Modeling PRPF31 retinitis pigmentosa using iPSC-derived retinal organoids and retinal pigmented epithelium and rescue by gene augmentation strategy. Modeling PRPF31 retinitis pigmentosa using iPSC-derived retinal organoids and retinal pigmented epithelium and rescue by gene augmentation strategy

Mutations in the ubiquitously expressed pre-mRNA processing factor (PRPF) 31, one of the most common cause of dominant form of Retinitis Pigmentosa (RP), lead to retina-specific phenotype. It is uncertain which retinal cell types are affected and animal models do not clearly present the RP phenotype observed in PRPF31 patients. Retinal organoids and retinal pigmented epithelial (RPE) cells derived from human induced pluripotent stem cells (iPSCs) provide potential opportunities for studying human PRPF31-related RP. We demonstrated that RPE cells carrying PRPF31 mutations present important morphological and functional changes and that PRPF31-mutated retinal organoids recapitulate the human RP phenotype, with a rod photoreceptor cell death followed by a loss of cones. The low level of PRPF31 expression may explain the defective phenotypes of PRPF31-mutated RPE and photoreceptor cells, which were not observed in cells derived from asymptomatic patients or after correction of the pathogenic mutation by CRISPR/Cas9. Transcriptome profiles revealed differentially expressed and mis-spliced genes belonging to pathways in line with the observed defective phenotypes. The rescue of RPE and photoreceptor defective phenotypes by PRPF31 gene augmentation, provide the proof of concept for future therapeutic strategies. Overall design: Comparative gene expression profiling analysis of RNA-seq data from retinal organoids and retinal pigmented epithelial (RPE) cells derived from patient induced pluripotent stem cells (iPSCs). Human iPSCs were derived from control and familly members carrying PRPF31 mutations who are asymptomatic or symprtomatic with photoreceptor degeneration.

泛表达的前mRNA剪接因子31（PRPF31）的突变是显性遗传性色素性视网膜炎（RP）最常见的致病原因之一，却仅会引发视网膜特异性表型。目前尚未明确具体受累及的视网膜细胞类型，且现有动物模型无法复现PRPF31突变患者所观察到的RP表型。由人类诱导多能干细胞（iPSCs）分化得到的视网膜类器官与视网膜色素上皮（RPE）细胞，为研究PRPF31相关RP提供了理想的实验模型。本研究证实，携带PRPF31突变的RPE细胞会出现显著的形态与功能异常；携带PRPF31突变的视网膜类器官则可复现人类RP的典型表型，表现为杆状光感受器细胞先发生死亡，随后出现视锥细胞丢失。PRPF31的低表达水平或可解释PRPF31突变RPE细胞与光感受器细胞的缺陷表型：这一表型在无症状患者来源的细胞中未被观测到，经CRISPR/Cas9校正致病突变后亦未出现。转录组测序分析显示，差异表达基因与异常剪接基因所富集的通路，与本研究观测到的缺陷表型高度契合。通过PRPF31基因增补可挽救RPE细胞与光感受器细胞的缺陷表型，这为未来的治疗策略提供了概念验证依据。实验设计概述：对患者诱导多能干细胞（iPSCs）分化得到的视网膜类器官与视网膜色素上皮（RPE）细胞的RNA-seq数据进行比较基因表达谱分析。本研究中的人类iPSCs源自健康对照组及携带PRPF31突变的家族成员，其中部分成员无临床症状，部分则出现光感受器退行性病变。