# US 2023/0407331 A1 · Miniaturized dystrophins having spectrin fusion domains and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2023/0407331 A1 - Document ID: US20230407331A1 - Status: Published application - Kind: A1 - Application number: 18/051,936 - Filed: 2022-11-02 - Published: 2023-12-21 - Earliest claimed: 2020-04-29 - Assignees: Bristol-Myers Squibb Company - Applicants: Bristol-Myers Squibb Company - Inventors: Glen B. Banks; Jonathan Harry Davis; Paul Charles Levesque - Web page: https://patentgrep.com/patent/US20230407331A1 ## CPC - C07K 14/4708: {Duchenne dystrophy} - C12N 15/86: Viral vectors - A61P 21/00: Drugs for disorders of the muscular or neuromuscular system - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C07K 14/47: from mammals - C12N 2750/14143: viral genome or elements thereof as genetic vector - C12N 2830/50: regulating RNA stability, not being an intron, e.g. poly A signal - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Disclosed herein are nucleic acid molecules, polypeptides, cells, vectors, and pharmaceutical compositions relating to miniaturized dystrophin. Methods of production and methods of therapeutic use of the miniaturized dystrophin are also disclosed. ## Claims ### Claim 1 A nucleic acid molecule comprising a nucleotide sequence, which encodes a miniaturized dystrophin polypeptide comprising a modified spectrin repeat 16 (R16) domain, wherein a part of spectrin repeat 16 (R16) domain is replaced by a corresponding part of a different spectrin repeat domain. ### Claim 2 (depends on 1) The nucleic acid molecule of claim 1, wherein the different spectrin repeat domain is spectrin repeat 2 (R2) domain. ### Claim 3 (depends on 1, 2) The nucleic acid molecule of claim 1 or 2, wherein the modified R16 domain comprises an amino acid sequence 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 about 100% identical to a sequence selected from the group consisting of SEQ ID NO: 68, 69, 70 and 71. ### Claim 4 (depends on 1, 2, 3) The nucleic acid molecule of any one of claims 1 to 3, wherein the miniaturized dystrophin polypeptide comprises from N terminus to C terminus a hinge 1 (H1) domain, a spectrin repeat 1 (R1) domain, the modified R16 domain, a spectrin repeat 17 (R17) domain, a hinge 3 (H3) domain, a spectrin repeat 23 (R23) domain, a spectrin repeat 24 (R24) domain, and a hinge 4 (H4) domain of dystrophin. ### Claim 5 (depends on 4) The nucleic acid molecule of claim 4, wherein (i) the H1 domain and the R1 domain are fused directly, (ii) the R1 domain and the modified R16 domain are fused directly, (iii) the modified R16 domain and the R17 domain are fused directly, (iv) the R17 domain and the H3 domain are fused directly, (v) the H3 domain and the R23 domain are fused directly, (vi) the R23 domain and the R24 domain are fused directly, or (vii) the R24 domain and the H4 domain are fused directly, or (vii) any combination thereof. ### Claim 6 (depends on 1, 2, 3, 4, 5) The nucleic acid molecule of any one of claims 1 to 5, wherein the miniaturized dystrophin polypeptide does not comprise a spectrin repeat 2 (R2) domain, spectrin repeat 3 (R3) domain, spectrin repeat 4 (R4) domain, spectrin repeat 5 (R5) domain, spectrin repeat 6 (R6) domain, spectrin repeat 7 (R7) domain, spectrin repeat 8 (R8) domain, spectrin repeat 9 (R9) domain, spectrin repeat 10 (R10) domain, spectrin repeat 11 (R11) domain, spectrin repeat 12 (R12) domain, spectrin repeat 13 (R13) domain, spectrin repeat 14 (R14) domain, spectrin repeat 15 (R15) domain, spectrin repeat 18 (R18) domain, spectrin repeat 19 (R19) domain, spectrin repeat 20 (R20) domain, spectrin repeat 21 (R21) domain, and/or spectrin repeat 22 (R22) domain. ### Claim 7 (depends on 1, 2, 3, 4, 5, 6) The nucleic acid molecule of any one of claims 1 to 6, wherein the miniaturized dystrophin polypeptide further comprises an ABD1 domain and/or a CR domain. ### Claim 8 (depends on 7) The nucleic acid molecule of claim 7, wherein the miniaturized dystrophin polypeptide consists essentially of or consists of, from N terminus to C terminus, the ABD1 domain, the H1 domain, the R1 domain, the modified R16 domain, the R17 domain, the H3 domain, the R23 domain, the R24 domain, the H4 domain, and the CR domain of dystrophin. ### Claim 9 (depends on 4, 5, 6, 7, 8) The nucleic acid molecule of any one of claims 4 to 8, wherein the H1 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 74. ### Claim 10 (depends on 4, 5, 6, 7, 8, 9) The nucleic acid molecule of any one of claims 4 to 9, wherein the R1 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 75. ### Claim 11 (depends on 4, 5, 6, 7, 8, 9, 10) The nucleic acid molecule of any one of claims 4 to 10, wherein the modified R16 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 76. ### Claim 12 (depends on 4, 5, 6, 7, 8, 9, 10, 11) The nucleic acid molecule of any one of claims 4 to 11, wherein the R17 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 77. ### Claim 13 (depends on 4, 5, 6, 7, 8, 9, 10, 11, 12) The nucleic acid molecule of any one of claims 4 to 12, wherein the H3 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 78. ### Claim 14 (depends on 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) The nucleic acid molecule of any one of claims 4 to 13, wherein the R23 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 79. ### Claim 15 (depends on 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) The nucleic acid molecule of any one of claims 4 to 14, wherein the R24 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 80. ### Claim 16 (depends on 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) The nucleic acid molecule of any one of claims 4 to 15, wherein the H4 domain is an amino acid sequence 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 about 100% identical to SEQ ID NO: 81. ### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) The nucleic acid molecule of any one of claims 1 to 16, wherein the miniaturized dystrophin polypeptide further comprises at the N terminus an amino acid sequence 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 about 100% identical to SEQ ID NO: 73. ### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) The nucleic acid molecule of any one of claims 1 to 17, wherein the miniaturized dystrophin polypeptide further comprises at the C terminus an amino acid sequence 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 about 100% identical to SEQ ID NO: 82. ### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The nucleic acid molecule of any one of claims 1 to 18, wherein the miniaturized dystrophin polypeptide comprises an amino acid sequence 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 about 100% identical to SEQ ID NO: 83. ### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The nucleic acid molecule of any one of claims 1 to 18, wherein the miniaturized dystrophin polypeptide comprises an amino acid sequence 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 about 100% identical to SEQ ID NO: 84. ### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The nucleic acid molecule of any one of claims 1 to 18, wherein the miniaturized dystrophin polypeptide comprises an amino acid sequence 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 about 100% identical to SEQ ID NO: 85. ### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The nucleic acid molecule of any one of claims 1 to 18, wherein the miniaturized dystrophin polypeptide comprises an amino acid sequence 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 about 100% identical to SEQ ID NO: 86. ### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) The nucleic acid molecule of any one of claims 1 to 18, wherein the miniaturized dystrophin polypeptide comprises an amino acid sequence 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 about 100% identical to SEQ ID NO: 87. ### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) The nucleic acid molecule of any one of claims 1 to 23, wherein the miniaturized dystrophin polypeptide exhibits a higher expression of the miniaturized dystrophin polypeptide than BXA-212372 (SEQ ID NO: 88). ### Claim 25 (depends on 24) The nucleic acid molecule of claim 24, wherein the miniaturized dystrophin polypeptide expression is at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold. at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold or at least about 3 fold higher than the BXA-212372 polypeptide (SEQ ID NO: 88) expression. ### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) The nucleic acid molecule of any one of claims 1 to 25, which further comprises a promoter. ### Claim 27 (depends on 26) The nucleic acid molecule of claim 26, wherein the promoter is a tissue-specific promoter. ### Claim 28 (depends on 26, 27) The nucleic acid molecule of claim 26 or 27, wherein the promoter drives expression of the therapeutic protein in muscle cells, hepatocytes, endothelial cells, neuronal cells, sinusoidal cells, or any combination thereof. ### Claim 29 (depends on 26, 27, 28) The nucleic acid molecule of any one of claims 26 to 28, wherein the promoter is selected from the group consisting of a C5-12(T) promoter, an MLC2v-cTNT455 promoter, a mouse thyretin promoter (mTTR), an endogenous human factor VIII promoter (F8), a human alpha-1-antitrypsin promoter (hAAT), a human albumin minimal promoter, a mouse albumin promoter, a tristetraprolin (TTP) promoter, a CASI promoter, a synapsin 1 gene promoter, a CAG promoter, a cytomegalovirus (CMV) promoter, α1-antitrypsin (AAT), muscle creatine kinase (MCK), myosin heavy chain alpha (αNMC), myoglobin (MB), desmin (DES), SPc5-12, 2R5Sc5-12, dMCK, tMCK, and a phosphoglycerate kinase (PGK) promoter. ### Claim 30 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) The nucleic acid molecule of any one of claims 1 to 29, which further comprises an intronic sequence. ### Claim 31 (depends on 30) The nucleic acid molecule of claim 30, wherein the intronic sequence is positioned 5′ to the nucleotide sequence encoding the miniaturized dystrophin polypeptide. ### Claim 32 (depends on 30) The nucleic acid molecule of claim 30, wherein the intronic sequence is positioned 3′ to the promoter. ### Claim 33 (depends on 30, 31, 32) The nucleic acid molecule of any one of claims 30 to 32, wherein the intronic sequence comprises a synthetic intronic sequence. ### Claim 34 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33) The nucleic acid molecule of any one of claims 1 to 33, which further comprises a post-transcriptional regulatory element. ### Claim 35 (depends on 34) The nucleic acid molecule of claim 34, wherein the post-transcriptional regulatory element is positioned 3′ to the nucleotide sequence encoding the miniaturized dystrophin polypeptide. ### Claim 36 (depends on 34, 35) The nucleic acid molecule of claim 34 or 35, wherein the post-transcriptional regulatory element comprises a mutated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a microRNA binding site, or a DNA nuclear targeting sequence, or any combination thereof. ### Claim 37 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) The nucleic acid molecule of any one of claims 1 to 36, which further comprises a 3′UTR poly(A) tail sequence. ### Claim 38 (depends on 37) The nucleic acid molecule of claim 37, wherein the 3′UTR poly(A) tail sequence is selected from the group consisting of dystrophin poly(A), bGH poly(A), actin poly(A), hemoglobin poly(A), and any combination thereof. ### Claim 39 (depends on 37, 38) The nucleic acid molecule of claim 37 or 38, wherein the 3′UTR poly(A) tail sequence comprises dystrophin poly(A). ### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) The nucleic acid molecule of any one of claims 1 to 39, which further comprises an enhancer sequence. ### Claim 41 (depends on 40) The nucleic acid molecule of claim 40, which further comprises a first ITR and/or a second ITR. ### Claim 42 (depends on 41) The nucleic acid molecule of claim 41, wherein the first ITR and the second ITR are identical. ### Claim 43 (depends on 41, 42) The nucleic acid molecule of claim 41 or 42, wherein the first ITR and/or the second ITR are derived from adeno-associated virus. ### Claim 44 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) The nucleic acid molecule of any one of claims 1 to 43, which comprises a sequence encoding a heterologous moiety. ### Claim 45 (depends on 44) The nucleic acid molecule of claim 44, wherein the heterologous moiety is selected from the group consisting of albumin or a fragment thereof, an immunoglobulin Fc region, the C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin, a PAS sequence, a HAP sequence, a transferrin or a fragment thereof, an albumin-binding moiety or a derivative thereof, and any combination thereof. ### Claim 46 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) A vector comprising the nucleic acid molecule of any one of claims 1 to 45. ### Claim 47 (depends on 46) The vector of claim 46, which is selected from the group consisting of a adenoviral vector, a retroviral vector, poxvirus vector, a baculovirus vector, a herpes viral vector. ### Claim 48 (depends on 46) The vector of claim 46, which is an adeno-associated virus (AAV) vector. ### Claim 49 (depends on 48) The vector of claim 48, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. ### Claim 50 (depends on 48) The vector of claim 48, wherein the AAV vector is AAV8 or AAV9. ### Claim 51 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) The nucleic acid molecule of any one of claims 1 to 45 or vector of any one of claims 46 to 50, wherein the nucleic acid molecule or the vector is formulated with a delivery agent. ### Claim 52 (depends on 51) The nucleic acid molecule or vector of claim 51, wherein the delivery agent comprises a lipid nanoparticle. ### Claim 53 (depends on 51) The nucleic acid molecule or vector of claim 51, wherein the delivery agent is selected from the group consisting of liposomes, non-lipid polymeric molecules, endosomes, and any combination thereof. ### Claim 54 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) The nucleic acid molecule of any one of claims 1 to 45, the vector of any one of claims 46 to 50, or the nucleic acid molecule or vector of any one of claims 51 to 53, which is formulated for intravenous, transdermal, intradermal, subcutaneous, pulmonary, or oral delivery, or any combination thereof. ### Claim 55 (depends on 54) The nucleic acid molecule or vector of claim 54, which is formulated for intravenous delivery. ### Claim 56 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55) A polypeptide encoded by the nucleic acid molecule of any one of claims 1 to 45, the vector of any one of claims 46 to 50, or the nucleic acid molecule or the vector of any one of claims 51 to 55. ### Claim 57 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) A host cell comprising the nucleic acid molecule of any one of claims 1 to 45 or vector of any one of claims 46 to 50. ### Claim 58 (depends on 57) The host cell of claim 57, wherein the cell is a CHO cell, a HEK293 cell, a HBK cell, a COS cell, a NSO cell, or a HT1080 cell. ### Claim 59 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58) A pharmaceutical composition comprising (a) the nucleic acid of any one of claims 1 to 45, the vector of any one of claims 46 to 50, the nucleic acid molecule or the vector of any one of claims 51 to 55, the polypeptide of claim 56, or the host cell of claim 57 or 58; and (b) a pharmaceutically acceptable excipient. ### Claim 60 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59) A kit, comprising the nucleic acid of any one of claims 1 to 45, the vector of any one of claims 46 to 50, the nucleic acid molecule or the vector of any one of claims 51 to 55, the polypeptide of claim 56, the host cell of claim 57 or 58, or the pharmaceutical composition of claim 59, and instructions for administering the nucleic acid molecule, the vector, the polypeptide, the host cell, or the pharmaceutical composition to a subject in need thereof. ### Claim 61 (depends on 57, 58) A method of producing a miniaturized dystrophin polypeptide, comprising: culturing the host cell of claim 57 or 58 under suitable conditions and recovering the miniaturized dystrophin polypeptide. ### Claim 62 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 59) A method of expressing a miniaturized dystrophin polypeptide in a subject in need thereof, comprising administering to the subject the nucleic acid of any one of claims 1 to 45, the vector of any one of claims 46 to 50, the nucleic acid molecule or the vector of any one of claims 51 to 55, the host cell of claim 57 or 58, or the pharmaceutical composition of claim 59. ### Claim 63 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59) A method of treating a subject having a disease or condition comprising administering to the subject the nucleic acid of any one of claims 1 to 45, the vector of any one of claims 46 to 50, the nucleic acid molecule or the vector of any one of claims 51 to 55, the polypeptide of claim 56, the host cell of claim 57 or 58, or the pharmaceutical composition of claim 59. ### Claim 64 (depends on 63) The method of claim 63, wherein the disease or condition is a disease caused by dystrophin deficiency. ### Claim 65 (depends on 64) The method of claim 64, wherein the disease is Sarcopenia, a heart disease, cachexia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), X-linked dilated cardiomyopathy (XLDC), facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, distal muscular dystrophy, and/or congenital muscular dystrophy. ### Claim 66 (depends on 62, 63, 64, 65) The method of any one of claims 62 to 65, wherein the nucleic acid molecule, the vector, the polypeptide, the host cell, or the pharmaceutical composition is administered intravenously, transdermally, intradermally, subcutaneously, orally, or pulmonarily, or any combination thereof. ### Claim 67 (depends on 63, 64, 65, 66) The method of any one of claims 63 to 66, further comprising administering to the subject a second agent. ### Claim 68 (depends on 62, 63, 64, 65, 66, 67) The method of any one of claims 62 to 67, wherein the subject is a human. ### Claim 69 (depends on 62, 63, 64, 65, 66, 67, 68) The method of any one of claims 62 to 68, wherein the administration of the nucleic acid molecule, the vector, the polypeptide, the host cell, or the pharmaceutical composition to the subject results in increased dystrophin protein expression, relative to dystrophin protein expression in the subject prior to the administration, wherein the dystrophin protein expression is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold. ### Claim 70 A nucleic acid molecule comprising a nucleotide sequence, which encodes a miniaturized dystrophin polypeptide comprising an amino acid sequence of SEQ ID NO: 83. ### Claim 71 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69) The nucleic acid molecule of any one of claims 1 to 45, the vector of any one of claims 46 to 50, the nucleic acid molecule or vector of any one of claims 51 to 55, the polypeptide of claim 56, the host cell of claim 57 or 58, the pharmaceutical composition of claim 59, the kit of claim 60, or the method of any one of claims 61 to 69, comprising a nucleotide sequence encoding a miniaturized dystrophin polypeptide comprising an amino acid sequence of SEQ ID NO: 83. ### Claim 72 A nucleic acid molecule comprising a nucleotide sequence comprising in order a C5-12(T) promoter of SEQ ID NO: 109, an SV40 intron of SEQ ID NO: 110, a coding sequence for miniaturized dystrophin BXA-220931 of SEQ ID NO: 111, a 3′ UTR of SEQ ID NO: 112, and a polyA sequence of SEQ ID NO: 113. ### Claim 73 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69) The nucleic acid molecule of any one of claims 1 to 45, the vector of any one of claims 46 to 50, the nucleic acid molecule or vector of any one of claims 51 to 55, the polypeptide of claim 56, the host cell of claim 57 or 58, the pharmaceutical composition of claim 59, the kit of claim 60, or the method of any one of claims 61 to 69, comprising a nucleotide sequence comprising in order a C5-12(T) promoter of SEQ ID NO: 109, an SV40 intron of SEQ ID NO: 110, a coding sequence for miniaturized dystrophin BXA-220931 of SEQ ID NO: 111, a 3′ UTR of SEQ ID NO: 112, and a polyA sequence of SEQ ID NO: 113