Modeling Enhanced S-cone Syndrome Using Patient-Specific iPSC-derived Retinal Eyecups

Abstract

Purpose : NR2E3 is a retinal transcription factor that is required for the suppression of cone-specific genes during photoreceptor development. NR2E3-associated disease is characterized by congenital night blindness, increased sensitivity to short wavelength light due to a larger population of S-cones and varying degrees of medium (M-cone)- and long (L-cone)-mediated vision. The purpose of this study is to generate patient-specific iPSCs and iPSC-derived 3D retinal eyecups to model disease pathophysiology of enhanced S-cone syndrome, caused by mutations in the gene NR2E3.

Methods : iPSCs were generated from two patients with molecularly confirmed mutations in NR2E3 via viral transduction of OCT4, SOX2, KLF4 and c-MYC in an FDA-registered cGMP facility. iPSCs were differentiated via suspension culture to induce the formation of 3D retinal eyecups. Eyecups were evaluated for retinal-specific and photoreceptor-specific markers via indirect immunofluorescence and confocal microscopy throughout development. CRISPR/Cas9 technology was also tested using small guide RNAs specifically targeting the NR2E3 locus in patient iPSCs.

Results : We have successfully generated two lines of clinical-grade NR2E3 patient-specific iPSCs in a cGMP manner. Early NR2E3 patient eyecups form polarized neural epithelial folds that express the early retinal progenitor markers SOX2, PAX6, VSX2/CHX10 and OTX2. As eyecups mature, dense pockets of OTX2-positive photoreceptor precursor cells arise. Later, eyecups begin to express photoreceptor-specific transcription factors CRX and NRL, as well as the phototransduction protein, recoverin. Compared to controls, NR2E3 eyecups exhibit an increased abundance of SW-opsin-positive blue cone photoreceptors, recapitulating the developmental phenotype observed in enhanced S-cone syndrome patients.

Conclusions : We have demonstrated that we can generate patient-specific iPSCs and iPSC-derived retinal photoreceptor precursor cells from patients with NR2E3-associated enhanced S-cone syndrome. These patient-derived cells recapitulate the retinal phenotype observed in patients and can be used to interrogate the pathophysiology of this rare disease. In particular, we will use CRISPR-mediated genome correction to correct the NR2E3 locus in patient iPSCs and rescue the enhanced blue cone phenotype and restore the downstream development of rod, M- and L-cone photoreceptors.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.