In vivo photoreceptor base editing ameliorates rhodopsin-E150K autosomal- recessive retinitis pigmentosa in mice

• 成果类型: Article
• 署名作者: Du, Samuel W.; Newby, Gregory A.; Salom, David; Gao, Fangyuan; Menezes, Carolline Rodrigues; Suh, Susie; Choi, Elliot H.; Chen, Paul Z.; Liu, David R.; Palczewski, Krzysztof
• 署名单位: University of California System; University of California Irvine; University of California System; University of California Irvine; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Harvard University; Harvard University; Johns Hopkins University; Johns Hopkins University; Massachusetts Institute of Technology (MIT); Massachusetts Institute of Technology (MIT); University of California System; University of California Irvine; University of California System; University of California Irvine
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9481
• DOI: 10.1073/pnas.2416827121
• 发表日期: 2024-11-26
• 关键词: outer segments; retinal rod; Mutation; e150k; gene; quantification; architecture; TRAFFICKING; expression; families

摘要

Rhodopsin, the prototypical class- A G- protein coupled receptor, is a highly sensitive receptor for light that enables phototransduction in rod photoreceptors. Rhodopsin plays not only a sensory role but also a structural role 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, including the autosomal recessive E150K mutation, result in rod dysfunction and death. Therefore, correction of deleterious rhodopsin mutations could rescue inherited retinal degeneration, as demonstrated for other visual genes such as RPE65 and PDE6B. In this study, we describe a CRISPR/Cas9 adenine base editing strategy to correct the E150K mutation and demonstrate precise in vivo editing in a Rho- E150K mouse model of autosomal recessive retinitis pigmentosa (RP). Using ultraviolet- visible spectroscopy, mass spectrometry, and the G- protein activation assay, we characterized wild- type rhodopsin and rhodopsin variants containing bystander base edits. Subretinal injection of dual- adeno- associated viruses delivering our base editing strategy yielded up to 44% Rho correction in homozygous Rho- E150K mice. Injection at postnatal day 15, but not later time points, restored rhodopsin expression, partially rescued retinal function, and partially preserved retinal structure. These findings demonstrate that in vivo base editing can restore the function of mutated structural and functional proteins in animal models of disease, including rhodopsin- associated RP and suggest that the timing of gene- editing is a crucial determinant of successful treatment outcomes for degenerative genetic diseases.