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 method of treating otoferlin related hearing loss, comprising administering to a target cell a plurality of AAV vectors comprising:
a) a first AAV vector comprising a promoter operably linked to a portion of a gene encoding an otoferlin polypeptide, a first splicing sequence, a 5′ inverted terminal repeat (ITR), and a 3′ ITR; and
b) a second AAV vector comprising a second splicing sequence, a portion of the gene encoding the otoferlin polypeptide, a polyadenylation signal, a 5′ ITR, and a 3′ ITR;
wherein the first AAV vector and the second AAV vector are capable of constituting a full-length otoferlin messenger RNA in the target cell, and wherein the otoferlin related hearing loss is associated with reduced expression, lack of expression, or dysfunction of otoferlin.
2. The method of claim 1 , wherein the plurality of AAV vectors is administered into a cochlea of a subject.
3. The method of claim 1 , wherein the otoferlin polypeptide is human otoferlin isoform 5 polypeptide.
4. The method of claim 1 , wherein the first and second vectors independently contain packaging capacity of less than about 6 kb.
5. The method of claim 1 , wherein the 5′ and 3′ ITRs of the first and the second AAV vectors are AAV2 ITRs.
6. The method of claim 1 , wherein the first and second AAV vectors are encapsulated by AAV capsids.
7. The method of claim 6 , wherein the AAV capsids are AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8, rh10, rh39, rh43, or Anc80.
8. The method of claim 6 , wherein the first and second AAV vectors are encapsulated by Anc80 capsids.
9. The method of claim 1 , wherein the target cell is a mammalian cell.
10. The method of claim 1 , wherein the first and second AAV vectors comprise a region of homology.
11. The method of claim 1 , wherein the first AAV vector and the second AAV vector undergo (i) concatamerization, (ii) concatamerization and trans-splicing, (iii) concatamerization and homologous recombination, or (iv) concatamerization, trans-splicing, and homologous recombination to reconstitute the full-length gene encoding the otoferlin polypeptide.
12. A plurality of AAV vectors comprising:
a) a first AAV vector comprising a promoter operably linked to a portion of a gene encoding an otoferlin polypeptide, a 5′ inverted terminal repeat (ITR), and a 3′ ITR; and
b) a second AAV vector comprising a portion of the gene encoding the otoferlin polypeptide, a 5′ ITR, and a 3′ ITR;
wherein the first AAV vector and the second AAV vector are capable of constituting a full-length otoferlin messenger RNA in a target cell, wherein the otoferlin polypeptide is human otoferlin isoform 5 polypeptide.
13. The plurality of AAV vectors of claim 12 , wherein the first AAV vector further comprises a splicing sequence and the second AAV vector further comprises a splicing sequence and a polyadenylation signal.
14. The plurality of AAV vectors of claim 12 , 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.
15. The plurality of AAV vectors of claim 12 , wherein the first and second AAV vectors are encapsulated by Anc80 capsids.
16. The plurality of AAV vectors of claim 12 , wherein the first and second AAV vectors comprise a region of homology.
17. The plurality of AAV vectors of claim 12 , wherein the first and second AAV vectors each comprise a splicing sequence.
18. The plurality of AAV vectors of claim 17 , wherein the first AAV vector and the second AAV vector undergo (i) concatamerization, (ii) concatamerization and trans-splicing, (iii) concatamerization and homologous recombination, or (iv) concatamerization, trans-splicing, and homologous recombination to reconstitute the full-length otoferlin gene.
19. The plurality of AAV vectors of claim 17 , wherein the first and second AAV vectors comprise a region of homology.
20. A pharmaceutical composition comprising the plurality of AAV vectors of claim 12 .
21. A plurality of AAV vectors comprising:
a) a first AAV vector comprising a promoter operably linked to a portion of a gene encoding an otoferlin polypeptide, a 5′ inverted terminal repeat (ITR), and a 3′ ITR; and
b) a second AAV vector comprising a portion of the gene encoding the otoferlin polypeptide, a 5′ ITR, and a 3′ ITR;
wherein the first AAV vector and the second AAV vector are capable of constituting a full-length otoferlin messenger RNA in a target cell, wherein the first and second AAV vectors are encapsulated by Anc80 capsids.
22. The plurality of AAV vectors of claim 21 , wherein the first AAV vector further comprises a splicing sequence and the second AAV vector further comprises a splicing sequence and a polyadenylation signal.
23. The plurality of AAV vectors of claim 21 , wherein the first and second AAV vectors comprise a region of homology.
24. The plurality of AAV vectors of claim 23 , wherein the first AAV vector and the second AAV vector undergo (i) concatamerization, (ii) concatamerization and trans-splicing, (iii) concatamerization and homologous recombination, or (iv) concatamerization, trans-splicing, and homologous recombination to reconstitute the full-length otoferlin gene.
25. A pharmaceutical composition comprising the plurality of AAV vectors of claim 21 .