Karl J. Wahlin, Ph.D. - US grants

DESCRIPTION (provided by applicant): 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.

SUMMARY Vision loss in glaucoma and optic neuropathies results from the loss of retinal ganglion cells (RGCs) that is irreversible. Regardless of etiology, once cells are lost, RGCs cannot regrow and blindness is considered permanent. Retinas derived from pluripotent stem cells (PSCs) offer a source of tissue to address some of the mechanism(s) of human optic nerve cell loss at the level of the retina, however, one limitation to laboratory grown retinas is that they lack integration with higher order Lateral geniculate nucleus (LGN) and Superior colliculus (SC) neurons which ultimately connect with the visual cortex, thus they are not physiologic and retinal synapses might not form properly. As a first step towards understanding how to repopulate the eye with new RGCs and make those new connections, we propose to develop an optic nerve model to study axon outgrowth and pathfinding which will lead to improved engraftment of projections to the brain. Therefore, we propose to (1) develop a 3D printed scaffold that is permissive to axonal outgrowth, (2) improve the optic nerve by reconstructing the cellular components of the optic nerve, and (3) study cues to control proper connections through the optic chiasm. The optic chiasm represents the first relay station in the transmission of visual signals to the brain and thus is an important first step. We hypothesize that many developmental features of non- human mammalian models will also apply to developing human retinas. These features include neurite outgrowth and guidance towards the optic nerve head (stage I), axon guidance towards the optic chiasm (stage II), and decussation of ipsilateral and contra-lateral RGC axons [1-4]. We further hypothesize that human RGC axons emanating from PSC derived 3D organoids in hydrogel scaffolds will recapitulate these important features and thereby provide an experimentally tractable model for the study of glaucoma and other optic neuropathies. More importantly it will also provide a critical readout for testing therapeutic approaches aimed at restoring vision through cell replacement. We propose to develop a microprinted scaffold for 3D retinal organoids that will facilitate retinal ganglion cell (RGC) axon outgrowth and targeting and improve the organization of optic nerve cells and axon outgrowth by reconstructing the cellular components (including oligodendrocytes, astrocytes and microglial cells) of the human optic nerve.

SUMMARY Retinal degenerative (RD) diseases, such as Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), cause dysfunction and cell death of photoreceptor (PR) cells, ultimately leading to blindness. LCA is the leading cause of inherited childhood blindness resulting in a loss of vision at or soon after birth. Though this is considered to be quite rare, these blinding diseases are devastating for those affected. Current efforts are being made to develop gene-therapies aimed at correcting some of the genes affected in RD and this approach has shown some promise in animals and humans for restoring RPE65 gene expression, but there are many other causes of RD for which there is no cure. In addition, due to the many mutations involved in RD, there are significant gaps in our understanding of how PR loss occurs. To address this, we will use human pluripotent stem cell (PSC) based retinal cell-reporter lines with RD-associated alleles to help explore the mechanisms of PR cell death. Given the typically long period of time required to generate human retinas in the laboratory, the severity and rapid onset of degeneration in LCA makes it an attractive experimental model to study human RD and to develop potential therapies. We will study the aryl hydrocarbon receptor interacting protein-like1 (AIPL1) gene to explore three functional domains that harbor naturally occurring mutations in patients with LCA and cone-rod dystrophy (CORD). A comparative analysis of different mutations might lead to a better understanding of how rods and cones die and greater insight into other more common forms of PR degeneration, such as age- related macular degeneration (AMD). A central hypothesis is that human PSC derived 3D retina organoids with AIPL1 mutations will recapitulate human retinal dystrophy resulting in PR loss. This hypothesis is supported by our recent work, and others, showing that human PSCs can be coaxed into becoming retinal eyecup-like structures with PRs, a laminar morphology and outer segment structures that are similar to an actual retina. This proposal will bridge two innovative technologies; (1) genome-editing to generate genetically matched retinal reporter PSC derived retinas with disease-associated mutations and (2) gene-correction to repair genetic defects and promote PR cell survival. Given the very early onset of LCA it is important to define the appropriate windows of time for such treatment options. Not only will these studies lead to new insights into the biology of RD disease, but could also provide an innovative resource to develop therapies for the treatment of RD.