OR12

Retinitis pigmentosa (RP), the predominant cause of genetic blindness, is inherited in an autosomal dominant manner (adRP) in 35% of cases. Mutations in the RHO gene, which encodes rhodopsin, account for the majority of adRP cases. In those cases, gene addition therapy is hindered by the toxic effects of mutant rhodopsin. Instead, "Suppression and Replacement" strategies, independent of specific mutations and employing CRISPR/Cas9 targeting RHO combined with cDNA delivery, have demonstrated efficacy in animal models harbouring the most prevalent mutation observed in US patients, linked with a mild phenotype. In an attempt to investigate the applicability of this strategy to models with a more severe phenotype, we aim to perform this approach on an adRP mouse characterised by one of the most recurrent mutations in Europe and Asia. We first conducted in vitro guide RNA (gRNA) screening on the RHO gene. The optimal gRNA was administered in vivo via dual-AAV-encoding Cas9 and gRNA in adRP mice. Next-generation sequencing (NGS) showed modest 0.5% editing efficiency on the whole retina. To enhance editing efficiency, a non-viral approach was explored, injecting naked Cas9 ribonucleoproteins (RNPs) into adRP mouse retinas, revealing a total of 2% editing via NGS analysis. In evaluating safety, Cas9 RNP injection induced retinal damage, signifying in vivo RNP toxicity. Our preliminary results offer insights into AAV vs. RNP-associated gene editing and in vivo toxicity for adRP treatment. These findings suggest the need for optimising delivery strategies, notably in degenerating models, to increase editing efficiency and decrease toxicity.