Genome-wide Screening Identifies Novel Genetic and Epigenetic Factors for the Rejuvenation and Enhancement of RPE to Alleviate Age-related Macular Degeneration

Abstract

Purpose : Age-related macular degeneration (AMD) impacts millions of patients globally, with dry AMD presenting a challenge due to its prevalence and the absence of effective therapies. Innovative approaches are needed to address the oxidative stress and aging components of AMD progression. Here, we conducted a genome-scale overexpression screen to systematically identify potent genetic factors (e.g. transcription factors) that offer oxidative protection to RPE cells. We also examined a novel epigenetic reprogramming strategy using OSK (Oct4, Sox2, Klf4) to counteract RPE aging.

Methods : A library of over 12,000 different ORFs from the human genome, each with unique barcodes, was introduced into ARPE-19 cells at a low MOI. Following maturation in culture, live cells that survived the potent oxidant, NaIO3, were sequenced for their barcodes and compared with the barcode representation in dead or undamaged cells. ORFs enriched in live cells, as well as with the epigenetic reprogramming factors OSK, were cloned into inducible lentiviral vectors to infect RPE cells and their oxidative protective effects were tested. The validated ORFs were further cloned into AAV and delivered to the RPE layer of mice through subretinal injection. Three in vivo mouse models were employed to assess the effects of AAV-mediated overexpression of ORFs: (1) acute RPE degeneration model induced by IP NaIO3; (2) aged mice at 12-24 months old; and (3) aged mice carrying the human CFH risk allele on a high-fat diet (HFD). Visual acuity and morphological assessment of RPE layers were performed.

Results : A genome-wide ORF screen revealed key factors that may be implicated in AMD pathogenesis, while also highlighting novel transcription factors that enhance RPE oxidative resistance in vitro and in an acute RPE degeneration model. The OSK epigenetic reprogramming factors provided RPE with resistance to oxidative stress, stabilized their epigenome, restored youthful retinal structure and visual acuity in aged mice, and reduced AMD-like RPE dysmorphia in aged CFH mice on HFD.

Conclusions : A genetic screen, coupled with epigenetic reprogramming, systematically addresses AMD pathogenesis by identifying genetic factors enhancing RPE oxidative stress resistance and rejuvenating aging processes.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.