AMD-Patient-Derived hiPSC-RPE: Gateway for Assessing Novel and Emerging Modulators of Autophagy

AMD 患者衍生的 hiPSC-RPE：评估新型和新兴自噬调节剂的网关

• 批准号: 10283447
• 负责人: Zubair M. Ahmed
• 金额: $ 23.18万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2023-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10283447
• 关键词: Affect; Age related macular degeneration; Aging; Antibodies; Atrophic; Attenuated; Autophagocytosis; Autophagosome; Basic Science; Blood Vessels; Cell Line; Cell physiology; Cells; Cellular biology; Characteristics; Choroid; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Complement; Cultured Cells; Data; Disease; Disease Progression; Disease model; Elderly; Enhancers; Eye diseases; Female; Goals; Grant; Homeostasis; Human; Ii-Key; Impairment; Inherited; Injections; Knowledge; Lead; Libraries; Life; Light; Link; Lucentis; Mediating; Mitotic; Modeling; Molecular; Mus; Nonexudative age-related macular degeneration; Organelles; Pathology; Patients; Pattern; Phagocytes; Phagocytosis; Phase; Phenotype; Photoreceptors; Play; Population; Prevalence; Process; Proteins; Publishing; Raloxifene; Rattus; Reporting; Research; Research Methodology; Research Personnel; Resistance; Retina; Retinal Degeneration; Risk Factors; Role; Sampling; Screening procedure; Signal Transduction; Sirolimus; Stains; Structure of retinal pigment epithelium; Technical Expertise; Testing; Thapsigargin; Therapeutic; Thinness; Tissues; Translational Research; Validation; Vision; Vision Disorders; Visual Acuity; Visual impairment; age related; aged; base; bevacizumab; cell type; complement pathway; experience; genome wide association study; in vitro Assay; induced pluripotent stem cell; male; mouse model; neovascular; novel; prevent; programs; protein biomarkers; screening; small molecule; therapeutic development; therapy development; translational impact; wortmannin

Dr. Sethna: Project Summary Dr. Sethna is a new investigator seeking to establish himself as an independent researcher in vision field. He has extensive experience in retinal pigment epithelium (RPE) cell biology, autophagy, age-related macular degeneration (AMD), and the research methods described in the grant. Hence, he is the ideal candidate to conduct the study. The project described for the R21 Exploratory grant in the application will be critical for developing rationale treatment options for AMD patients. AMD is a progressive degenerative disease of the retina and the largest cause of vision problems in the elderly. Anti-VEGF antibodies are exceptionally efficacious against neovascular ‘wet’ AMD. Dry AMD occurs in ~90% of the cases, however, as of now there are no treatment options for preventing or attenuating the disease progression. Dry AMD originates primarily in the RPE and choroid, secondarily impairing photoreceptor function and integrity. RPE plays an integral role in photoreceptor survival by canonical and non-canonical autophagy. Previous studies, including ours, have implicated impaired canonical and non-canonical autophagy within the RPE in AMD patient samples. Upregulating autophagy flux in RPE, hence, is a promising yet unexplored avenue for therapeutic development. The discovery and validation of small molecular therapeutics for elderly people with AMD is the main objective of this project. We propose to perform a high content screen (HCS) for autophagy flux enhancers using RPE, the cell type primarily affected by dry AMD. Our exciting preliminary data using small molecule screen and RPE cell line uncovered three candidate autophagy modulators, and thus support the feasibility of our proposed studies. Further, we were able to generate and characterize hiPSC- RPE in our lab. To that end, in Aim 1, we will generate CRISPR-mediated endogenously tagged LC3 and p62/SQSTM1 and perform a HCS to uncover additional autophagy flux enhancers and validate them in an orthogonal secondary screen. In Aim 2, we will validate the leads in AMD- patient-derived human induced pluripotent stem cell-derived RPE (AMD- hiPSC-RPE). Mice do not recapitulate the AMD phenotype, hence using AMD hiPSC-RPE as the disease relevant model to validate lead compounds is crucial for treatment options. My long-term goal is establishing an independent research program with clear translational impact on autophagy and age-related vision disorders. The project described herein will open up new avenues for basic and translational research, and will equip me with additional new technical skills, which are imperative for my future research goals.

Sethna 博士：项目摘要 Sethna 博士是一名新调查员，寻求将自己确立为一名独立研究人员 他在视网膜色素上皮（RPE）细胞生物学方面拥有丰富的经验， 自噬、年龄相关性黄斑变性 (AMD) 以及中描述的研究方法 因此，他是开展 R21 项目的理想人选。 申请中的探索性资助对于制定合理的治疗方案至关重要 AMD 患者是一种进行性视网膜退行性疾病，也是导致视网膜退化的最大原因。 抗 VEGF 抗体对治疗老年人的视力问题特别有效。 约 90% 的病例发生新生血管性“湿性”AMD。 预防或减轻干性 AMD 的治疗选择。 主要影响 RPE 和脉络膜，其次损害光感受器功能和完整性。 通过规范和非规范自噬在光感受器存活中发挥着不可或缺的作用。 以前的研究，包括我们的研究，都表明规范和非规范受损 AMD 患者样本中 RPE 内的自噬 上调 RPE 中的自噬通量，因此， 是一种有前景但尚未探索的治疗开发途径。 针对老年 AMD 患者的小分子疗法是该项目的主要目标。 我们建议使用 RPE 对自噬通量增强剂进行高内涵筛选 (HCS)， 我们使用小分子获得的令人兴奋的初步数据。 筛选和 RPE 细胞系发现了三种候选自噬调节剂，从而支持 此外，我们能够生成并表征 hiPSC-。 为此，我们的实验室将在目标 1 中生成 CRISPR 介导的内源标记。 LC3 和 p62/SQSTM1 并执行 HCS 以发现其他自噬通量增强剂和 在正交辅助屏幕中验证它们 在目标 2 中，我们将验证 AMD 中的线索。 患者来源的人类诱导多能干细胞衍生的 RPE (AMD-hiPSC-RPE) 小鼠。 不能概括 AMD 表型，因此使用 AMD hiPSC-RPE 作为疾病相关 验证先导化合物的模型对于治疗选择至关重要。 建立一个对自噬具有明显转化影响的独立研究计划 本文描述的项目将为基本治疗开辟新途径。 和转化研究，并将为我提供额外的新技术技能，这些技能是 对我未来的研究目标至关重要。