Exploring Mechanisms in Retinal Development/Homeostasis, Retinal Immune Surveillance and Diabetic Retinopathy Using Forward Genetics

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Project Summary Defects in the carefully orchestrated processes of retinal development, homeostasis and retinal immune surveillance lead or contribute to a wide range of diseases. It is now clear that genetics not only play a role in these processes but may also modulate diabetic retinopathy. Our short-term goal is to identify and characterize gene/protein defects and molecular pathways that lead to abnormal retinal development/homeostasis, altered retinal immune surveillance and modulation of diabetic retinopathy. The long-term goal is to leverage our research discoveries to understand retinal disease processes, and to identify novel therapeutic opportunities. We propose that a high-throughput and unbiased strategy provides an ideal approach to discovery of gene/phenotype associations in this setting. In collaboration with Nobel laureate Bruce Beutler, we will employ a robust state-of-the-science and unbiased forward genetics approach, in which thousands of new random mutations are generated and mice demonstrating retinal anomalies are identified by screening using fundus photographs and OCT. Our approach has significant advantages compared to other existing protocols. Most importantly, ours is the first and only protocol in which all mice have been pre-genotyped at all mutant loci. In addition, the large scale of our system and the large pedigree size will also add to the discovery power. Together, these advantages will allow us to identify and pursue novel gene/phenotype associations related to retinal development, homeostasis and disease. We have identified over 43 gene-phenotype associations after covering just 8% of the mouse genome. Of these, 12 genes have weak associations to the retina in the literature, and another 20 genes have not been reported in association to the retina. This is strong evidence that expanding our screening to include the remaining 92% of the mouse genome will yield many more gene-phenotype associations related to retina development, homeostasis and immune surveillance. Of note, our proposal starts by selecting a few of the most promising genes we have already identified for further study. We will harness the power of CRISPR/Cas9 gene editing, single cell RNA sequencing, co-immunoprecipitation experiments with highly sensitive mass spectrometry and proteomics analysis, our recently published light injury model and other techniques to explore the mechanisms of these associations. We will also apply the streptozotocin model of diabetic retinopathy to our OCT retinal imaging pipeline to identify genes that can modulate early diabetic retinopathy. This proposed research will advance our knowledge of retinal health and disease, and we anticipate that it will lead to the identification of new therapeutic avenues.