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 composition comprising a first adeno-associated virus (AAV) vector and a second AAV vector, wherein:
a) the first AAV vector comprises a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin polypeptide positioned 3′ of the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a coding sequence encoding a destabilization domain, wherein the destabilization domain is 3′ to the first coding sequence and 3′ to the splicing donor signal sequence; and
b) the second AAV vector comprises a splicing acceptor signal sequence, a second coding sequence that encodes a C-terminal portion of the otoferlin polypeptide positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3′ end of the second coding sequence;
wherein the first and/or second AAV vector comprises a stuffer sequence comprising the full-length nucleotide sequence set forth in any one of SEQ ID NOs: 54-58, 90 and 91, wherein the stuffer sequence is 5′ to the promoter or 3′ to the polyadenylation sequence;
wherein when introduced into amammalian cell the first and second AAV vectors undergo intermolecular concatamerization and trans-splicing, thereby forming a recombined nucleic acid that encodes a full-length otoferlin polypeptide.
2 . A plurality of adeno-associated virus (AAV) vectors comprising a first AAV vector and a second AAV vector, wherein:
a) the first AAV vector comprises a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin polypeptide positioned 3′ of the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a coding sequence encoding a destabilization domain, wherein the destabilization domain is 3′ to the first coding sequence and 3′ to the splicing donor signal sequence; and
b) the second AAV vector comprises a splicing acceptor signal sequence, a second coding sequence that encodes a C-terminal portion of the otoferlin polypeptide positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence at the 3′ end of the second coding sequence;
wherein the first and/or second AAV vector comprises a stuffer sequence comprising the full-length nucleotide sequence set forth in any one of SEQ ID NOs: 54-58, 90 and 91, wherein the stuffer sequence is 5′ to the promoter or 3′ to the polyadenylation sequence;
wherein when introduced into amammalian cell the first and second AAV vectors undergo intermolecular concatamerization and trans-splicing, thereby forming a recombined nucleic acid that encodes a full-length otoferlin polypeptide.
3 . The composition of claim 1 , wherein the destabilization domain comprises a mutant dihydrofolate reductase (DHFR) or a mutant FK-506 binding protein (FKBP).
4 . The composition of claim 1 , wherein the destabilization domain comprises the sequence of SEQ ID NO: 53.
5 . The plurality of AAV vectors of claim 2 , wherein the destabilization domain comprises a mutant dihydrofolate reductase (DHFR) or a mutant FK-506 binding protein (FKBP).
6 . The plurality of AAV vectors of claim 2 , wherein the destabilization domain comprises the sequence of SEQ ID NO: 53.
7 . The composition of claim 1 , wherein the destabilization domain comprises the CL1 degradation sequence of SEQ ID NO: 71.
8 . The plurality of AAV vectors of claim 2 , wherein the destabilization domain comprises the CL1 degradation sequence of SEQ ID NO: 71.