Allele-specific gene-editing approach for vision loss restoration in <i>RHO</i>-associated retinitis pigmentosa.

Abstract

Mutant <i>RHO</i> is the most frequent genetic cause of autosomal dominant retinitis pigmentosa (adRP). Here, we developed an allele-specific gene-editing therapeutic drug to selectively target the human T17M <i>RHO</i> mutant allele while leaving the wild-type <i>RHO</i> allele intact for the first time. We identified a <i>Staphylococcus aureus</i> Cas9 (SaCas9) guide RNA that was highly active and specific to the human T17M <i>RHO</i> allele. <i>In vitro</i> experiments 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 single guide RNA (sgRNA) to the retinas of the <i>RHO</i> humanized mice showed that this therapeutic drug targeted the mutant allele selectively, thereby downregulating the mutant <i>RHO</i> mRNA 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 heterozygous mutant humanized mice. Our study demonstrated a dose-dependent therapeutic effect <i>in vivo</i>. 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 treat <i>RHO</i>-T17M-associated adRP, also offers a generalizable framework for developing gene-editing medicine. Furthermore, our success in restoring the vision loss in the suffering <i>RHO</i> humanized mice verifies the feasibility of allele-specific CRISPR/Cas9-based medicines for other autosomal dominant inherited retinal dystrophies.