In vivo photoreceptor base editing for the rhodopsin-E150K autosomal-recessive retinitis-pigmentosa mouse model

Rhodopsin, the prototypical class A G-protein coupled receptor, is a highly sensitive receptor for light and enables phototransduction in rod photoreceptors. Rhodopsin plays not only a sensory role, but also a structural one as a major component of the rod outer segment disc, comprising over 90% of the protein content of the disc membrane. Mutations in RHO which lead to structural or functional abnormalities result in rod photoreceptor dysfunction and death. Therefore, correction of rhodopsin mutations which are causative for inherited retinal degenerations could result in a potential therapeutic, as demonstrated for other visual proteins such as RPE65 and PDE6B. In this study, we report the identification and first application of a CRISPR/Cas9 adenine base-editing strategy to correct a mutation in Rho. The E150K mutation was first identified in families with autosomal recessive retinitis pigmentosa, and a knock-in mouse model recapitulates many of the clinical phenotypes. Using UV-vis spectroscopy, mass spectrometry, and the G-protein activation assay, we characterized rhodopsin and its variants generated by bystander base editing. We treated homozygous Rho-E150K mice at post-natal day 15 by subretinal injection of ABEmax in dual-AAV8s, efficiently and precisely edited Rho up to 44.2%, partially rescued visual function, and partially preserved the structure of the retina. We hypothesize that even robust editing of Rho was able only to partially restore vision due to the challenging dual nature of Rho as a receptor and structural scaffold. This study provides proof-of-concept of base editing for treating rhodopsin-associated retinitis pigmentosa and indicates that the timing of therapy may be crucial for optimal success in patients.