AAV-mediated delivery of therapeutic antibodies to the inner ear
Provided herein are methods that include introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; (b) a polypeptide including an antigen-binding antibody fragment operably linked to a signal peptide; or (c) a soluble vascular endothelial growth factor receptor operably linked to a signal peptide.
1. An adeno-associated virus (AAV) vector comprising a nucleotide sequence which comprises:
(i) a first coding sequence that encodes a first polypeptide comprising an antibody heavy chain variable domain operably linked to a first signal peptide, and a second coding sequence that encodes a second polypeptide comprising an antibody light chain variable domain operably linked to a second signal peptide,
wherein the sequence encoding the antibody heavy chain variable domain consists of SEQ ID NO: 52, and the sequence encoding the antibody light chain variable domain consists of SEQ ID NO: 53, and
(ii) a sequence encoding a thosea asigna virus 2A (T2A) peptide that is present between the first coding sequence and the second coding sequence,
wherein the first and second polypeptides together specifically bind to one or more mammalian VEGF proteins.
2. A kit comprising the adeno-associated virus (AAV) vector of claim 1 .
3. A composition comprising an adeno-associated virus (AAV) vector and a pharmaceutically acceptable carrier,
wherein the AAV vector comprises a nucleotide sequence comprising:
(i) a first coding sequence that encodes a first polypeptide comprising an antibody heavy chain variable domain operably linked to a first signal peptide, and a second coding sequence that encodes a second polypeptide comprising an antibody light chain variable domain operably linked to a second signal peptide,
wherein the sequence encoding the antibody heavy chain variable domain consists of SEQ ID NO: 52, and the sequence encoding the antibody light chain variable domain consists of SEQ ID NO: 53, and
(ii) a sequence encoding a thosea asigna virus 2A (T2A) peptide that is present between the first coding sequence and the second coding sequence,
wherein the first and second polypeptides together specifically bind to one or more mammalian VEGF proteins, and
wherein the composition is formulated for intra-cochlear administration.
4. The AAV vector of claim 1 , wherein the nucleotide sequence comprises one or both of (i) a promoter and (ii) a Kozak sequence.
5. The AAV vector of claim 4 , wherein the promoter is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.
6. The AAV vector of claim 5 , wherein the constitutive promoter is a CAG promoter, a CBA promoter, or a CMV promoter.
7. The AAV vector of claim 1 , wherein the nucleotide sequence comprises a polyadenylation signal sequence.
8. The AAV vector of claim 1 , wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 54.
9. The AAV vector of claim 1 , wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 7.
10. The AAV vector of claim 1 , wherein one or both of the first and second signal peptides comprise an IL2 signal peptide.
11. The AAV vector of claim 1 , wherein the nucleotide sequence further comprises two AAV inverted terminal repeats (ITRs), wherein the two AAV ITRs flank both of the coding sequences.
12. The AAV vector of claim 11 , wherein the two AAV ITRs are AAV2 ITRs, or are derived from AAV2 ITRs.
13. The AAV vector of claim 11 , wherein one of the two ITRs comprises the nucleotide sequence as set forth in SEQ ID NO: 36.
14. The AAV vector of claim 11 , wherein one of the two ITRs comprises the nucleotide sequence as set forth in SEQ ID NO: 48.
15. The composition of claim 3 , wherein the nucleotide sequence comprises one or both of (i) a promoter and (ii) a Kozak sequence.
16. The composition of claim 15 , wherein the promoter is an inducible promoter, a constitutive promoter, or a tissue-specific promoter.
17. The composition of claim 16 , wherein the constitutive promoter is a CAG promoter, a CBA promoter, or a CMV promoter.
18. The composition of claim 3 , wherein the nucleotide sequence comprises a polyadenylation signal sequence.
19. The composition of claim 3 , wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 54.
20. The composition of claim 3 , wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 7.
21. The composition of claim 3 , wherein one or both of the first and second signal peptide comprises an IL2 signal peptide.
22. The composition of claim 3 , wherein the nucleotide sequence further comprises two AAV inverted terminal repeats (ITRs), wherein the two AAV ITRs flank both of the coding sequences.
23. The composition of claim 22 , wherein the two AAV ITRs are AAV2 ITRs, or are derived from AAV2 ITRs.
24. The composition of claim 22 , wherein one of the two ITRs comprises the nucleotide sequence as set forth in SEQ ID NO: 36.
25. The composition of claim 22 , wherein one of the two ITRs comprises the nucleotide sequence as set forth in SEQ ID NO: 48.