Gene-edited mouse models and human retinal organoids to study the function of CERKL, NR2E3 and ATXN3 genes in inherited retinal dystrophies

Abstract

Purpose : Inherited retinal dystrophies (IRDs) are a group of genetically heterogeneous rare diseases affecting 1:3000 people worldwide. The precise physiological function of many causative IRD genes is yet to be determined. We aimed to elucidate the functional role of CERKL, NR2E3 and ATXN3 using: 1) primary cultures of retinal cells, 2) retinas from knockout and CRISPR-edited mice and 3) retinal organoids derived from human patient and control.

Methods : Human retinal cups have been generated from iPSCs derived from a Retinitis Pigmentosa patient and a control sibling to study the effect of the p.R257X (p.R283X) CERKL nonsense mutation. CRISPR-edited and a conventional knockout mice have been generated and analyzed to study CERKL, NR2E3 and ATXN3 contribution to retinal degeneration, while specific phenotypic traits have been studied in cultured cells by gene overexpression or silencing assays. Human organoid, murine retinas and cellular models have been phenotypically characterized by immunofluorescence detection, transmission electron microscopy and biochemical assays. Transcriptomic and electrophysiological analyses are underway.

Results : Our results show that CERKL contributes to retinal cell resilience in front of oxidative/luminic stress by formation of RNA stress-granules and regulation of mitochondrial dynamics, while its depletion causes retinal degeneration. Some gene-edited NR2E3 alleles differentially express shorter isoforms with a gain of function, which result in a significant increase of cone photoreceptors in the mouse retina. On the other hand, both ATXN3 depletion and overexpression studies indicate that ATXN3 modulates ciliary trafficking and microtubule-mediated transport in photoreceptors and the retinal pigment epithelium.

Conclusions : Overall, our studies on human retinal organoids, retinas from CRISPR-edited and knockout mice, and retinal cultured cells confirm the relevant role of CERKL, NR2E3 and ATXN3 in the development, physiology, homeostasis, and resilience to stress of the mammal retina. Mutations in CERKL and NR2E3 have already been shown to cause inherited retinal dystrophies although the physiological function of these genes need further dissection. We also propose ATXN3 as a good candidate to regulate key retinal functions such as cilium formation and microtubule-mediated cargo transport.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.