muscle tissuespecific promoter.

212. The DNA plasmid of claim 211, wherein the muscle tissue-specific promoter is a MHCK7 promoter.

213. The DNA plasmid of claim 212, wherein the MHCK7 promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

214. The DNA plasmid of any one of claims 208-213, wherein the 5’-ITRis a 5’ AAV2 ITR.

215. The DNA plasmid of claim 214, wherein the 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

216. The DNA plasmid of any one of claims 208-215, wherein the 3 ’-ITR is a 3’ AAV2 ITR.

217. The DNA plasmid of claim 216, wherein the 3 ’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.

218. The DNA plasmid of any one of claims 208-217, wherein the poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.

219. The DNA plasmid of any one of claims 208-218, wherein the Kozak sequence comprises a nucleic acid sequence of SEQ ID NO: 3.

220. A DNA plasmid comprising an N-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’:305.(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;306.(ii) a promoter sequence of SEQ ID NO: 2;307.(iii) a Kozak sequence of SEQ ID NO: 3;308.(iv) an N-terminal coding sequence of SEQ ID NO: 9 encoding an N-terminal portion of a mid-length dystrophin protein, said N-terminal coding sequence being operably linked to and under control of said promoter;309.(v) a splicing donor sequence of SEQ ID NO: 10;310.(vi) a dimerization domain sequence of SEQ ID NO: 11;311.(vii) a poly(A) signal sequence of SEQ ID NO: 13; and312.(viii) a 3 ’-ITR sequence of SEQ ID NO:

14.

221. A DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’:313.(i) a 5’-ITR sequence;314.(ii) a promoter sequence;315.(iii) a dimerization domain sequence, said dimerization domain being operably linked to and under control of said promoter;316.(iv) a splicing acceptor sequence;317.(v) a C-terminal coding sequence encoding a C-terminal portion of a midlength dystrophin protein;318.(vi) a poly(A) signal sequence; and319.(vii) a 3 ’ -ITR sequence;320.wherein the C-terminal coding sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 21.

222. The DNA plasmid of claim 221, wherein the splicing acceptor comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

223. The DNA plasmid of claim 221 or claim 222, wherein the dimerization domain comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15.

224. The DNA plasmid of any one of claims 221-223, wherein the promoter is a muscle tissue-specific promoter.

225. The DNA plasmid of claim 224, wherein the muscle tissue-specific promoter is a MHCK7 promoter.

226. The DNA plasmid of claim 225, wherein the MHCK7 promoter comprises the nucleic acid sequence of SEQ ID NO: 2.

227. The DNA plasmid of any one of claims 221-226, wherein the 5 ’-ITR is a 5’ AAV2 ITR.

228. The DNA plasmid of claim 227, wherein the 5’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1.

229. The DNA plasmid of any one of claims 221-228, wherein the 3’ -ITR is a 3’ AAV2 ITR.

230. The DNA plasmid of claim 229, wherein the 3’-ITR comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.

231. The DNA plasmid of any one of claims 221-230, wherein the poly(A) signal sequence comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.

232. A DNA plasmid comprising a C-terminal transgene construct, wherein the transgene construct comprises from 5’ to 3’:331.(i) a 5 ’ -ITR sequence of SEQ ID NO: 1;332.(ii) a promoter sequence of SEQ ID NO: 2;333.(iii) a dimerization domain sequence of SEQ ID NO: 15, said dimerization domain being operably linked to and under control of said promoter;334.(iv) a splicing acceptor sequence of SEQ ID NO: 16;335.(v) a C-terminal coding sequence of SEQ ID NO: 21 encoding a C-terminal portion of a mid-length dystrophin protein;336.(vi) a poly(A) signal sequence of SEQ ID NO: 13; and337.(vii) a 3 ’-ITR sequence of SEQ ID NO: 14.

233. A packaging cell comprising one or more DNA plasmids of any one of claims 208-232.

234. The packaging cell of claim 233, comprising the DNA plasmid comprising an N-terminal transgene construct and the DNA plasmid comprising a C-terminal transgene construct.

235. The packaging cell of claim 233 or claim 234, further comprising a helper plasmid containing adenoviral components necessary for recombinant adeno-associated virus production.

236. The packaging cell of any one of claims 233-235, further comprising a plasmid comprising AAV replication (rep) and capsid (cap) genes.

237. The packaging cell of any one of claims 233-236, wherein the packaging cell is a HEK293 cell.

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