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.
Retinal photoreceptors differentiated from human induced pluripotent stem cells (hiPSCs) could offer a reliable model for understanding human retinal degenerative eye disease and are likely to enable translational studies aimed at identifying neuroprotective compounds. In this application we propose to use a genome-editing approach to establish hiPSC reporter lines that will help us standardize 3D retina cultures and enable a small molecule chemical screen to explore pathways that promote PR differentiation. We will further modify hiPSC reporters to generate mutant alleles and develop a human disease model with which we can potentially identify pathways important in maintaining and/or restoring photoreceptors.