Production of large-sized microdystrophins in an AAV-based vector configuration
An adeno-associated viral (AAV) vector containing an expression construct, wherein: the expression construct comprises a nucleic acid sequence which encodes a microdystrophin (MD); and the nucleic acid sequence encoding the MD has a size of at least 4.1 kb.
1. An adeno-associated viral (AAV) vector comprising an expression construct, wherein:
the expression construct comprises a nucleic acid sequence which encodes a microdystrophin (MD);
the nucleic acid sequence encoding the MD has a size of at least 4.1 kb;
the expression construct has a size of less than 6 kb; and
the MD contains a partial or full-length C terminal domain of dystrophin.
2. The AAV vector according to claim 1 , wherein the expression construct comprises two ITR sequences, and wherein the expression construct and the two ITR sequences together have a size of at least 5.1 kb.
3. The AAV vector according to claim 2 , wherein the expression construct comprises two ITR sequences, and wherein the expression construct comprises or consists of the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 18.
4. The AAV vector according to claim 2 , wherein the expression construct has a size of at least 5.2 kb.
5. The AAV vector according to claim 2 , wherein the expression construct has a size of at least 5.45 kb.
6. The AAV vector according to claim 1 , wherein the AVV vector is an AAV vector of serotype 8 (AAV8) or 9 (AAV9).
7. The AAV vector according to claim 6 , wherein the AAV vector is an AAV 2/8 or an AAV 2/9 vector.
8. The AAV vector according to claim 1 , wherein the expression construct further comprises a muscle-specific promoter which is operably linked to the nucleic acid sequence encoding the MD.
9. The AAV vector according to claim 8 , wherein the muscle-specific promoter is a Spc5-12 promoter.
10. The AAV vector according to claim 1 , wherein said MD comprises or consists of the sequence of SEQ ID NO: 13 or SEQ ID NO: 16, or a variant thereof having at least 80% sequence identity thereto.
11. A composition comprising the AAV vector according to claim 1 and a pharmaceutically acceptable carrier.
12. A method for treating a subject with a dystrophic disease, comprising administering to the subject an effective amount of the composition of claim 11 , thereby treating the dystrophic disease.
13. The method of claim 12 , wherein the subject is a human.
14. The method of claim 13 , wherein the dystrophic disease is Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).
15. The method of claim 12 , wherein said MD comprises a central rod domain and a partial or full C-terminal domain, and wherein said MD has a deletion ΔR4-23 in the central rod domain and wherein the partial or full-length C terminal domain is encoded by exons 70 to 75, or exons 70 to 79, respectively.
16. The AAV vector according to claim 1 , wherein said MD comprises or consists of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 16 or a variant thereof having at least 90% sequence identity thereto.
17. The AAV vector according to claim 16 , wherein said MD comprises or consists of the sequence of SEQ ID NO: 13 or SEQ ID NO: 16.
18. An adeno-associated viral (AAV) vector comprising an expression construct, wherein:
the expression construct comprises a nucleic acid sequence which encodes a microdystrophin (MD);
the nucleic acid sequence encoding the MD has a size of at least 4.1 kb;
the MD contains a partial or full length C terminal domain of dystrophin, and
said MD comprises a central rod domain and has a deletion ΔR4-23 in the central rod domain and wherein the partial or full-length C terminal domain is encoded by exons 70 to 75 or exons 70 to 79, respectively.
19. A method for treating a subject with a dystrophic disease, comprising administering to the subject an effective amount of the composition of claim 18 , thereby treating the dystrophic disease.