Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generated retinal organoids carrying retinal disease–causing variants in NR2E3 , as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing (scRNA-Seq) across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNA-Seq data, we reconstruct the rod photoreceptor developmental lineage and identify a branch point unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different from those observed in Nr2e3 rodent models. Together, these data provide a road map of human photoreceptor development and leverage patient induced pluripotent stem cells to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

Autologous photoreceptor cell replacement is one of the most promising strategies currently being developed for the treatment of patients with inherited retinal degenerative blindness. Induced pluripotent stem cell–derived (iPSC-derived) retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of transplantable photoreceptors required for these therapies. However, retinal organoids contain other retinal cell types, including bipolar, horizontal, and amacrine cells, which are unneeded and may reduce the potency of the final therapeutic product. Therefore, approaches for isolating fate-committed photoreceptor cells from dissociated retinal organoids are desirable. In this work, we present partial dissociation, a technique that leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. We demonstrate up to 90% photoreceptor cell purity by simply selecting cell fractions liberated from retinal organoids during enzymatic digestion in the absence of mechanical dissociation. Since the presented approach relies on the use of standard plasticware and commercially available current good manufacturing practice–compliant reagents, we believe that it is ideal for use in the preparation of clinical photoreceptor cell replacement therapies.