Methods of treating non-syndromic sensorineural hearing loss

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 a stereocilin 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 a stereocilin 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 sequence of each of the encoded portions do not overlap;
• wherein no single vector of the two different vectors encodes a full-length stereocilin protein;
• and
• when introduced into a mammalian cell, splicing occurs between the splicing donor signal sequence and the splicing acceptor signal sequence, thereby forming a recombined nucleic acid that encodes a full-length stereocilin protein.

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 a stereocilin 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 of the two different nucleic acid vectors comprises 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 a stereocilin 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 amino acid sequence of each of the encoded portions do not overlap;
• wherein no single vector of the two different vectors encodes a full-length stereocilin protein;
• and
• when introduced into a mammalian cell, splicing occurs between the splicing donor signal and the splicing acceptor signal, thereby forming a recombined nucleic acid that encodes a full-length stereocilin protein.

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 a stereocilin 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 of the two different nucleic acid vectors comprises a F1 phage recombinogenic region, a splicing acceptor signal sequence positioned 3′ of the F1 phage recombinogenic region, a second coding sequence that encodes a C-terminal portion of a stereocilin 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 amino acid sequence of each of the encoded portions do not overlap;
• wherein no single vector of the two different vectors encodes a full-length stereocilin protein;
• and
• when introduced into a mammalian cell, splicing occurs between the splicing donor signal and the splicing acceptor signal, thereby forming a recombined nucleic acid that encodes a full-length stereocilin protein.

A composition comprising:

• a Cas9 nuclease;
• a guide RNA that includes at the 5′ end of the guide RNA a complementary region consisting of 20 nucleotides that are complementary to 20 consecutive nucleotides within positions 13955-14151 of SEQ ID NO:5, that can be used in CRISPR/Cas9 RNA-guided genome editing to remove a nonsense mutation at a predetermined site in the endogenous stereocilin pseudogene sequence of SEQ ID NO:
• 5;
• and
• when introduced into a mammalian cell, a nucleic acid encoding a full-length stereocilin protein is reconstituted at the locus of the stereocilin pseudogene.

A composition comprising at least one nucleic acid vector, wherein the at least one nucleic acid vector comprises an adeno-associated virus (AAV) vector that comprises an antisense oligonucleotide that is at least 80% complementary to a contiguous nucleotide sequence of SEQ ID NO:

• 5 at positions 13955-14151,
• wherein the antisense oligonucleotide is between 15-30 nucleotides in length;
• and
• when introduced into a mammalian cell, a nucleic acid encoding a full-length stereocilin protein is generated at the locus of the stereocilin pseudogene.