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

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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.