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

Sethna博士：项目总结 博士Sethna是一名新的调查员，他试图将自己定位为一名独立的研究人员， 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、年龄相关性黄斑变性（AMD）以及在 格兰特.因此，他是进行这项研究的理想人选。R21的项目描述 申请中的探索性补助金对于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变性疾病，是导致视网膜病变的最大原因。 老年人的视力问题。抗VEGF抗体对抗VEGF异常有效。 新生血管性“湿性”AMD。干性AMD发生在约90%的病例中，然而，截至目前， 用于预防或减轻疾病进展的治疗选择。干性AMD起源于 主要在RPE和脉络膜中，其次损害光感受器功能和完整性。RPE 通过典型和非典型自噬在光感受器存活中起着不可或缺的作用。 以前的研究，包括我们的研究，都涉及到受损的典型和非典型 AMD患者样本中RPE内的自噬。上调RPE中的自噬通量，因此， 是一种有前途但尚未开发的治疗发展途径。发现组和验证 本项目的主要目的是研究老年AMD患者的小分子治疗方法。 我们建议使用RPE对自噬通量增强剂进行高含量筛选（HCS）， 主要受干性AMD影响的细胞类型。我们令人兴奋的初步数据使用小分子 筛选和RPE细胞系发现了三种候选的自噬调节剂，因此支持 我们建议的研究的可行性。此外，我们能够生成和表征hiPSC- 在我们的实验室里。为此，在目标1中，我们将产生CRISPR介导的内源性标记的 LC 3和p62/SQSTM 1，并进行HCS以发现额外的自噬通量增强剂， 在正交的二级屏幕中验证它们。在目标2中，我们将验证AMD中的潜在客户- 患者来源的人诱导多能干细胞来源的RPE（AMD-hiPSC-RPE）。老鼠会 不概括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方案至关重要。我的长期目标是 建立一个独立的研究计划，对自噬有明确的翻译影响， 与年龄有关的视力障碍。本文所述的项目将为基础教育开辟新的途径。 和翻译研究，并将装备我额外的新技术技能，这是 对我未来的研究目标至关重要。

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.