Miniaturized dystrophins and uses thereof
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.
1 . A nucleic acid molecule comprising a nucleotide sequence, which encodes a miniaturized dystrophin polypeptide comprising from N terminus to C terminus hinge 1 (H1) domain, spectrin repeat 1 (R1) domain, spectrin repeat 3 (R3) domain, hinge 2 (H2) domain, spectrin repeat 16 (R16) domain, spectrin repeat 17 (R17) domain, spectrin repeat 24 (R24) domain, and hinge 4 (H4) domain of dystrophin, wherein the miniaturized dystrophin polypeptide does not comprise spectrin repeat 2 (R2) of dystrophin, wherein the R1 domain and the R3 domain are fused by amino acids ARGVAL (RV) and wherein the H2 domain and the R16 domain are fused by the amino acid sequence set forth in SEQ ID NOs: 74 or 75, and wherein (i) the H1 domain and the R1 domain are fused directly, (ii) the R3 domain and the H2 domain are fused directly, (iii) the R16 and R17 domains are fused directly, (iv) the R17 and R24 domains are fused directly, and (v) the R24 and H4 domains are fused directly.
2 . The nucleic acid molecule of claim 1 , wherein the miniaturized dystrophin polypeptide further comprises an ABD1 domain and/or a CR domain.
3 . The nucleic acid molecule of claim 2 , wherein the miniaturized dystrophin polypeptide consists of, from N terminus to C terminus, the ABD1 domain, the H1 domain, the R1 domain, amino acids RV, the R3 domain, the H2 domain, the amino acid sequence as set forth in SEQ ID NOs: 74 or 75, the R16 domain, the R17 domain, the R24 domain, the H4 domain, and the CR domain of dystrophin.
4 . The nucleic acid molecule of claim 3 , wherein the miniaturized dystrophin polypeptide comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 118.
5 . The nucleic acid molecule of claim 4 , wherein the nucleotide sequence comprises a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 133.
6 . The nucleic acid molecule of any one of claim 5 , wherein the miniaturized dystrophin polypeptide comprises the amino acid sequence of SEQ ID NO: 118.
7 . A vector comprising the nucleic acid molecule of claim 1 .
8 . The vector of claim 7 , which is an adeno-associated virus (AAV) vector, wherein the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.
9 . The vector of claim 8 , wherein the AAV vector is AAV9.
10 . The vector of claim 8 , wherein the AAV vector is AAV8.
11 . A host cell comprising the vector of claim 7 .
12 . A pharmaceutical composition comprising the vector of claim 7 and a pharmaceutically acceptable excipient.
13 . A polypeptide encoded by the nucleic acid molecule of claim 1 .
14 . A pharmaceutical composition comprising the polypeptide of claim 13 and a pharmaceutically acceptable excipient.
15 . A host cell comprising the nucleic acid molecule of claim 1 .
16 . A pharmaceutical composition comprising the host cell of claim 15 and a pharmaceutically acceptable excipient.
17 . A pharmaceutical composition comprising the nucleic acid molecule of claim 1 and a pharmaceutically acceptable excipient.
18 . A recombinant adeno-associated virus (rAAV) vector particle comprising an AAV capsid and a nucleic acid molecule comprising a nucleotide sequence, which encodes a miniaturized dystrophin polypeptide comprising the amino acid sequence of SEQ ID NO: 118.
19 . The rAAV vector particle of claim 18 , comprising a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 133.
20 . The rAAV vector particle of claim 19 , wherein the nucleic acid molecule further comprises a first ITR and a second ITR both from AAV.
21 . The rAAV vector particle of claim 20 , wherein the first ITR and the second ITR are from the genome of AAV2.
22 . The rAAV vector particle of claim 21 , wherein the AAV capsid is from AAV9.
23 . The rAAV vector particle of claim 21 , wherein the AAV capsid is from AAV8.
24 . The rAAV vector particle of claim 18 , wherein the nucleic acid molecule further comprises a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR) both from adeno-associated virus (AAV).
25 . The rAAV vector particle of claim 24 , wherein the first ITR and the second ITR are from the genome of AAV2.
26 . The rAAV vector particle of claim 25 , wherein the AAV capsid is from AAV9.
27 . The rAAV vector particle of claim 25 , wherein the AAV capsid is from AAV8.