Characterizing AMD risk genes in an iRPE functional genetic screening platform

Abstract

Purpose :
Age-related macular degeneration (AMD) is the third leading cause of vision loss and is
characterized by lipid-rich extracellular deposits and localized inflammation in the macula. Retinal Pigment Epithelial (RPE) cells progressively lose their ability to phagocytose photoreceptor outer segments, maintain tight-barrier function and become inflamed resulting in changes in morphology and function during the intermediate and later stages of AMD. Genome-wide association studies (GWAS) and transcriptomic profiling of human donor eyes strongly implicate RPEs in AMD risk, progression and pathogenesis. However, little is known about the gene programs driving AMD risk in the context of RPE biology.

Methods :
We have developed a functional genetic screening platform using human iPSC derived RPEs
(iRPEs). These cells recapitulate key RPE transcriptomic profiles when assessed via scRNA- and scATAC-seq. iRPEs also display functional aspects of RPEs such as pigmentation, phagocytosis, barrier function and secretion of immunomodulatory cytokines. To knock out any gene of interest we optimized a CRISPR-Cas9 mediated protocol which efficiently perturbed gene expression without compromising cell survival. We have thus developed an iRPE functional genetic screening platform which allows for scalable assessment of gene function (Figure 1).

Results :
To understand the gene programs driving AMD risk and progression we screened genes with population or functional genetic evidence of association with advanced or intermediate AMD, as well as the RPE transcription factor, MITF, as a positive control. MITF knockout resulted in a discernible change in the transcriptomic profile and dysregulation of pathways associated with melanogenesis, gap junction formation and MAPK signaling. Knockout of Nemo-like kinase (NLK) resulted in a significant reduction in the phagocytic activity of iRPE cells independent of changes in morphology or viability.

Conclusions : Together these data highlight the power of using functional genetic screens informed by human genetics in appropriate in vitro systems to uncover novel biology and for therapeutic target discovery. The intersection of human genetics and functional genetic screens provides a new paradigm for target prioritization and drug discovery.

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