Optimized AAV Vectors for TMC1 Gene Therapy in a Humanized Mouse Model of DFNB7/11.

Gene therapy for genetic hearing loss is an emerging therapeutic modality for hearing restoration. However, the approach has not yet been translated into clinical application. To further develop inner-ear gene therapy, we engineered a novel mouse model bearing a human mutation in the transmembrane channel-1 gene () and characterized the auditory phenotype of the mice. TMC1 forms the mechanosensory transduction channel in mice and humans and is necessary for auditory function. We found that mice harboring the equivalent of the human p.N199I mutation (p.N193I) had profound congenital hearing loss due to loss of hair cell sensory transduction. Next, we optimized and screened viral payloads packaged into AAV9-PHP.B capsids. The vectors were injected into the inner ears of mice and the new humanized -p.N193I mouse model. Auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), cell survival, and biodistribution were evaluated in the injected mice. We found broad-spectrum, durable recovery of auditory function in -p.N193I mice injected with AAV9-PHP.B-. ABR and DPOAE thresholds were equivalent to those of wild-type mice across the entire frequency range. Biodistribution analysis revealed viral DNA/RNA in the contralateral ear, brain, and liver but no overt toxicity. We conclude that the AAV9-PHP.B- construct may be suitable for further development as a gene therapy reagent for treatment of humans with genetic hearing loss due to recessive mutations.