Establishing an in vitro human Leber congenital amaurosis model system

Abstract

Purpose : Dominant cone-rod homeobox (CRX)-associated Leber congenital amaurosis (LCA7) is a severe retinal degenerative disease for which no treatments are currently available. Disease-causing variants in CRX typically result in the production of a dominant negative form of the protein, which disrupts normal photoreceptor maturation. To gain further insight into LCA7, we aimed to establish an in vitro model system to investigate CRX variant-specific disease mechanisms.

Methods : Human iPSC lines were generated from LCA7 patient whole blood samples, which harbor dominant mutations in CRX (CRX^T155ins4/+ and CRX^K88Q/+). The patient-derived hiPSC lines were differentiated to generate retinal organoids, and immunohistochemistry, TEM, qPCR, and single-cell RNA sequencing were utilized to characterize photoreceptor cell development and maturation up to day 240 (D240) of differentiation.

Results : Retinal organoids were successfully generated from patient iPSCs, and both lines exhibit retinal tissue thickness and photoreceptor cell numbers comparable to those of CRX^WT organoids throughout differentiation. Morphologically, both CRX^T155ins4/+ and CRX^K88Q/+ organoids remain identical to CRX^WT until D240, when they fail to develop outer segments, similar to what has been reported in LCA7 patients and mouse models. Although both CRX^T155ins4/+ and CRX^K88Q/+ organoids exhibit similar levels of OTX2 protein and mRNA compared to control, CRX^T155ins4/+ organoids show an increase in total CRX, while CRX^K88Q/+ organoids show a decrease. CRX^T155ins4/+ and CRX^K88Q/+ organoids both reveal a reduction in RCVRN, AIPL1, ARR3, RHO, OPN1SW, OPN1MW, and OPN1LW compared to CRX^WT organoids (p<0.05), suggesting a decrease in expression of key photoreceptor genes. Variant-specific differences in levels of NRL, NR2E3, SAG, and cone opsins were also observed through D240 of differentiation, highlighting intriguing differences in disease mechanisms between variants.

Conclusions : Here, we have established an early photoreceptor cell-specific LCA phenotype in our patient organoids, and these data provide promise for a reliable in vitro model system which can be used to study variant-specific disease mechanisms. Future work will focus on studying disease mechanisms and therapeutic approaches in our organoid model system.

This is a 2021 ARVO Annual Meeting abstract.

 

Phase-contrast images (A-C) and immunofluorescence staining (D-I) of hiPSC-derived retinal organoids at D240 of differentiation. Scale bars = 100µm.