Modeling Photoreceptor Development and Disease Using Human Pluripotent Stem Cells

使用人类多能干细胞模拟光感受器发育和疾病

• 批准号: 8764475
• 负责人: KARL J WAHLIN
• 金额: $ 9万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8764475
• 关键词: Address; Alleles; Area; Bioinformatics; Biological Assay; Biological Models; Biology; Blindness; Cell Death; Cell Differentiation process; Cell Line; Cells; Cellular Stress; Cessation of life; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Cone; DNA Sequence; Data Set; Development; Disease; Disease model; Eye Development; Eye diseases; Functional disorder; Future; GRB10 gene; Gene Expression; Generations; Genes; Genome; Goals; Human; Immunohistochemistry; In Vitro; Knock-in Mouse; Knowledge; Label; Leber' s amaurosis; Measures; Mentors; Modeling; Molecular; Molecular Profiling; Morphology; Mutation; Opsin; Optic vesicle; Parents; Pathway interactions; Phase; Phenotype; Photoreceptors; Pluripotent Stem Cells; Positioning Attribute; Process; Proteins; Protocols documentation; Rare Diseases; Reporter; Research; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinal Photoreceptors; Retinitis Pigmentosa; Rhodopsin; SHH gene; Signal Pathway; Signal Transduction; Staging; Structure; Technology; Terminator Codon; Therapeutic Intervention; Therapeutics for Rare and Neglected Diseases; Time; Training; United States National Institutes of Health; Variant; Work; base; cell type; gene therapy; human disease; human stem cells; in vitro Assay; induced pluripotent stem cell; innovative technologies; insight; mutant; novel; optic cup; photoreceptor degeneration; public health relevance; repaired; screening; skills; small molecule; small molecule libraries; tool; transcriptome sequencing; translational study

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.

描述(申请人提供)：视网膜退行性疾病，如孤儿疾病视网膜色素变性(RP)和Lebers先天性黑色素沉着(LCA)，导致光感受器(PR)细胞功能障碍和细胞死亡导致失明。据估计，这些致盲疾病分别影响着10万人和3000人，对那些遭受痛苦的人来说是毁灭性的。美国国立卫生研究院已经认识到有必要通过其“罕见和被忽视疾病的治疗”(TRND)倡议来解决罕见疾病。尽管针对一种特殊形式的LCA的基因治疗显示出希望，但对于其他视网膜退行性变，还没有治愈的方法，而且我们对PR丢失如何发生的理解存在重大差距。为了解决这一问题，我们将开发基于转基因人诱导多能干细胞(HiPSC)的视网膜细胞报告系和RD相关的hiPSC，这将帮助我们利用促进视网膜眼球分化的细胞信号通路，并揭示潜在参与PR细胞死亡的途径。一个中心假设是，人类干细胞来源的视网膜视杯将重现视网膜的发育和/或退化。这一假设得到了我们最近的工作的支持，该工作表明，HiPSCs可以被诱导成具有PR的视网膜眼杯样结构，以及类似于成熟视网膜的板层形态。这项提议将架起三项创新技术的桥梁：(1)HiPSCs产生3D分化的视网膜，(2)使用CRISPR技术进行基因组编辑，产生基因匹配的视网膜记者和基于疾病的突变HiPSCs，以及(3)小分子化学筛选，以确定增加PR产生的途径。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高内容筛选(HCS)中心接受培训，在那里PI将能够筛选小分子化学物质，以探测与视网膜和PR发育相关的信号通路。指导阶段将辅以生物信息学专家蒋谦博士的培训，他将提供与PR发展(指导阶段)和退化期间(独立阶段)相关的NextGen测序数据集分析方面的培训。这个项目不仅将通过在全新领域的培训来提高公关的技术技能，而且可以确定公关发展的新机制，并提供对公关退化的机械性洞察。因此，该项目指导阶段的目标是发现新的机制，可以提高PR/眼罩生成的效率和速度，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，将在该项目的独立阶段用于开发疾病模型。这些目标意义重大，因为识别这样的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。