Allele-specific gene editing approach for vision loss restoration inRHO-associated Retinitis Pigmentosa

MutantRHOis the most frequent genetic cause of autosomal dominant retinitis pigmentosa. Here, we developed an allele-specific gene editing therapeutic drug to selectively target the human T17MRHOmutant allele while leaving the wild-typeRHOallele intact for the first time. We identified aStaphylococcus aureusCas9 (SaCas9) guide RNA that was highly active and specific to the human T17MRHOallele.In vitroexperiments using HEK293T cells and patient-specific induced pluripotent stem cells (iPSCs) demonstrated active nuclease activity and high specificity. Subretinal delivery of a single adeno-associated virus serotype 2/8 packaging SaCas9 and sgRNA to the retinas of theRHOhumanized mice showed that this therapeutic drug targeted the mutant allele selectively, thereby downregulating the mutantRHOmRNA expression. Administration of this therapeutic drug resulted in a long-term (up to 11 months after treatment) improvement of retinal function and preservation of photoreceptors in the mutant humanized heterozygous mice. Our study demonstrated a dose-dependent therapeutic effectin vivo.Unwanted off-target effects were not observed at the whole-genome sequencing level. Our study provides strong support for the further development of this effective therapeutic drug to treatRHO-T17M associated autosomal dominant retinitis pigmentosa (adRP), also offers a generalizable framework for developing gene editing medicine. Furthermore, our success in retoring the vision loss in the sufferingRHOhumanized mice verifies the feasibility of allele-specific CRISPR/Cas9-based medicines for other autosomal dominant inherited retinal dystrophies.