Compositions and methods for expressing otoferlin
Inventors
Boye, Sanford L. • Dyka, Frank M. • Hauswirth, William W. • Akil, Omar
Assignees
University of Florida Research Foundation Inc • University of California San Diego UCSD
Publication Number
US-12359221-B2
Publication Date
2025-07-15
Expiration Date
2038-05-04
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Abstract
Provided herein are methods and compositions for expressing Otoferlin, e.g., utilizing adeno-associated viral (AAV) particles. Such methods and compositions may be useful for treatment of diseases such as Deafness, Autosomal Recessive 9 (DFNB9).
Core Innovation
The invention provides methods and compositions for expressing Otoferlin in cells or subjects using adeno-associated viral (AAV) particles. Specifically, the invention employs a dual AAV system wherein two different AAV particles each carry partial coding sequences of the OTOF cDNA, along with regions of homology and splicing elements, which facilitate recombination and splicing in vivo to produce a full-length Otoferlin polypeptide. This approach has been demonstrated to rescue hearing in Otoferlin knock-out mice to near wild-type levels.
The problem addressed by the invention arises from mutations in the OTOF gene that cause Deafness, Autosomal Recessive 9 (DFNB9), a form of nonsyndromic deafness due to defective or absent Otoferlin protein. Existing treatments for DFNB9, such as cochlear implants or hearing aids, rely heavily on electronic devices and have limitations. There is a need for alternative therapeutic approaches that restore hearing by molecular means without depending extensively on electronic prostheses.
Claims Coverage
The patent contains one independent claim directed to a method of treating hearing loss by administering two AAV particles carrying distinct portions of an Otoferlin expression cassette. The main inventive features involve the specific components and organization of the polynucleotides within the AAV vectors, their homology regions, splicing sites, promoter choice, and viral serotype.
Dual AAV particles for expressing full-length Otoferlin
Administering to inner hair cells two AAV particles, one carrying a polynucleotide with a promoter, an N-terminal partial coding sequence of an Otoferlin polypeptide, a splice donor site, and a first region of homology; and the other carrying a polynucleotide with a second region of homology, a splice acceptor site, a C-terminal partial coding sequence of Otoferlin, and a polyadenylation signal sequence, all flanked by inverted terminal repeats.
Region of homology length
The first and second regions of homology each have a length between 50 and 300 nucleotides to facilitate homologous recombination between the two polynucleotides.
Sequence identity of homology regions
The regions of homology comprise the nucleotide sequence of SEQ ID NO: 3, ensuring homology between the two vectors for effective recombination.
Use of chimeric CMV chicken β actin promoter
The promoter used in the first polynucleotide is a chimeric cytomegalovirus (CMV) chicken β actin promoter or its truncated form, comprising the sequence of SEQ ID NO: 4.
Splice donor and acceptor site sequences
The method employs specific splice donor and splice acceptor sites comprising the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
Use of AAV2 inverted terminal repeat sequences
The inverted terminal repeat (ITR) sequences flanking the expression cassettes are derived from AAV2.
AAV2 serotype for both particles
Both the first and second AAV particles are of the AAV2 serotype.
The claims cover a gene therapy method using a dual AAV vector system with specific promoter, splice sites, homology regions, and AAV2 serotype to deliver split Otoferlin coding sequences to inner hair cells for treating hearing loss associated with DFNB9 mutations.
Stated Advantages
Delivery of the OTOF cDNA via a dual AAV system effectively rescues hearing in Otoferlin knock-out mice to near wild-type levels.
The dual vector strategy overcomes the packaging size limitations of AAVs, enabling delivery of the large OTOF cDNA by splitting it into two complementary vectors.
The treatment offers an alternative to cochlear implants and hearing aids, potentially reducing dependency on electronic devices for restoring hearing.
The restored hearing in treated animals was maintained long term, demonstrating durable therapeutic effect.
Documented Applications
Treatment of hearing loss caused by mutations in the OTOF gene associated with Deafness, Autosomal Recessive 9 (DFNB9) in human subjects.
Increasing expression of Otoferlin protein in cells or tissues, including inner ear hair cells, via administration of AAV particles encoding split Otoferlin cDNA.
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