Résumé
Purpose : Mutations in USH2A are the most frequent cause of Usher syndrome type 2 (USH2) and autosomal recessive non-syndromic retinitis pigmentosa (arRP). Furthermore, two recurrent USH2A mutations (c.2276G>T; p.Cys759Phe and c.2299delG; p.Glu767fs) account for approximately half the patient cohorts. Currently, there is no available treatment for patients with mutations in USH2A. Similarly, disease models to study the pathogenicity of these two mutations are not available. Here, induced pluripotent stem cells (iPSC) from patients with USH2A mutations were generated and a CRISPR/Cas9 strategy has been developed as a possible future therapeutic approach.Methods : Two patients were included in this study: The first presented with USH2 and carried the c.2299delG in the homozygous state; the second presented with non-syndromic arRP and was compound heterozygous for c.2276G>T and c.2299delG. Patients’ fibroblasts were cultured from a skin biopsy and reprogrammed into iPSC. The iPSC generated were characterized for pluripotency. The differentiation potential was studied by an embryoid body (EB) formation assay. A CRISPR/Cas9 approach using a high-fidelity Cas9 was validated in HEK293 cells. The most efficient gRNAs were selected and single stranded oligonucleotides (ssODN) were designed as a repair template. Single cell sorting and sequencing of the CRISPR-iPSC clones was performed for mutation verification correction.Results : The iPSC generated were positive for the pluripotency markers NANOG, OCT3/4, LIN28a and SOX2 as assessed by RT-qPCR and immunofluorescence staining. The iPSC were able to differentiate into the three germ layers: mesoderm, ectoderm and endoderm as assessed by SMA, GFAP and AFP immunofluorescence staining, respectively. The gRNAs selected introduced INDELs at the desired regions in both, HEK293 cells and iPSC. Correction of the c.2299delG mutation in iPSC after homologous directed repair (HDR) has been confirmed. Preliminary data for the c.2276G>T mutation, indicates that this mutation can also be successfully corrected using CRISPR/Cas9.Conclusions : The results presented here provide hope for a future therapy applicable to a large number of patients. In addition, the generation of iPSC from patients with USH2A mutations presenting with USH2 or non-syndromic arRP will provide insights regarding the pathogenesis of syndromic vs. non-syndromic RP.