Gene Correction Reverses Ciliopathy and Photoreceptor Loss in iPSC-Derived Retinal Organoids from Retinitis Pigmentosa Patients.

Wen-Li Deng,Yan-Ping Li,Tian Xue,Xi-Xi Xia,Ji-Neng Lv,Kai-Wen He,Mei-Ling Gao,Yu-Chen Chen,Xin-Lan Lei,Deng Pan,Huan Zhao,Zi-Bing Jin,Ling-Yun Li

doi:10.1016/j.stemcr.2018.02.003

Abstract

SummaryRetinitis pigmentosa (RP) is an irreversible, inherited retinopathy in which early-onset nyctalopia is observed. Despite the genetic heterogeneity of RP, RPGR mutations are the most common causes of this disease. Here, we generated induced pluripotent stem cells (iPSCs) from three RP patients with different frameshift mutations in the RPGR gene, which were then differentiated into retinal pigment epithelium (RPE) cells and well-structured retinal organoids possessing electrophysiological properties. We observed significant defects in photoreceptor in terms of morphology, localization, transcriptional profiling, and electrophysiological activity. Furthermore, shorted cilium was found in patient iPSCs, RPE cells, and three-dimensional retinal organoids. CRISPR-Cas9-mediated correction of RPGR mutation rescued photoreceptor structure and electrophysiological property, reversed the observed ciliopathy, and restored gene expression to a level in accordance with that in the control using transcriptome-based analysis. This study recapitulated the pathogenesis of RPGR using patient-specific organoids and achieved targeted gene therapy of RPGR mutations in a dish as proof-of-concept evidence.

Highlights

Retinitis pigmentosa (RP) is a leading cause of blindness worldwide
Generation and Characterization of RPGR PatientDerived induced pluripotent stem cells (iPSCs) Urinary cells were isolated from 100–300 mL of urine from three male patients with childhood night blindness who were subsequently diagnosed with RP (Figure S1)
Patient 1 harbored a mutation in exon 14 of RPGR gene with c.1685_1686delAT, while patients 2 and 3 had mutations in ORF15 of RPGR gene with c.2234_2235delGA and c.2403_2404delAG

Summary

Introduction

Retinitis pigmentosa (RP) is a leading cause of blindness worldwide. this disease is still incurable due to its extreme heterogeneity and unclear mechanisms. More than 85 genes involved in RP have been identified (Daiger et al, 2013; Ran et al, 2014; Huang et al, 2015a) Among these genes, the RPGR gene, which was discovered two decades ago (Meindl et al, 1996; Roepman et al, 1996), is one of the most prevalent causative genes, accounting for approximately 16% of RP patients (Vervoort et al, 2000; Hartong et al, 2006; Jin et al, 2006a; Huang et al, 2015b). The RPGR gene is located in the X chromosome, containing 19 exons and one open reading frame (ORF15) (Meindl et al, 1996; Vervoort et al, 2000). The function of ORF15 consisting of glutamic acid/glycinerich domain is unknown

Results

Discussion

Conclusion