Compositions and methods for treating non-age-associated hearing impairment in a human subject

Abstract

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.

What is claimed is: 
     
         1 . A composition comprising at least two different nucleic acid vectors, wherein:
 each of the at least two different vectors comprises a coding sequence that encodes a different portion of an otoferlin protein, each of the encoded portions being at least 30 amino acid residues in length, wherein the amino acid sequence of each of the encoded portions may optionally partially overlap with the amino acid sequence of a different one of the encoded portions;   no single vector of the at least two different vectors encodes a full-length otoferlin protein;   at least one of the coding sequences comprises a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons; and   when introduced into a mammalian cell the at least two different vectors undergo concatamerization or homologous recombination with each other, thereby forming a recombined nucleic acid that encodes a full-length otoferlin protein.   
     
     
         2 . The composition of  claim 1 , wherein each of the at least two different vectors is a plasmid, a transposon, a cosmid, an artificial chromosome, or a viral vector. 
     
     
         3 . The composition of  claim 1 , wherein each of the at least two different vectors is a human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or a P1-derived artificial chromosome (PAC). 
     
     
         4 . The composition of  claim 1 , wherein each of the at least two different vectors is a viral vector selected from an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, or a retrovirus vector. 
     
     
         5 . The composition of  claim 1 , wherein each of the at least two different vectors is an AAV vector. 
     
     
         6 . The composition of any one of  claims 1 - 5 , wherein the amino acid sequence of one of the encoded portions overlaps with the amino acid sequence of a different one of the encoded portions. 
     
     
         7 . The composition of  claim 6 , wherein the amino acid sequence of each of the encoded portions partially overlaps with the amino acid sequence of a different encoded portion. 
     
     
         8 . The composition of  claim 7 , wherein the overlapping amino acid sequence is between about 30 amino acid residues to about 1000 amino acid residues in length. 
     
     
         9 . The composition of any one of  claims 1 - 5 , wherein the vectors include two different vectors, each of which comprises a different segment of an intron, wherein the intron comprises the nucleotide sequence of an intron that is present in otoferlin genomic DNA, and wherein the two different segments overlap in sequence by at least 100 nucleotides. 
     
     
         10 . The composition of  claim 9 , wherein the two different segments overlap in sequence by about 100 nucleotides to about 800 nucleotides. 
     
     
         11 . The composition of any one of  claims 1 - 10 , wherein the nucleotide sequence of each of the at least two different vectors is between about 500 nucleotides to about 10,000 nucleotides in length. 
     
     
         12 . The composition of  claim 11 , wherein the nucleotide sequence of each of the at least two different vectors is between 500 nucleotides to 5,000 nucleotides in length. 
     
     
         13 . The composition of any one of  claims 1 - 12 , wherein the number of different vectors in the composition is two. 
     
     
         14 . The composition of  claim 13 , wherein a first of the two different vectors comprises a coding sequence that encodes an N-terminal portion of the otoferlin protein. 
     
     
         15 . The composition of  claim 14 , wherein the N-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length. 
     
     
         16 . The composition of  claim 15 , wherein the N-terminal portion of the otoferlin protein is between 200 amino acids to 1500 amino acids in length. 
     
     
         17 . The composition of any one of  claims 14 - 16 , wherein the first vector further comprises one or both of a promoter and a Kozak sequence. 
     
     
         18 . The composition of  claim 17 , wherein the first vector comprises a promoter that is an inducible promoter, a constitutive promoter, or a tissue-specific promoter. 
     
     
         19 . The composition of any one of  claims 14 - 18 , wherein the first vector further comprises a coding sequence encoding a destabilization domain, wherein the destabilization domain is 3′ to the coding sequence that encodes the N-terminal portion of the otoferlin protein. 
     
     
         20 . The composition of  claim 19 , wherein the coding sequence that encodes the N-terminal portion of the otoferlin protein comprises exons 1-21 of isoform 5 of the human otoferlin gene. 
     
     
         21 . The composition of any one of  claims 14 - 20 , wherein the second of the two different vectors comprises a coding sequence that encodes a C-terminal portion of the otoferlin protein. 
     
     
         22 . The composition of  claim 21 , wherein the C-terminal portion of the otoferlin protein is between 30 amino acids to 1600 amino acids in length. 
     
     
         23 . The composition of  claim 22 , wherein the C-terminal portion of the otoferlin protein is between 200 amino acids to 1500 amino acids in length. 
     
     
         24 . The composition of any one of  claims 21 - 23 , wherein the second vector further comprises a poly(dA) sequence. 
     
     
         25 . The composition of  claim 21 , wherein the coding sequence that encodes the C-terminal portion of the otoferlin protein comprises exons 22-47 of isoform 5 of the human otoferlin gene. 
     
     
         26 . The composition of any one of  claims 1 - 25 , further comprising a pharmaceutically acceptable excipient. 
     
     
         27 . A kit comprising a composition of any one of  claims 1 - 26 . 
     
     
         28 . A kit of  claim 27 , further comprising a pre-loaded syringe comprising the composition. 
     
     
         29 . A method comprising:
 introducing into a cochlea of a mammal a therapeutically effective amount of the composition of any one of  claims 1 - 26 .   
     
     
         30 . The method of  claim 29 , wherein the mammal is a human. 
     
     
         31 . The method of  claim 29  or  30 , wherein the mammal has been previously identified as having a defective otoferlin gene. 
     
     
         32 . A method of increasing expression of a full-length otoferlin protein in a mammalian cell, the method comprising introducing the composition of any one of  claims 1 - 26  into the mammalian cell. 
     
     
         33 . The method of  claim 32 , wherein the mammalian cell is an inner hair cell. 
     
     
         34 . The method of  claim 32  or  33 , wherein the mammalian cell is a human cell. 
     
     
         35 . The method of any one of  claims 32 - 34 , wherein the mammalian cell has previously been determined to have a defective otoferlin gene. 
     
     
         36 . A method of increasing expression of a full-length otoferlin protein in an inner hair cell in a cochlea of a mammal, the method comprising:
 introducing into the cochlea of the mammal a therapeutically effective amount of the composition of any one of  claims 1 - 26 .   
     
     
         37 . The method of  claim 36 , wherein the mammal has been previously identified as having a defective otoferlin gene. 
     
     
         38 . The method of  claim 36  or  37 , wherein the mammal is a human. 
     
     
         39 . A method of treating non-symptomatic sensorineural hearing loss in a subject identified as having a defective otoferlin gene, the method comprising:
 administering a therapeutically effective amount of a composition of any one of  claims 1 - 26  into the cochlea of the subject.   
     
     
         40 . The method of  claim 39 , wherein the subject is a human. 
     
     
         41 . The method of  claim 39  or  40 , further comprising, prior to the administering step, determining that the subject has a defective otoferlin gene. 
     
     
         42 . A composition comprising two different nucleic acid vectors, wherein:
 a first nucleic acid vector of the two different nucleic acid vectors comprises a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin protein positioned 3′ of the promoter, and a splicing donor signal sequence positioned at the 3′ end of the first coding sequence; and   a second nucleic acid vector of the two different nucleic acid vectors comprises a splicing acceptor signal sequence, a second coding sequence that encodes a C-terminal portion of an otoferlin protein 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 each of the encoded portions is at least 30 amino acid residues in length, wherein the amino acid sequences of the encoded portions do not overlap, wherein no single vector of the two different vectors encodes a full-length otoferlin protein, and, when the coding sequences are transcribed in a mammalian cell, to produce RNA transcripts, splicing occurs between the splicing donor signal sequence on one transcript and the splicing acceptor signal sequence on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length otoferlin protein.   
     
     
         43 . The composition of  claim 42 , wherein the coding sequence of at least one of the vectors comprises a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons. 
     
     
         44 . A composition comprising:
 a first nucleic acid vector comprising a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin protein positioned 3′ of the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a first detectable marker gene positioned 3′ of the splicing donor signal sequence; and   a second nucleic acid vector, different from the first nucleic acid vector, comprising a second detectable marker gene, a splicing acceptor signal sequence positioned 3′ of the second detectable marker gene, a second coding sequence that encodes a C-terminal portion of an otoferlin protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3′ end of the second coding sequence;   wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the respective amino acid sequences of the encoded portions do not overlap with each other, wherein no single vector of the two different vectors encodes a full-length otoferlin protein, and, when the coding sequences are transcribed in a mammalian cell to produce RNA transcripts, splicing occurs between the splicing donor signal on one transcript and the splicing acceptor signal on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length otoferlin protein.   
     
     
         45 . The composition of  claim 44 , wherein the coding sequence of at least one of the vectors comprises a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons. 
     
     
         46 . The composition of  claim 44 , wherein the first or second detectable marker gene encodes alkaline phosphatase. 
     
     
         47 . The composition of  claim 44  or  46 , wherein the first and second detectable marker genes are the same. 
     
     
         48 . A composition comprising:
 a first nucleic acid vector comprising a promoter, a first coding sequence that encodes an N-terminal portion of an otoferlin protein positioned 3′ to the promoter, a splicing donor signal sequence positioned at the 3′ end of the first coding sequence, and a F1 phage recombinogenic region positioned 3′ to the splicing donor signal sequence; and   a second nucleic acid vector, different from the first nucleic acid vector, comprising a second F1 phage recombinogenic region, a splicing acceptor signal sequence positioned 3′ of the second F1 phage recombinogenic region, a second coding sequence that encodes a C-terminal portion of an otoferlin protein positioned at the 3′ end of the splicing acceptor signal sequence, and a polyadenylation sequence positioned at the 3′ end of the second coding sequence;   wherein each of the encoded portions is at least 30 amino acid residues in length, wherein the respective amino acid sequences of the encoded portions do not overlap with each other, wherein no single vector of the two different vectors encodes a full-length otoferlin protein, and, when the coding sequences are transcribed in a mammalian cell to produce RNA transcripts, splicing occurs between the splicing donor signal one transcript and the splicing acceptor signal on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length otoferlin protein.   
     
     
         49 . The composition of  claim 48 , wherein the coding sequence of at least one of the vectors comprises a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA, and lacks an intronic sequence between the two neighboring exons. 
     
     
         50 . A kit comprising a composition of any one of  claims 42 - 49 . 
     
     
         51 . A kit of  claim 50 , wherein the composition is pre-loaded in a syringe. 
     
     
         52 . A method comprising introducing into a cochlea of a mammal a therapeutically effective amount of the composition of any one of  claims 42 - 49 . 
     
     
         53 . The method of  claim 52 , wherein the mammal is a human. 
     
     
         54 . The method of  claim 52  or  53 , wherein the mammal has been previously identified as having a defective otoferlin gene. 
     
     
         55 . A method of increasing expression of a full-length otoferlin protein in a mammalian cell, the method comprising introducing the composition of any one of  claims 42 - 49  into the mammalian cell. 
     
     
         56 . The method of  claim 55 , wherein the mammalian cell is a cochlear inner hair cell. 
     
     
         57 . The method of  claim 55  or  56 , wherein the mammalian cell is a human cell. 
     
     
         58 . The method of any one of  claims 55 - 57 , wherein the mammalian cell has previously been determined to have a defective otoferlin gene. 
     
     
         59 . A method of increasing expression of a full-length otoferlin protein in an inner hair cell in a cochlea of a mammal, the method comprising introducing into the cochlea a therapeutically effective amount of the composition of any one of  claims 39 - 46 . 
     
     
         60 . The method of  claim 59 , wherein the mammal has been previously identified as having a defective otoferlin gene. 
     
     
         61 . The method of  claim 59  or  60 , wherein the mammal is a human. 
     
     
         62 . A method of treating non-symptomatic sensorineural hearing loss in a subject identified as having a defective otoferlin gene, the method comprising administering a therapeutically effective amount of a composition of any one of  claims 42 - 49  into a cochlea of the subject. 
     
     
         63 . The method of  claim 62 , wherein the subject is a human. 
     
     
         64 . The method of  claim 62  or  63 , further comprising, prior to the administering step, determining that the subject has a defective otoferlin gene. 
     
     
         65 . The composition of  claim 13 , wherein the one of the two vectors comprises SEQ ID NO: 39 and the second of the two vectors comprises SEQ ID NO: 40. 
     
     
         66 . The composition of  claim 13 , wherein the one of the two vectors comprises SEQ ID NO: 41 and the second of the two vectors comprises SEQ ID NO: 42. 
     
     
         67 . The composition of  claim 13 , wherein one of the two vectors comprises SEQ ID NO:84 and the second of the two vectors comprises SEQ ID NO: 85. 
     
     
         68 . The composition of  claim 1 , wherein one of the at least two different vectors comprises a sequence encoding a NTF3 protein. 
     
     
         69 . The composition of  claim 68 , wherein the sequence encoding a NTF3 protein is at least 90% identical to SEQ ID NO: 78.