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: 9197794
• 负责人: KARL J WAHLIN
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197794
• 关键词: Address; Alleles; Area; Bioinformatics; Biological Assay; Biological Models; Biology; Blindness; CRISPR/Cas technology; 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; Gene Expression; Generations; Genes; Genome; Goals; Health; Human; Immunohistochemistry; In Vitro; Knock-in; 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; Technical Expertise; Terminator Codon; Therapeutic Intervention; Therapeutics for Rare and Neglected Diseases; Time; Training; United States National Institutes of Health; Variant; Work; base; cell type; differential expression; gene therapy; genome editing; human disease; human stem cells; in vitro Assay; induced pluripotent stem cell; innovative technologies; insight; interstitial retinol-binding protein; mutant; next generation sequencing; novel; optic cup; photoreceptor degeneration; repaired; screening; small molecule; small molecule libraries; tool; transcription activator-like effector nucleases; 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（由申请人提供）：视网膜退行性疾病，例如孤儿疾病色素性视网膜炎（RP）和Lebers先天性amaurosis（LCA），导致光感受器（PR）细胞的功能障碍和细胞死亡，导致失明。这些盲目疾病分别遭受了估计的100,000和3,000人的困扰，对患病者来说是毁灭性的。 NIH已意识到有必要通过其“稀有和被忽视的疾病的疗法”（TRND）倡议来解决罕见疾病。尽管一种特定形式的LCA基因疗法显示出希望，但对于其他视网膜变性，我们对PR损失的发生时没有治愈的治愈且存在明显的差距。为了解决这个问题，我们将开发基于人类诱导的多能干细胞（HIPSC）基于视网膜细胞培养基系和RD相关的HIPSC，这将有助于我们利用细胞信号途径，这些途径促进视网膜眼球分化，并揭开潜在参与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网膜将概括视网膜发育和/或变性。我们最近的工作表明，可以将HIPSC哄骗到具有PRS和类似于成熟视网膜类似的层状形态的视网膜眼状结构的结构来支持这一假设。该提议将桥接三种创新技术。 （1）HIPSC生成3D分化的视网膜，（2）使用CRISPR技术进行基因组编辑，以生成遗传匹配的视网膜报告和基于疾病的突变体HIPSC和（3）小分子化学筛选，以识别增加PR产生的途径。在指导阶段（AIMS1-2），PI将进行基因组编辑的工作，并在Donald Zack博士的实验室中获得进一步的专业知识，并将在Wilmer High-Content筛选（HCS）中心获得培训，PI将能够在其中筛选小分子化学品以探测与视网膜和Retrinal和PR开发相关的信号通路。指导阶段将通过与生物信息学专家的江安·齐安（Jiang Qian）博士进行培训，他将在与公关开发（指导阶段）和变性期间（独立阶段）的NextGen测序数据集进行分析中提供培训。该项目不仅可以通过在全新的领域进行培训来提高PI的技术技能，而且可以确定公关开发的新型机制，并提供对公关退化的机械洞察力。因此，该项目的指导阶段的目的是揭示可能提高PR/Eyecup发电的效率和节奏的新机制，从而洞悉眼睛发育的生物学，并提供一种实用的研究工具，该工具将在该项目的独立阶段中利用以开发疾病模型。这些目标是重要的，因为对这种机制的识别将有助于填补我们关于人类PRS如何发展和退化的重要差距，并且可以发现治疗干预的新目标。