Creating an in vitro Leber congenital amaurosis model to investigate disease mechanisms

Abstract

Purpose : Dominant cone-rod homeobox (CRX)-associated Leber congenital amaurosis (LCA) 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 development. To gain further insight into CRX-associated LCA (LCA7), we aimed to establish an in vitro model system to investigate CRX variant-specific disease mechanisms.

Methods : Peripheral blood mononuclear cells (PBMCs) isolated from whole blood samples donated by patients with CRX-associated LCA (CRX^T155ins4/+ and CRX^K88Q/+), were reprogrammed using the Sendai virus to generate stable induced pluripotent stem cell (iPSC) lines (n=2 clones per line). Each iPSC line was then differentiated to generate retinal organoids, and immunohistochemistry and qPCR were utilized to characterize photoreceptor development and maturation.

Results : Retinal organoids were successfully generated from our patient iPSC lines, and both lines exhibit similar amounts of retinal tissue compared to CRX^WT organoids. For CRX^T155ins4/+ organoids, we see an increase in total CRX protein levels compared to the CRX^WT and CRX^K88Q/+ organoids, with a drastic decrease in wildtype CRX immunolabeling. Furthermore, both CRX^T155ins4/+ and CRX^K88Q/+ organoids exhibit elevated OTX2 and NRL protein and mRNA, compared to control organoids, possibly indicating a compensatory response to a decrease in wildtype CRX activity. In contrast, CRX^T155ins4/+ and CRX^K88Q/+ organoids show a reduction in recoverin and cone arrestin compared to CRX^WT organoids, suggesting a decrease in expression of key photoreceptor genes which are controlled by CRX. Finally, we observe mislocalization of photoreceptor cells in our patient organoids, even by day 120 of differentiation.

Conclusions : While additional characterization is required, we have begun to establish 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 mechanism. Future work will focus on studying allele-specific CRISPR/Cas9 editing to alleviate LCA-associated phenotypes in our organoid model system.

This is a 2020 ARVO Annual Meeting abstract.

 

Immunofluorescence staining of human induced pluripotent stem cell-derived retinal organoids at day 120 of differentiation. Scale bar = 100µm.