1. Department of Otorhinolaryngology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
2. Institute for Yonsei Ear Science, Seoul 03722, Republic of Korea
3. Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
4. Department of Pharmacology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul 03722, Republic of Korea
5. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic of Korea
6. Center for Nanomedicine, Institute for Basic Science, Seoul 03722, Republic of Korea
7. Yonsei-IBS Institute, Yonsei University, Seoul 03722, Republic of Korea
*These authors contributed equally.
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.
Methods: We generated a mouse model harboring Kcnq4W276S/+ 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 in vivo gene editing strategy to disrupt the dominant-negative allele in Kcnq4 and prevent progressive hearing loss.
Results: In vivo 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.
Conclusion: 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.
Keywords: DFNA2, outer hair cell, KCNQ4, Cas9, gene editing