Genetic Approach to Therapy for DFNA9

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ABSTRACT/PROJECT SUMMARY Impressive advances in gene discovery in the auditory system have occurred in the last decades, making specific targeted approaches for therapeutics realistic goals of great interest in the field of hearing and deafness. Hearing loss (HL) is an increasingly significant health problem in populations worldwide, with a substantial proportion due to genetic causes. Given the health burden and ongoing rapid discoveries, it is now essential to pursue new strategies for specific early interventions that could prevent or mitigate severity and progression of HL. One such disorder is DFNA9, an adult-onset sensorineural HL with balance dysfunction, caused by mutations in COCH, encoding cochlin, the most abundantly detected protein in the inner ear. This disease model is similar to and representative of the majority of genetic HL disorders with a dominant mode of inheritance and with a deleterious gain-of-function of the mutant protein. Aims 1 and 2 of this proposal involve utilization of the powerful and versatile CRISPR-Cas9 gene editing technology for specific targeting and disruption of the dominant missense pathogenic COCH variants p.G88E and p.A449T. We will utilize human fibroblasts from patients with these two variants, and derived pluripotent stem cells and organoids. These biological resources will serve as tools for the development of effective methods for allele-specific gene disruption of the COCH pathogenic variants, while leaving the normal allele intact and functional. The organoids will be assessed as a possible in vitro system for elucidating the biology of COCH aggregates pathognomonic of DFNA9 temporal bones. Furthermore, we will utilize two Coch knock-in (KI) mouse models with these variants for implementation of somatic tissue gene targeting in the inner ear. These approaches will establish methodologies for gene editing not only for DFNA9, but also for a broader category of other HL disorders with a dominant gain-of-function mechanism of pathology, with the ultimate goal of translation to clinical trials and therapeutic intervention.