Mutation repair reverses ciliopathy and photoreceptor loss in iPSC-derived retinal organoids from retinitis pigmentosa patients

Abstract

Purpose : Retinitis pigmentosa (RP) is an irreversible, inherited retinopathy in which nyctalopia is observed at early onset, eventually followed by complete blindness as the disease progresses. Despite the genetic heterogeneity of RP, Retinitis Pigmentosa GTPase Regulator (RPGR) mutations are still the most common causes of this disease in patients. However, mechanisms of RPGR mutations remain largely unclear.

Methods : We generated induced pluripotent stem cells (iPSCs) from three RP patients with different frameshift mutations in the RPGR gene and five normal controls. The mutation in one patient iPSCs was repaired with CRISPR/Cas9. Each iPSCs were differentiated into 3D retinal organoids (ROs). Compared patient’ 3D ROs with correction and control ones in photoreceptor specific protein and transcriptional expression using immunostaining and RNA-seq respectively. The expression of glial fibrillary acidic protein (GFAP) in those organoids was also tested by Western blot. Patch clamp of control, correction and patient-ROs were recorded. Also the cilia length in control, correction and patient- iPSCs, ROs and RPE cells were measured.

Results : Patient, control and correction-iPSCs could be differentiated into well-structured ROs after more than 35 weeks culture. However, we observed significant defects in photoreceptor development in terms of photoreceptor morphology, localization and transcriptional profiling in patient ROs, and in those highly expressed GFAP. Furthermore, shorted cilia were found in patient iPSCs, RPE cells and 3D ROs. The specific signal of photoreceptor could almost not be recorded with patch clamp in patient ROs. CRISPR-Cas9-mediated correction of the mutation not only rescued photoreceptor development but also reversed the observed ciliopathy. Additionally transcriptome-based analysis revealed that gene expression in the mutation-corrected ROs was in accordance with that in the normal controls. And the GFAP expression level in correction organoids was also reduced close to normal level. The patch clamp recordings of photoreceptors in correction ROs were just as same as in control ones.

Conclusions : Collectively, this study recapitulated the pathogenesis of RPGR using patient-specific 3D retinal organoids for the first time and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.