Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor

Programmable RNA editing tools enable the reversible correction of mutant transcripts, reducing the potential risk associated with permanent genetic changes associated with the use of DNA editing tools. However, the potential of these RNA tools to treat disease remains unknown. Here, we evaluated RNA correction therapy with Cas13-based RNA base editors in the myosin VI p.C442Y heterozygous mutation ( Myo6 C442Y/+ ) mouse model that recapitulated the phenotypes of human dominant-inherited deafness. We first screened several variants of Cas13-based RNA base editors and guide RNAs (gRNAs) targeting Myo6 C442Y in cultured cells and found that mini dCas13X.1-based adenosine base editor (mxABE), composed of truncated Cas13X.1 and the RNA editing enzyme adenosine deaminase acting on RNA 2 deaminase domain variant (ADAR2dd E488Q ), exhibited both high efficiency of A > G conversion and low frequency of off-target edits. Single adeno-associated virus (AAV)–mediated delivery of mxABE in the cochlea corrected the mutated Myo6 C442Y to Myo6 WT allele in homozygous Myo6 C442Y/C442Y mice and resulted in increased Myo6 WT allele in the injected cochlea of Myo6 C442Y/+ mice. The treatment rescued auditory function, including auditory brainstem response and distortion product otoacoustic emission up to 3 months after AAV-mxABE- Myo6 injection in Myo6 C442Y/+ mice. We also observed increased survival rate of hair cells and decreased degeneration of hair bundle morphology in the treated compared to untreated control ears. These findings provide a proof-of-concept study for RNA editing tools as a therapeutic treatment for various semidominant forms of hearing loss and other diseases.