Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation

Mutations in microRNA-96 (MIR96) cause dominant delayed onset hearing loss DFNA50 without treatment. Genome editing has shown efficacy in hearing recovery by intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications, which has not been done. Here, we developed a genome editing therapy for Mir96 mutation 14C>A by screening genome editors and optimizing Cas9 expression and sgRNA scaffold for efficient and specific mutation editing. By AAV delivery in pre-symptomatic (3-week-old) and symptomatic (6-week-old) adult Mir9614C>A/+ mutant mice, hair cell on-target editing significantly improved hearing long-term, with efficacy increased by injection in younger age. Adult inner ear delivery resulted in transient Cas9 expression without evidence of AAV genomic integration, which greatly improves the safety profile of in vivo editing. We developed an AAV-sgmiR96-master system by one AAV carrying sgRNAs against all known human MIR96 mutations. As mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lays the foundation for the development of treatment of DFNA50 patients caused by MIR96 mutations. We established a feasibility of in vivo genome editing as a viable approach for treating hearing loss caused by genetic mutations in adult animals, offering insights into the potential clinical applications of this technology for treating inherited hearing disorders.