Résumé
PurposeMutations 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 (c.2276G>T and c.2299delG), which account for approximately half the patient cohorts. Currently, there is no available treatment and disease models for these mutations are not available. We previously reported seamless CRISPR/Cas9-mediated gene correction of these two recurrent mutations in the iPSC of two patients: one presenting with USH2 and the other with arRP. Here, we differentiated the isogenic corrected and non-corrected iPSC lines into retinal organoids to functionally validate the gene-correction strategy and to obtain, for the first time, mature disease models for these two mutations.MethodsIsogenic corrected and non-corrected iPSC lines together with a wild-type (WT) iPSC line were differentiated onto retinal organoids using a combinatory two-dimensional (2D) and 3D protocol. iPSC-derived retinal organoids were maintained as floating cultures in retinal differentiation medium until processing of the samples. Mature retinal organoids were analyzed by immunofluorescence studies for the expression of common photoreceptor markers. Photoreceptor ultrastructure was also investigated using electron microscopy (EM).ResultsThe generated iPSC-derived retinal organoids express common photoreceptor markers such as CRX, Recoverin, Rhodopsin, Rhodopsin-kinase, Opsins, Cone-arrestin. Moreover, the photoreceptors within the retinal organoids present key features of photoreceptor maturation, such as a connecting cilium and outer segments (OS) as observed by either immunofluorescence or ultrastructure analysis studies. Characterization of the isogenic corrected and non-corrected retinal organoids show a reversion of the aberrant phenotype in the CRISPR-corrected organoids compared to the USH2A mutant organoids. Our preliminary data suggest that iPSC-derived USH2 and arRP retinal organoids show a differential phenotype.ConclusionThe results presented here provide hope for a future therapy applicable to a large number of patients carrying the recurrent USH2A mutations. The validation of the CRISPR strategy in retinal organoids brings this therapy one step closer to clinical translation. In addition, insights into the differential pathophysiology of USH2 and arRP will further our understanding on the pathophysiology associated to USH2A mutations.