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，例如色素性视网膜炎（RP）和莱伯斯先天性黑蒙（LCA）等孤儿病，会导致感光（PR）细胞功能障碍和细胞死亡，从而导致失明。这些致盲疾病估计分别影响 100,000 人和 3,000 人，对患者来说是毁灭性的。美国国立卫生研究院 (NIH) 认识到需要通过其“罕见和被忽视疾病治疗”(TRND) 计划来解决罕见疾病问题。尽管针对一种特定形式的 LCA 的基因疗法显示出希望，但对于其他视网膜变性，无法治愈，而且我们对 PR 丧失如何发生的理解存在重大差距。为了解决这个问题，我们将开发基于转基因人类诱导多能干细胞 (hiPSC) 的视网膜细胞报告系和 RD 相关 hiPSC，这将帮助我们利用促进视网膜眼杯分化的细胞信号传导途径，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/或退化。这一假设得到了我们最近的研究的支持，该研究表明 hiPSC 可以被诱导成为视网膜眼杯状结构，具有 PR 和类似于成熟视网膜的层状形态。该提案将连接三项创新技术； (1) hiPSC 生成 3D 分化的视网膜，(2) 使用 CRISPR 技术进行基因组编辑，生成基因匹配的视网膜报告基因和基于疾病的突变 hiPSC，以及 (3) 小分子化学筛选，以确定增加 PR 生成的途径。在指导阶段 (AIMS1-2)，PI 将在 Donald Zack 博士的实验室开展基因组编辑工作并获得进一步的专业知识，并将在 Wilmer 高内涵筛选 (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江谦博士的培训，他将提供与 PR 开发（指导阶段）和退化期间（独立阶段）相关的 NextGen 测序数据集分析的培训。该项目不仅将通过全新领域的培训来提高 PI 的技术技能，而且可以确定 PR 发展的新机制，并提供对 PR 退化的机制洞察。因此，该项目指导阶段的目标是发现新的机制，可以提高 PR/eyecup 生成的效率和速度，从而深入了解眼睛发育的生物学，并提供可用于开发疾病模型的实用研究工具在该项目的独立阶段。这些目标意义重大，因为识别此类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空白，并可能发现治疗干预的新目标。