Abstract
Purpose :
Despite the recent success of gene supplementation therapy for monogenic recessive diseases, therapeutic approaches to treat dominantly inherited diseases fall behind. Mutation-specific knockdown/out of the disease alleles is largely limited by off-target effects of RNA interference and the availability of PAM sites. Base editor or prime editor enables precise repair of the disease allele but is not versatile for the diseases with high mutation heterogeneity. Here, we present a new gene knock-in (KI) therapy to integrate the wild-type coding sequence (CDS) into the 5' untranslated region (UTR), more specifically immediately upstream of the Kozak sequence, of the disease gene.
Methods :
AAV-Cas9-mediated gene knock-in (KI) was targeted to the 5' untranslated region (UTR) of the Rho gene by homology-independent targeted integration (HITI). Two AAV vectors, one packaging hRK-Cas9 and the other packaging hRK-mCherry, pU6-gRNA1 and the HITI donor sequence, were subretinally injected into neonatal mouse eyes. We tested this approach in a mouse model of autosomal dominant Retinitis Pigmentosa (adRP) to demonstrate its safety and efficacy.
Results :
We showed that HITI-AAVs mediate highly efficient gene insertion in mouse Rho 5' UTR in vivo. NGS results showed 43% alleles with successful 5' UTR Rho KI, 44% alleles with 5' UTR INDELs, and 14% unmodified alleles in the purified AAV-transduced rods. By applying this strategy to the RhoP23H/wt mice, which carry the most common dominant point mutation found in all adRP patients, we showed that the KI treatment significantly prolonged photoreceptor survival and visual function in the mouse disease model. As expected, the control treatment that causes 5' UTR INDELs only did not rescue or deteriorate the retinal degeneration phenotypes. Our in vivo data showed that the 5' UTR Rho KI treatment is safe and efficient to improve the retinal phenotypes.
Conclusions :
In summary, we designed a new 5' UTR-targeting gene KI therapy for RHO-associated RP. The 5' UTR gene KI approach can be applied to the other common adRP genes as well as other dominantly inherited diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.