Generation and characterization of human USH2A retinal disease model

Abstract

AbstractPurpose: Mutations in USH2A are the most common cause of autosomal recessive retinitis pigmentosa (RP) and syndromic RP in combined deaf‐blindness, Usher syndrome type 2 (USH2). In both cases, the effective treatment is lacking. The failure of retinal phenotype in available animal models warrants the generation of human tissue mimics in which to explore the underlying molecular mechanism and test available therapeutic modalities. To model this disease in vitro, we generated iPSC‐derived retinal organoids from the USH2 patient and isogenic control. We studied cellular and morphological alterations in retinal organoids and the effectiveness of gene editing to rescue retinal cell phenotypes. We describe the precise gene editing approach to genetically correct the p.Cys759Phe mutation in USH2A and hence generate the isogenic iPSC line.Methods: The editing machinery was introduced by delivery of recombinant Cas9 and synthetic crRNA into the iPSCs derived from the patient bearing p.Cys759Phe mutation in USH2A. A single stranded oligonucleotide donor (ssODN) template carrying the wild‐type sequence was used to drive the mutation correction. The single cell clones were obtained and their edited genotype confirmed. Patient and isogenic control were subjected toward retinal organoid differentiation and analysed by fluorescence microscopy throughout the protocol, stage specific retinal cell markers were identified.Results: The CRISPR/Cas9 gene editing approach generated homo‐and heterozygously corrected clones with acceptable efficiency. Patient and isogenic control lines were induced toward retinal organoids with all mayor retinal cell types. We observe reduced efficiency in neural retina formation and paucity in cellular laminar arrangement of patients' cells. The isogenic control was able to revert disease related cellular phenotypes and subcellular features.Conclusions: Our study corroborates the feasibility of CRISPR/Cas9 editing of genetic lesion in monogenic disease bearing hiPSCs further supporting the applicability of CRISPR/Cas9 machinery in in vivo settings. We have identified retinal cell phenotype in USH2A p.Cys759Phe disease background which allows us to study disease mechanism and test novel treatment options.