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

塞纳纳博士：项目总结 塞纳纳博士是一名新的调查员，试图将自己确立为 视野。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、老年性黄斑变性(AMD)以及 格兰特。因此，他是进行这项研究的理想人选。为R21描述的项目 应用程序中的探索性拨款将对制定合理的治疗方案至关重要 AMD患者。AMD是一种进行性视网膜退行性疾病，是 老年人的视力问题。抗血管内皮生长因子抗体对 新生血管“湿性”AMD。干性AMD发生在约90%的病例中，然而，到目前为止还没有 预防或减缓疾病进展的治疗方案。干性AMD起源于 主要是RPE和脉络膜，继而损害光感受器的功能和完整性。RPE 通过典型和非典型自噬在感光细胞存活中起着不可或缺的作用。 以前的研究，包括我们的研究，已经牵涉到规范和非规范受损 AMD患者样本中RPE内的自噬。因此，上调RPE中的自噬通量， 是一条很有前途但尚未被探索的治疗发展途径。发现和验证 老年AMD小分子疗法的研究是本项目的主要目标。 我们建议使用RPE对自噬通量增强剂进行高内容筛选(HCS)， 主要受干性AMD影响的细胞类型。我们使用小分子的激动人心的初步数据 Screen和RPE细胞株发现了三个候选的自噬调节器，从而支持 我们建议的研究的可行性。此外，我们能够产生并表征HiPSC- 我们实验室里的RPE。为此，在目标1中，我们将生成CRISPR介导的内源标记 Lc3和p62/SQSTM1，并进行HCS以发现其他自噬通量增强剂和 在一个正交的二级屏幕中验证它们。在目标2中，我们将验证AMD中的销售线索- 患者来源的人诱导多能干细胞来源的RPE(AMD-hiPSC-RPE)。老鼠做的事 不能概括AMD的表型，因此使用AMD hiPSC-RPE作为疾病相关 验证先导化合物的模型对治疗方案至关重要。我的长期目标是 建立对自噬和自噬有明显翻译影响的独立研究计划 与年龄相关的视力障碍。这里描述的项目将开辟新的途径， 和翻译研究，并将使我掌握更多新的技术技能，这些技能是 对我未来的研究目标来说是必不可少的。

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.