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

项目摘要 视网膜发育、动态平衡和视网膜免疫过程中精心安排的缺陷 监测导致或促成了广泛的疾病。现在很清楚的是，遗传不仅在 但这些过程也可能调节糖尿病视网膜病变。我们的短期目标是识别和描述 导致视网膜发育/动态平衡异常的基因/蛋白质缺陷和分子通路改变 视网膜免疫监测与糖尿病视网膜病变的调控。我们的长期目标是利用我们的 研究发现，以了解视网膜疾病过程，并确定新的治疗机会。 我们认为，高通量和不偏不倚的策略为发现 在这个环境中的基因/表型关联。与诺贝尔奖获得者布鲁斯·比特勒合作，我们将聘用 一种强大的科学状态和公正的前向遗传学方法，在这种方法中，数千名新的随机 产生突变，并通过眼底筛查识别出表现出视网膜异常的小鼠 照片和OCT。 与现有的其他协议相比，我们的方法具有显著的优势。最重要的是，我们的 这是第一个也是唯一一个在所有突变基因座对所有小鼠进行预基因分型的方案。此外，大型 我们系统的规模和庞大的谱系规模也将增加发现能力。总而言之，这些优势 将使我们能够识别和寻求与视网膜发育相关的新的基因/表型关联， 动态平衡和疾病。在仅涵盖8%的基因-表型关联之后，我们已经确定了超过43个基因-表型关联 小鼠基因组。在这些基因中，有12个基因与文献中的视网膜有微弱的关联，另有20个基因 尚未有与视网膜相关的报道。这是强有力的证据表明，将我们的检查范围扩大到 包括其余92%的小鼠基因组将产生更多与以下相关的基因-表型关联 视网膜发育、动态平衡和免疫监视。值得注意的是，我们的建议从以下几个方面开始 我们已经确定的最有希望进行进一步研究的基因。我们将利用 CRISPR/Cas9基因编辑、单细胞RNA测序、免疫共沉淀实验 敏感质谱学和蛋白质组学分析，我们最近发表的光损伤模型和其他 探索这些关联的机制的技术。我们还将应用链脲佐菌素模型 糖尿病视网膜病变到我们的OCT视网膜成像管道，以确定可以调节早期糖尿病的基因 视网膜病变。这项拟议的研究将促进我们对视网膜健康和疾病的了解，我们预计 这将导致新的治疗途径的确定。

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.