CRISPR-mediated photoreceptor reprogramming reduces degeneration in an optogenetic mouse model of geographic atrophy in AMD

Abstract

Purpose : Age-related macular degeneration (AMD) is the predominant cause of vision loss in older adults. Geographic atrophy (GA) in advanced AMD exhibits retinal pigment epithelium (RPE) damage which can result in the death of photoreceptors, causing irreversible vision loss. We hypothesize that reprogramming vulnerable rods into more resilient cone cells using an adeno-associated viral (AAV)-mediated CRISPR system to ablate the neural retina leucine zipper (Nrl) rod-fate transcription factor gene may protect them against photoreceptor degeneration in GA in advanced AMD.

Methods : We subretinally injected a dual AAV8 system into eyes of wild-type C57BL/6J mice with 2 vectors to express (1) SpCas9 under a photoreceptor-specific rhodopsin kinase (RK) promoter and (2) mCherry under an RPE-specific Bestrophin (VMD2) promoter with a mitochondrial targeting sequence and single guide RNA (gRNA) sequences to target Nrl or scrambled control under a U6 promoter. A custom-built scanning-laser ophthalmoscopy (SLO) / optical coherence tomography (OCT) system operating at 561 nm and 700 μW power was used to scan a small region of mouse retina to activate the reactive oxygen species (ROS)-generating mCherry chromophores and induce mitochondrial oxidative stress to selectively ablate RPE, resembling GA in AMD. Anatomic changes were measured longitudinally in vivo using the same SLO/OCT system, followed by collection of retinal tissues for deep sequencing to measure gene editing rate and immunohistochemistry to assess photoreceptor structure.

Results : Immunohistochemistry showed localization of Cas9 in photoreceptors and mCherry in RPE mitochondria. Photoactivation of RPE-specific mCherry in mouse eyes using a custom SLO/OCT system enabled photochemical ablation of RPE with secondary photoreceptor dysfunction and atrophy resembling GA. CRISPR-mediated targeting of Nrl generated 5.89 ± 5.83% editing rate in retinal tissues with reduction in outer retinal layer disruption, but did not completely prevent photoreceptor degeneration compared to scrambled gRNA controls.

Conclusions : Reprogramming rods into cone-like cells by CRISPR-mediated ablation of Nrl may protect against an optogenetic model of outer retinal and RPE atrophy in mouse eyes, and serve as a novel therapeutic strategy to preserve vision in patients with atrophic AMD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.