Résumé
Purpose : 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 (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 successful CRISPR/Cas9 correction of these two recurrent mutations in patient iPSC. Here, we differentiated isogenic corrected and non-corrected iPSC lines onto retinal organoids to functionally validate the strategy and to obtain a disease model of these two recurrent mutations.Methods : Isogenic 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 until processing of the sample. Four different time points (56, 100, 150, 225 days) were analyzed by qPCR and immunofluorescence studies for the expression of common photoreceptor markers. Photoreceptor ultrastructure was also investigated using electron microscopy (EM).Results : The generated iPSC-derived retinal organoids express common photoreceptor markers such as CRX, Recoverin, Rhodopsin, Rhodopsin-kinase, Opsins, Cone-arrestin. In addition, they express outer segments (OS) photoreceptor markers such as ABCA4, PDE6B and PRPH2. The photoreceptors within the retinal organoids present key features of photoreceptor maturation, such as a connecting cilium and OS, as observed by EM. Characterization of corrected and non-corrected retinal organoids show a clear reversion of the aberrant phenotype in the CRISPR-corrected organoids compared to the USH2A mutant organoids, functionally validating our CRISPR strategy.Conclusions : The results presented here provide hope for a future therapy applicable to a large number of patients carrying USH2A mutations. The functional 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 have been obtained. 2021-05-01