Keratoconus (KC), a common corneal dystrophy that affects young people, causes progressive thinning, scarring and loss of corneal shape, which can ultimately lead to loss of vision. Crosslinking of collagens in the cornea can stiffen and delay its weakening, but severe cases require corneal transplantation. Although KC has a strong genetic component, its etiology is complex, polygenic and multifactorial. There is an urgent need to understand its etiology for developing early diagnosis and treatment strategies for KC. To address this, our competitive renewal application focuses on identifying cellular defects, biomarkers and the genetic causes of KC. Beyond obvious familial KC, the vast majority are isolated where disease likely results from rare pathogenic coding sequence variants and genome-wide common noncoding variants that increase one's susceptibility. Elucidating the underlying genetic defects in these “isolated KC” requires a range of biological evidence. Our recent studies and preliminary data provide this biological foundation for the current proposal. First, by whole- exome sequencing of KC families, we identified rare pathogenic variants in genes related to cell stress, cytoskeleton and extracellular matrix (ECM), which are now prioritized as candidate genes and networks for the isolated KC studies. Second, our transcriptomic and proteomic characterizations of KC and control donor corneas identified significant dysregulation in the NRF2-antioxidant program that is crucial for corneal cell survival and its functions. Finally, we developed corneal cell culture models that mimic key KC features, from oxidative stress to ECM insufficiency, and assays to measure these. We further developed the first cornea organoids from human induced pluripotent stem cells that will allow functional studies of genes and therapeutic agents in a physiological, cornea-like setting and in organoid-derived epithelial and stromal cell cultures. Importantly, this approach will yield cell culture disease models from genetically defined patient blood cells. These cell culture disease surrogates are particularly important, as there are no animal models that can capture the polygenic complexity of KC. In Aim 1 we will assess potential NRF2-regulated antioxidants as tear fluid biomarkers for KC, and investigate this network in corneal cell cultures. In Aim 2 we will identify rare pathogenic variants and common noncoding variants that increase disease susceptibility in isolated KC cases using the 1000Genome and the UK Biobank databases as controls. In Aim 3 we will functionally test the concept that a rare pathogenic variant (e.g., our published c.G12982A HSPG2), will cause cellular disease surrogates when CRISPR-edited into cells derived from KC individuals with high polygenic and not controls with low polygenic scores. Our findings will lead to potential anti-oxidant biomarkers, development of NRF2- activators for KC treatments, genetically defined KC cell culture models and insights into the complex genetic architecture of KC. Our studies are highly relevant to the goals of the NEI in understanding the complex genetics of eye diseases, treatments and reversing vision loss.

Keratoconus (KC) is a common vision-debilitating corneal dystrophy with an ill-defined polygenic basis and a complex etiopathology. We identified poor protective antioxidant responses and gene networks that regulate matrix and cellular functions in KC. Based on these premises, the current study will identify antioxidant biomarkers, disease-predisposing DNA variants by next generation sequencing, and establish patient-derived cell culture models to probe disease mechanisms and therapeutic agents.