Abstract
Mutations in
(
) cause autosomal dominant deafness-50 (DFNA50), a form of delayed-onset hearing loss. Genome editing has shown efficacy in hearing recovery through 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 the
mutation 14C>A by screening different CRISPR systems and optimizing Cas9 expression and the sgRNA scaffold for efficient and specific mutation editing. AAV delivery of the KKH variant of
Cas9 (SaCas9-KKH) and sgRNA to the cochleae of presymptomatic (3-week-old) and symptomatic (6-week-old) adult
mutant mice improved hearing long term, with efficacy increased by injection at a younger age. Adult inner ear delivery resulted in transient Cas9 expression without evidence of AAV genomic integration, indicating the good safety profile of our in vivo genome editing strategy. We developed a dual-AAV system, including an AAV-sgmiR96-master carrying sgRNAs against all known human
mutations. Because mouse and human
sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lay the foundation for the development of treatment for patients with DFNA50 caused by
mutations.