Résumé
Inherited retinal dystrophies (IRDs) are a group of neurodegenerative disorders characterized by progressive photoreceptor degeneration and vision loss. Mutations in USH2A are the most frequent cause of the syndromic IRD Usher syndrome type 2 (USH2), and the non-syndromic autosomal recessive retinitis pigmentosa (arRP). There are two recurrent USH2A mutations, which account for approximately half the patient cohorts. Currently, there is no available treatment and disease models for these mutations are not available. Due to the large size of the USH2A cDNA, gene augmentation therapy is inaccessible. However, CRISPR/Cas9-mediated genome editing is a viable alternative. We used, for the first time, eSpCas9 to successfully achieve seamless correction of the two most prevalent USH2A mutations in iPSC of patients with USH or arRP. Successful correction of the two mutations was efficiently achieved after nucleofection of the CRISPR components into the iPSC and single-cell sorting. Our results highlight features that promote high efficacy and specificity for eSpCas9-mediated correction. Consistently, whole exome sequencing did not identify any off-target mutagenesis in the corrected iPSC, which also retained pluripotency and genetic stability. Furthermore, analysis of USH2A mRNA expression levels in patient cells potentially identified a novel interactive regulation between the two recurrent mutations, which could account for their genotype-phenotype correlation. We are currently differentiating the parental and corrected iPSC into retinal organoids containing mature photoreceptors to obtain insights into the differential USH2 and arRP phenotypes. Taken together, our highly efficient CRISPR/Cas9-mediated mutation correction of USH2A brings hope for a potential treatment for USH and arRP patients.