Recreating pathophysiology of CLN2 disease and demonstrating reversion by TPP1 gene therapy in hiPSCs-derived retinal organoid and retina-on-chip

Mutations in tripeptidyl peptidase 1 (TPP1) gene lead to late infantile neuronal ceroid lipofuscinosis CLN2, characterized by lysosomal accumulation of lipofuscins predominantly found in brain and retina. The ocular phenotype is characterized by bilateral outer retinal degeneration that leads to complete vision loss. CLN2 animal models struggle in recapitulating the retinal phenotype observed in patients. Here, we leveraged human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs), retinal pigmented epithelial (RPE) cells, and retina-on-chip (RoC) technologies to model CLN2 disease in vitro in patient-specific microphysiological models of the human retina. Using these models, we were able to recreate the major clinical histological hallmark of CLN2 patients, namely the accumulation of lipofuscins containing subunit C of mitochondrial ATP synthase (SCMAS) and lipids mainly in the outer retina. Furthermore, single-cell RNA-sequencing of CLN2 ROs revealed a dysregulation of translational and mitochondrial function in cones. Finally, we demonstrate that an adeno-associated virus (AAV)-mediated TPP1 gene therapy was able to restore TPP1 expression and could decrease and even prevent SCMAS accumulations. In summary, our study produced novel human-relevant microphysiological retinal disease models, uncovered new mechanisms of CLN2 pathophysiology in the human retina, and demonstrated the immense potential of AAV9.hCLN2 gene therapy for CLN2 disease potentially treating and even curing blindness of affected individuals. Overall design: iPSC-derived retinal organoids (ROs) at d192 of two control lines (CTRL1, CTRL2) and two CLN2 Patients lines untreated (CLN2-1, CLN-2) and treated with AAV9-hCLN2 (AAV9.hCLN2-treated CLN2-1 and AAV9.hCLN2-treated CLN2-2) were subjected to single cell sequencing analysis (10X Genomics). The six samples were distributed into 4 runs and demultiplexed by SNPs using SoupOrCell v2.0 (Heaton et al 2020). Run 1 contained CTRL1 and CLN2-1, Run2 contained CTRL1 and AAV9.hCLN2-treated CLN2-1, Run3 contained CTRL2 and CLN2-2, Run4 contained CTRL2 and AAV9.hCLN2-treated CLN2-2.

三肽基肽酶1（tripeptidyl peptidase 1, TPP1）基因的突变会引发晚发型婴儿型神经元蜡样脂褐质沉积症CLN2，该病以脑与视网膜中主要蓄积脂褐素的溶酶体沉积为典型特征。其眼部表型以双侧视网膜外层变性为核心表现，最终可导致完全性视力丧失。现有CLN2动物模型难以重现患者体内观察到的视网膜病理表型。
本研究借助人类诱导多能干细胞（human induced pluripotent stem cell, hiPSC）来源的视网膜类器官（retinal organoids, ROs）、视网膜色素上皮（retinal pigmented epithelial, RPE）细胞及视网膜芯片（retina-on-chip, RoC）技术，在患者特异性的人类视网膜微生理模型中体外构建CLN2疾病模型。依托上述模型，我们成功重现了CLN2患者的主要临床组织病理学标志：即主要在视网膜外层蓄积含有线粒体ATP合酶C亚基（subunit C of mitochondrial ATP synthase, SCMAS）与脂质的脂褐素。
进一步对CLN2视网膜类器官开展单细胞RNA测序分析，结果显示视锥细胞的翻译与线粒体功能出现失调。最后，本研究证实腺相关病毒（adeno-associated virus, AAV）介导的TPP1基因疗法可恢复TPP1的表达，并能减少甚至阻断SCMAS的蓄积。
综上，本研究构建了新型的具有人类相关性的视网膜微生理疾病模型，揭示了人类视网膜中CLN2病理生理学的全新机制，并证明AAV9.hCLN2基因疗法治疗CLN2疾病具备巨大潜力，有望实现对受累个体失明症状的治疗乃至治愈。
整体实验设计：对2株对照细胞系（CTRL1、CTRL2）及2株未经处理的CLN2患者细胞系（CLN2-1、CLN2-2），与经AAV9-hCLN2处理的CLN2细胞系（AAV9.hCLN2处理的CLN2-1、AAV9.hCLN2处理的CLN2-2）在分化第192天的iPSC来源视网膜类器官进行单细胞测序分析（10X Genomics平台）。6个样本被分配至4个测序批次，通过SoupOrCell v2.0（Heaton等，2020）基于单核苷酸多态性（SNPs）进行解多路复用：批次1包含CTRL1与CLN2-1，批次2包含CTRL1与AAV9.hCLN2处理的CLN2-1，批次3包含CTRL2与CLN2-2，批次4包含CTRL2与AAV9.hCLN2处理的CLN2-2。