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.
PROJECT NARATIVE Human stem cell derived tissues offer new models for studying human retinal degenerative eye disease, potentially providing new insight into the biology of inherited retinal degenerations. In this application, we propose to use stem cell derived AIPL1 mutant reporter lines to explore how variations in different functional domains lead to the disease phenotypes observed in LCA and CORD. We propose to study the consequences of mutations in different functional domains in rods and cones and will develop a gene-therapy based approach to prevent retinal degenerative disease.