<i>In vivo</i> outer hair cell gene editing ameliorates progressive hearing loss in dominant-negative <i>Kcnq4</i> murine model

Abstract

Outer hair cell (OHC) degeneration is a major cause of progressive hearing loss and presbycusis. Despite the high prevalence of these disorders, targeted therapy is currently not available. <b>Methods:</b> We generated a mouse model harboring <i>Kcnq4</i>^W276S/+ to recapitulate DFNA2, a common genetic form of progressive hearing loss accompanied by OHC degeneration. After comprehensive optimization of guide RNAs, Cas9s, vehicles, and delivery routes, we applied <i>in vivo</i> gene editing strategy to disrupt the dominant-negative allele in <i>Kcnq4</i> and prevent progressive hearing loss. <b>Results:</b><i>In vivo</i> gene editing using a dual adeno-associated virus package targeting OHCs significantly improved auditory thresholds in auditory brainstem response and distortion-product otoacoustic emission. In addition, we developed a new live-cell imaging technique using thallium ions to investigate the membrane potential of OHCs and successfully demonstrated that mutant allele disruption resulted in more hyperpolarized OHCs, indicating elevated KCNQ4 channel activity. <b>Conclusion:</b> These findings can facilitate the development of targeted therapies for DFNA2 and support the use of CRISPR-based gene therapy to rectify defects in OHCs.