AAV-mediated base editing restores cochlear gap junction in GJB2 dominant-negative mutation-associated syndromic hearing loss model.

Mutations in the gap junction β2 (GJB2) gene, which encodes connexin 26, are the leading cause of genetic deafness. These mutations are characterized by the degeneration and fragmentation of gap junctions and gap junction plaques (GJPs) composed of connexin 26. Dominant-negative mutations of GJB2, such as R75W, cause syndromic hearing loss and palmoplantar keratoderma. We previously reported that the R75W mutation, a single-base substitution where C is replaced by T, causes fragmentation of GJPs. Therefore, an adenine base editor (ABE), which enables A-to-G base conversions, can potentially be useful for the treatment of this genetic disease. Here, we report that an all-in-one adeno-associated virus (AAV) vector, which includes a compact ABE (SaCas9-NNG-ABE8e) with broad targeting range, and a sgRNA targeting the R75W mutation in GJB2 corrected this pathogenic mutation and facilitated the recovery of the gap junction intercellular communication network of GJPs. In a transgenic mouse model with the GJB2 R75W mutation, AAV-mediated base editing also restored the fragmented GJPs to orderly outlines in cochlear supporting cells. Our findings suggest that an ABE-based base-editing strategy could be an optimal treatment for the dominant form of GJB2-related hearing loss, GJB2-related skin diseases, and other deafness-related mutations, especially single-base substitutions.