In vivo base editing rescues cone photoreceptors in a mouse model of early-onset inherited retinal degeneration

Abstract

Purpose : Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function, but also protects photoreceptors from further degeneration. Here, we sought to answer whether base editing could rescue cone photoreceptor’s survival and function.

Methods : We first screened for a combination of evolved ABE variants and sgRNAs to enhance the on-target correction rate and reduce bystander base editing. Then, we packaged the selected ABE variant and sgRNA into a lentivirus and subretinally delivered it to rd12 or rd12Gnat1^-/- mice at 3-weeks-old. We assessed the DNA correction, RPE65 rescue, survival of S-opsin- and M-opsin-positive cones (S-cones and M-cones), and localization of S-opsins and M-opsins on retinal wholemount and cross-section staining. Furthermore, we evaluated cone function in treated rd12Gnat1^-/- mice with electroretinography and visual cortex recordings. Lastly, we used single-cell RNA-sequencing to examine the impact of base editing on cone photoreceptors.

Results : Subretinal delivery of ABE and sgRNA corrected up to 40% of Rpe65 transcripts and preserved significantly higher numbers of S-opsin- and M-opsin-positive cones compared to the age-matched control group. The retinal cross-sections of the treated mice revealed correct localization of S-opsins and M-opsins in the cone outer segments. The photopic ERG waveforms from S- or M-cones from the treated rd12Gnat1^-/- mice exhibited a prominent b-wave, whereas untreated eyes did not respond. Long-term protection of cone photoreceptors and function was also evident in older mice at 6 months of age. Furthermore, single-cell RNA-seq of the treated retinas revealed rescue of the gene expressions associated with cone phototransduction and survival.

Conclusions : Our results show that base editing can rescue the function and survival of cone photoreceptors, which has been a major challenge in the treatment of inherited blindness. These findings provide a foundation for a potential one-time treatment for LCA that confers long-lasting retinal protection, and warrant development of base editing for clinical application.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.