Compositions and methods for treating non-age-associated hearing impairment in a human subject
Inventors
Simons, Emmanuel John • Reisinger, Ellen • KÜGLER, Sebastian • Al-Moyed, Hanan
Assignees
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Publication Number
US-11993777-B2
Publication Date
2024-05-28
Expiration Date
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.
Core Innovation
The invention relates to a composition comprising two different nucleic acid vectors for expressing otoferlin in mammalian cells. Each vector comprises a coding sequence encoding a different portion of an otoferlin protein, each encoded portion being at least 30 amino acid residues in length, and no single vector encodes a full-length otoferlin protein.
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. In one arrangement, a first vector comprises a promoter operably linked to a coding sequence encoding an N-terminal portion of the otoferlin protein, and at least one coding sequence spans two neighboring exons of otoferlin genomic DNA and lacks an intronic sequence between the two neighboring exons.
In another arrangement, one vector comprises a splicing donor signal sequence at the 3′ end of a first coding sequence encoding an N-terminal portion, and the other vector comprises a splicing acceptor signal sequence with a second coding sequence encoding a C-terminal portion plus a polyadenylation sequence. When transcribed in a mammalian cell, splicing occurs between the donor signal on one transcript and the acceptor signal on the other transcript, thereby forming a recombined RNA molecule that encodes a full-length otoferlin protein.
Claims Coverage
Two independent claims are present. They cover dual-vector splitting of otoferlin coding information into non-overlapping portions, with full-length otoferlin production achieved either by concatamerization or homologous recombination of nucleic acids or by trans-splicing of RNA transcripts.
Two-vector otoferlin portion coding without single full-length vector
A composition comprising two different nucleic acid vectors, wherein each vector comprises a coding sequence encoding a different portion of an otoferlin protein, each encoded portion being at least 30 amino acid residues in length, and no single vector encodes a full-length otoferlin protein.
Nucleic acid recombination or concatamerization to form full-length otoferlin
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.
Promoter-linked n-terminal portion vector with exon-spanning construct
A first vector comprises a promoter operably linked to a coding sequence that encodes an N-terminal portion of the otoferlin protein, and at least one coding sequence comprises a nucleotide sequence spanning two neighboring exons of otoferlin genomic DNA and lacks an intronic sequence between the two neighboring exons.
Trans-splicing between donor and acceptor transcripts to form full-length otoferlin
A composition comprising two different nucleic acid vectors in which, 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.
Non-overlapping n-terminal and c-terminal encoded portions
Each of the encoded portions is at least 30 amino acid residues in length, the amino acid sequences of the encoded portions do not overlap, and no single vector encodes a full-length otoferlin protein.
Overall claim coverage centers on dual-vector strategies that split the otoferlin protein into at least 30-amino-acid portions delivered by two different vectors, with full-length otoferlin produced in mammalian cells either through concatamerization or homologous recombination of nucleic acids or through trans-splicing of RNA transcripts.
Stated Advantages
Restoration of hearing and otoferlin function in Otof−/− mice is described in the partial content.
Documented Applications
In vivo large-animal testing using sheep with AAV-OTOF delivery via round-window membrane/trans-RWM infusion with ABR/DPOAE readouts and postmortem immunostaining/qPCR for OTOF distribution and toxicity assessment.
CRISPR/Cas9 generation and rescue of OTOF knockout transgenic sheep, including genotyping and otoferlin expression assays.
Pediatric human clinical delivery workflow using AAV-OTOF through stapes/RWM.
Downstream animal biology in Otof−/− mice demonstrating restoration of hearing and otoferlin function using dual AAV trans-splicing, including ABR wave restoration and full-length otoferlin expression restricted by immunohistochemistry, synapse preservation, and electrophysiology showing restored exocytosis/vesicle replenishment.
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