Compositions and methods for treating sensorineural hearing loss using otoferlin dual vector systems
The disclosure features compositions and methods for the treatment of sensorineural hearing loss and auditory neuropathy, particularly forms of the disease that are associated with mutations in otoferlin (OTOF), by way of OTOF gene therapy. The disclosure provides a variety of compositions that include a first nucleic acid vector that contains a polynucleotide encoding an N-terminal portion of an OTOF protein and a second nucleic acid vector that contains a polynucleotide encoding a C-terminal portion of an OTOF protein. These vectors can be used to increase the expression of OTOF in a subject, such as a human subject suffering from sensorineural hearing loss.
1. A composition comprising:
a first nucleic acid vector comprising a myosin 15 (Myo15) promoter having at least 98% sequence identity to the sequence of SEQ ID NO: 36 operably linked to a first coding polynucleotide that encodes an N-terminal portion of an otoferlin (OTOF) protein; and
a second nucleic acid vector comprising a second coding polynucleotide that encodes a C-terminal portion of an OTOF protein and a polyadenylation (poly(A)) sequence positioned at a 3′ end of the second coding polynucleotide,
wherein neither the first nor second nucleic acid vector encodes a full-length OTOF protein, and, when introduced into a mammalian cell, the first and second nucleic acid vectors undergo homologous recombination or concatemerization to form a recombined nucleic acid that encodes a full-length OTOF protein.
2. The composition of claim 1 , wherein
the first nucleic acid vector further comprises a splice donor signal sequence positioned at a 3 ′ end of the first coding polynucleotide; and
the second nucleic acid vector further comprises a splice acceptor signal sequence, wherein the second coding polynucleotide that encodes a C-terminal portion of an OTOF protein is positioned at a 3′ end of the splice acceptor signal sequence,
wherein the first coding polynucleotide and the second coding polynucleotide do not overlap.
3. The composition of claim 1 , wherein
the first nucleic acid vector further comprises a splice donor signal sequence positioned at a 3′ end of the first coding polynucleotide and a first recombinogenic region positioned 3′ of the splice donor signal sequence; and
the second nucleic acid vector further comprises a second recombinogenic region and a splice acceptor signal sequence positioned 3′ of the second recombinogenic region, wherein the second coding polynucleotide that encodes a C-terminal portion of an OTOF protein is positioned at a 3′ end of the splice acceptor signal sequence,
wherein the first coding polynucleotide and the second coding polynucleotide do not overlap.
4. The composition of claim 3 , wherein the first and second recombinogenic regions have the same nucleic acid sequence.
5. The composition of claim 3 , wherein the first nucleic acid vector further comprises a degradation signal sequence positioned 3′ of the recombinogenic region; and wherein the second nucleic acid vector further comprises a degradation signal sequence positioned between the recombinogenic region and the splice acceptor signal sequence.
6. The composition of claim 2 , wherein the division between the first and second coding polynucleotides is at an OTOF exon boundary.
7. The composition of claim 1 , wherein the first and second nucleic acid vectors are adeno-associated virus (AAV) vectors.
8. The composition of claim 1 , wherein the first and second coding polynucleotides that encode the OTOF protein do not comprise introns.
9. The composition of claim 1 , wherein the first and second nucleic acid vectors comprise an inverted terminal repeat (ITR) at each end of the nucleic acid sequence.
10. The composition of claim 1 , wherein the second nucleic acid vector comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
11. The composition of claim 1 , wherein the Myo15 promoter comprises the sequence of SEQ ID NO: 36.