Compositions and methods for treating non-age-associated hearing impairment in a human subject
Provided herein are compositions that include at least two different nucleic acid vectors, where each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, and the use of these compositions to treat hearing loss in a subject.
1. A plurality of AAV vectors comprising:
a) a first AAV vector comprising a 5′ inverted terminal repeat (ITR), a 3′ ITR, and a promoter operably linked to a portion of a gene encoding an otoferlin polypeptide; and
b) a second AAV vector comprising a 5′ ITR, a 3′ ITR, and a portion of the gene encoding an otoferlin polypeptide;
wherein the first AAV vector and the second AAV vector comprise a F1 phage recombinogenic region and are capable of constituting a full-length otoferlin messenger RNA, and
wherein the first and second AAV vectors each include a total number of nucleotides of up to about 5 kb.
2. The plurality of AAV vectors of claim 1 , wherein the otoferlin polypeptide is an otoferlin isoform 5 polypeptide.
3. The plurality of AAV vectors of claim 2 , wherein the otoferlin polypeptide is the human otoferlin isoform 5 polypeptide.
4. The plurality of AVV vectors of claim 1 , wherein the F1 phage recombinogenic region is a 77 base pair recombinogenic region from the F1 phage genome.
5. The plurality of AAV vectors of claim 1 , wherein the promoter is a CAG promoter or a CAG/CBA promoter.
6. The plurality of AAV vectors of claim 1 , wherein the first AAV vector further comprises a splicing donor signal sequence and the second AAV vector further comprises a splicing acceptor signal sequence and a polyadenylation signal.
7. The plurality of AAV vectors of claim 6 , wherein the polyadenylation signal is a bovine growth hormone polyadenylation sequence.
8. The plurality of AAV vectors of claim 6 , wherein the first AAV vector comprises the portion of the gene encoding the otoferlin polypeptide positioned 3′ to the promoter, the splicing donor signal sequence is positioned at the 3′ end of the first coding sequence, and the F1 phage recombinogenic region is positioned 3′ to the splicing donor signal sequence and the second AAV vector comprises the splicing acceptor signal sequence positioned 3′ to the F1 phage recombinogenic region, the portion of the gene encoding an otoferlin polypeptide is positioned at the 3′ end of the splicing acceptor signal sequence, and the polyadenylation sequence is positioned at the 3′ end of the second coding sequence.
9. The plurality of AAV vectors of claim 1 , wherein the 5′ and 3′ ITRs are AAV2 serotype ITRs.
10. The plurality of AAV vectors of claim 1 , wherein the first and second AAV vectors are encapsulated by AAV capsids and wherein the AAV capsids are AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rh10, rh39, rh43, or Anc80.
11. The plurality of AAV vectors of claim 10 , wherein the first and second AAV vectors are encapsulated by Anc80 capsids and wherein the Anc80 capsids are Anc80L65 capsids.
12. The plurality of AAV vectors of claim 1 , wherein the otoferlin coding sequence is split at the exon 21-exon 22 junction.
13. The plurality of AAV vectors of claim 1 , wherein each portion of the gene encoding the otoferlin polypeptide encodes up to 70% of SEQ ID NO: 5.
14. The plurality of AAV vectors of claim 1 , wherein the first AAV vector further comprises a splicing donor signal sequence and the second AAV vector further comprises a splicing acceptor signal sequence, and wherein the first and second AAV can undergo homologous recombination and splicing between the splicing donor signal sequence and the splicing acceptor signal sequence to reconstitute the full-length otoferlin messenger RNA.
15. A composition comprising the plurality of AAV vectors of claim 1 , and further comprising a synthetic perilymph solution, wherein the composition is formulated for intracochlear administration.
16. The plurality of AAV vectors of claim 1 , wherein the F1 phage recombinogenic region comprises the nucleotide sequence at positions 2-78 of SEQ ID NO: 39.
17. A plurality of AAV vectors comprising:
a) a first AAV vector comprising (i) a promoter, (ii) a portion of a gene encoding an otoferlin polypeptide, (iii) a splicing donor signal sequence, and (iv) 5′ and 3′ ITRs; and
b) a second AAV vector comprising (i) a splicing acceptor signal sequence, (ii) a portion of the gene encoding an otoferlin polypeptide, (iii) a polyadenylation signal, and (iv) 5′ and 3′ ITRs;
wherein the first AAV vector and the second AAV comprise a 77 base pair F1 phage recombinogenic region and are capable of constituting a full-length otoferlin messenger RNA;
wherein the first and second AAV vectors each include a total number of nucleotides of up to about 5 kb.
18. The plurality of AAV vectors of claim 17 , wherein the otoferlin polypeptide is an otoferlin isoform 5 polypeptide.
19. The plurality of AAV vectors of claim 18 , wherein the otoferlin polypeptide is the human otoferlin isoform 5 polypeptide.
20. The plurality of AAV vectors of claim 17 , wherein each portion of the gene encoding the otoferlin polypeptide encodes up to 70% of SEQ ID NO: 5.
21. The plurality of AAV vectors of claim 17 , wherein the otoferlin coding sequence is split at the exon 21-exon 22 junction.
22. A composition comprising the plurality of AAV vectors of claim 17 , and further comprising a synthetic perilymph solution, wherein the composition is formulated for intracochlear administration.
23. The plurality of AAV vectors of claim 17 , wherein the F1 phage recombinogenic region comprises the nucleotide sequence at positions 2-78 of SEQ ID NO: 39.
24. The plurality of AAV vectors of claim 17 , wherein the 5′ and 3′ ITRs are AAV2 serotype ITRs.
25. The plurality of AAV vectors of claim 17 , wherein the first and second AAV vectors are encapsulated by AAV capsids, and wherein the AAV capsids are AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rh10, rh39, rh43, or Anc80.
26. The plurality of AAV vectors of claim 25 , wherein the first and second AAV vectors are encapsulated by Anc80 capsids, and wherein the Anc80 capsids are Anc80L65 capsids.