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Kathleen R Chirco, Shereen Chew, Anthony T Moore, Jacque L Duncan, Renee Christine Ryals, Martha Neuringer, Deepak A Lamba; Using human LCA7 retinal organoids to establish allele-specific editing as a therapy. Invest. Ophthalmol. Vis. Sci. 2022;63(7):81 – A0054.
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© ARVO (1962-2015); The Authors (2016-present)
Dominant CRX-associated Leber congenital amaurosis (LCA7) is a severe retinal degenerative disease for which no treatments exist. Disease-causing variants in CRX typically result in production of a dominant negative form of the protein which disrupts normal photoreceptor maturation. We used an in vitro human retinal organoid model to evaluate gene editing of mutant CRX as a therapy for LCA7.
We utilized CRISPR/Cas9 tools to (a) correct the CRXK88Q/+ mutation in a patient-derived iPSC line, and (b) eliminate the mutant CRX allele, leaving the wildtype CRX allele intact. Rescue of LCA7 phenotypes was then compared in retinal organoids for both editing methods using brightfield images of the outer segments (OS), immunofluorescence (IF) staining, and qPCR.
Successful editing of patient iPSCs (CRXK88Q/+) to generate a corrected (CRX+/+) and an allelic knockout (CRX+/-) line was confirmed via Sanger sequencing. Retinal organoids were successfully generated for each of the three iPSC lines, and they exhibited comparable retinal tissue thickness and photoreceptor numbers throughout differentiation. Morphologically, all organoids remained identical until 180 days (D180), when differences in OS morphogenesis emerged. The CRX+/+ organoids produced longer OS-like projections by D180, compared to CRXK88Q/+ organoids, while CRX+/- organoids produced OS of intermediate length. mRNA (n=14 organoids per line) and protein (n=8 organoids per line) levels for early photoreceptor markers were comparable between CRX+/+ and CRX+/- lines, revealing a similar level of rescue at the cellular level. Morphological differences became less apparent by D240.
Here, we established that both gene correction (CRX+/+) and an allele-specific gene knockout strategy (CRX+/-) can rescue LCA7 phenotypes. Although there was a mild delay in OS morphogenesis for the CRX+/- line, this may be advantageous for therapy, since gene correction has lower efficiency compared to targeted mutagenesis. This work will guide future experiments focused on treating photoreceptor cells within growing retinal organoids.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
IF staining with anti-SAG (green; A-C), anti-RCVRN (red; D-F), and anti-ARR3 (green; G-I) are shown for CRX+/+; A,D,G), CRXK88Q/+ (B,E,H), and CRX+/- (C,F,I) retinal organoids at D180. Nuclei are counterstained with DAPI (blue). Scale bar=100µm. ONL=outer nuclear layer; INL/GCL=inner nuclear layer/ganglion cell layer.
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