Person:

Pan, Bifeng

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide permanent therapeutic restoration of gene function. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G, resulting in the substitution p.Y182C) in transmembrane channel-like 1 (Tmc1) that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction that causes profound deafness. To repair the Baringo mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual AAV vectors using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair-cell sensory transduction, hair-cell morphology, and partial low-frequency hearing four weeks post-injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.