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Kathleen R Chirco, Shereen Chew, Jacque L Duncan, Anthony T Moore, Deepak A Lamba; Establishing an in vitro human Leber congenital amaurosis model system. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1693.
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© ARVO (1962-2015); The Authors (2016-present)
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.
Human iPSC lines were generated from LCA7 patient whole blood samples, which harbor dominant mutations in CRX (CRXT155ins4/+ and CRXK88Q/+). 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.
Retinal organoids were successfully generated from patient iPSCs, and both lines exhibit retinal tissue thickness and photoreceptor cell numbers comparable to those of CRXWT organoids throughout differentiation. Morphologically, both CRXT155ins4/+ and CRXK88Q/+ organoids remain identical to CRXWT until D240, when they fail to develop outer segments, similar to what has been reported in LCA7 patients and mouse models. Although both CRXT155ins4/+ and CRXK88Q/+ organoids exhibit similar levels of OTX2 protein and mRNA compared to control, CRXT155ins4/+ organoids show an increase in total CRX, while CRXK88Q/+ organoids show a decrease. CRXT155ins4/+ and CRXK88Q/+ organoids both reveal a reduction in RCVRN, AIPL1, ARR3, RHO, OPN1SW, OPN1MW, and OPN1LW compared to CRXWT 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.
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.
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