Afanasyeva, TAV; Athanasiou, D; Perdigao, PRL; Whiting, KR; Duijkers, L; Astuti, GDN; Bennett, J; ... Collin, RWJ; + view all Afanasyeva, TAV; Athanasiou, D; Perdigao, PRL; Whiting, KR; Duijkers, L; Astuti, GDN; Bennett, J; Garanto, A; van der Spuy, J; Roepman, R; Cheetham, ME; Collin, RWJ; - view fewer (2023) CRISPR-Cas9 correction of a nonsense mutation in LCA5 rescues lebercilin expression and localization in human retinal organoids. Methods & Clinical Development , 29 pp. 522-531. 10.1016/j.omtm.2023.05.012.

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Abstract

Mutations in the lebercilin-encoding gene LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report on the generation of a patient-specific cellular model to study LCA5-associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in LCA5 (c.835C>T; p.Q279∗) in patient-derived induced pluripotent stem cells (iPSCs). The absence of off-target editing in gene-corrected (isogenic) control iPSCs was demonstrated by whole-genome sequencing. We differentiated the patient, gene-corrected, and unrelated control iPSCs into three-dimensional retina-like cells, so-called retinal organoids. We observed opsin and rhodopsin mislocalization to the outer nuclear layer in patient-derived but not in the gene-corrected or unrelated control organoids. We also confirmed the rescue of lebercilin expression and localization along the ciliary axoneme within the gene-corrected organoids. Here, we show the potential of combining precise single-nucleotide gene editing with the iPSC-derived retinal organoid system for the generation of a cellular model of early-onset retinal disease.

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