Mutation-independent gene knock-in therapy targeting 5 pam UTR for autosomal dominant retinitis pigmentosa

Despite the recent success of gene supplementation therapy for monogenic recessive diseases, therapeutic approaches to treat dominantly inherited diseases fall behind. Here, we present a new gene knock-in (KI) therapy which exploits AAV-Cas9-mediated homology-independent targeted integration (HITI) of the wild-type coding sequence (CDS) into the 5 pam untranslated region (UTR), more specifically immediately upstream of the Kozak sequence, of the disease gene. We tested this approach in the heterozygous RhoP23H/wt mice, which carry the most common dominant point mutation found in the autosomal dominant Retinitis Pigmentosa (adRP) patients. We show that HITI-AAVs can mediate highly efficient gene insertion in mouse Rho 5 pam UTR in vivo. NGS results showed 43% alleles with successful 5 pam UTR Rho KI, 44% alleles with 5 pam UTR INDELs, and 13% unmodified alleles in the purified AAV-transduced photoreceptors. The RhoP23H/wt mice had significantly prolonged photoreceptor survival and visual function following the 5 pam UTR gene KI treatment. In summary, we developed a mutation-independent gene KI approach that targets 5 pam UTR of the disease gene and demonstrated its therapeutic potential to treat dominant diseases.