Modeling Photoreceptor Development and Disease Using Human Pluripotent Stem Cells

描述（由申请人提供）：视网膜退行性疾病，如孤儿病色素性视网膜炎（RP）和莱伯斯先天性黑内障（LCA），会导致感光细胞功能障碍和细胞死亡，导致失明。据估计，这两种致盲疾病分别使10万人和3 000人患病，对患者来说是毁灭性的。美国国立卫生研究院已经认识到有必要通过其“罕见病和被忽视疾病的治疗”（TRND）倡议来解决罕见病问题。尽管对一种特定形式的LCA的基因治疗显示出希望，但对其他视网膜变性没有治愈方法，而且我们对PR丢失如何发生的理解存在重大差距。为了解决这个问题，我们将开发基于基因修饰的人类诱导多能干细胞（hiPSC）的视网膜细胞报告系和rd相关的hiPSC，这将有助于我们利用促进视网膜眼球分化的细胞信号通路，并揭示可能涉及PR细胞死亡的途径。一个中心假设是，人类干细胞衍生的视网膜视杯将重现视网膜发育和/或变性。我们最近的工作支持了这一假设，表明hiPSCs可以被诱导成具有pr的视网膜杯状结构和类似于成熟视网膜的层流形态。该提案将结合三种创新技术；(1)利用hiPSCs生成3d分化视网膜；(2)利用CRISPR技术进行基因组编辑，生成基因匹配的视网膜报告细胞和基于疾病的突变hiPSCs；(3)利用小分子化学筛选来识别增加PR生成的途径。在指导阶段（AIMS1- 2）， PI将进行基因组编辑工作，并在Donald Zack博士的实验室获得进一步的专业知识，并将在Wilmer高含量筛选（HCS）中心接受培训，PI将能够筛选小分子化学物质，以探测与视网膜和PR发展相关的信号通路。作为指导阶段的补充，生物信息学专家姜谦博士将提供与PR开发（指导阶段）和退化（独立阶段）相关的NextGen测序数据集分析的培训。这个项目不仅可以通过全新领域的培训来提高PI的技术技能，还可以确定公关发展的新机制，并为公关退化提供机制洞察力。因此，这个项目的指导阶段的目标是发现新的机制，可以提高PR/眼杯生成的效率和速度，从而深入了解眼睛发育的生物学，并提供一个实用的研究工具，将在这个项目的独立阶段用于开发疾病模型。这些目标意义重大，因为确定这些机制将有助于填补我们关于人类pr如何发展和退化的知识的主要空白，并可能发现治疗干预的新靶点。

DESCRIPTION (provided by applicant): Retinal degenerative diseases, such as the orphan diseases Retinitis pigmentosa (RP) and Lebers congenital amaurosis (LCA), cause dysfunction and cell death of photoreceptor (PR) cells leading to blindness. Afflicting an estimated 100,000 and 3,000 people respectively, these blinding diseases are devastating for those afflicted. The NIH has recognized a need to address rare diseases through its 'Therapeutics for Rare and Neglected Diseases' (TRND) initiative. Although gene-therapy for one specific form of LCA shows promise, for other retinal degenerations there is no cure and significant gaps exist in our understanding of how PR loss occurs. To address this we will develop genetically modified human induced pluripotent stem cells (hiPSC) based retinal cell-reporter lines and RD-associated hiPSCs that will help us exploit cell-signaling pathways that promote retinal eyecup differentiation and uncover pathways potentially involved in PR cell death. A central hypothesis is that human stem cell derived retinal optic cups will recapitulate retinal development and/or degeneration. This hypothesis is supported by our recent work showing that hiPSCs can be coaxed into becoming retinal eyecup-like structures with PRs and a laminar morphology similar to the mature retina. This proposal will bridge three innovative technologies; (1) hiPSCs to generate 3D-differentiatied retinas, (2) genome-editing using CRISPR technology to generate genetically matched retinal reporters and disease-based mutant hiPSCs and (3) a small molecule chemical screen to identify pathways that increase PR generation. In the mentored phase (AIMS1- 2), the PI will carry out genome-editing work and gain further expertise in Dr. Donald Zack's lab and will acquire training at the Wilmer high-content screening (HCS) center where the PI will be able to screen small molecule chemicals to probe for signaling pathways relevant to retinal and PR development. The mentored phase will be supplemented by training with Dr. Jiang Qian, an expert in bioinformatics, who will provide training in the analyses of NextGen sequencing datasets relevant to PR development (mentored phase) and during degeneration (independent phase). This project will not only enhance the PI's technical skills through training in completely new areas, but could identify novel mechanisms for PR development and provide mechanistic insight into PR degenerations. The goal of the mentored phase of this project is thus to uncover new mechanisms that could increase the efficiency and pace of PR/eyecup generation thus lending insight into the biology of eye development and provide a practical research tool that will be exploited to develop disease models during the independent phase of this project. These goals are significant because identification of such mechanisms will help to fill a major gap in our knowledge about how human PRs develop and degenerate and could uncover new targets for therapeutic intervention.