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

利用正向遗传学探索视网膜发育/稳态、视网膜免疫监视和糖尿病视网膜病变的机制

• 批准号: 10316653
• 负责人: Rafael Ufret-Vincenty
• 金额: $ 45.1万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10316653
• 关键词: Anatomy; Blindness; CRISPR/Cas technology; Cells; Co-Immunoprecipitations; Collaborations; Complex; Conceptions; Defect; Development; Diabetes Mellitus; Diabetic Retinopathy; Disease; Eligibility Determination; Ensure; Ethylnitrosourea; Functional disorder; Funding; Fundus; Gene Proteins; Generations; Genes; Genetic; Genome; Genotype; Goals; Health; Homeostasis; Immune; Immunologic Surveillance; Inflammatory; Injections; Injury; Investigation; Knowledge; Lead; Light; Literature; Maintenance; Mass Spectrum Analysis; Measurement; Mediating; Meiosis; Microglia; Modeling; Molecular; Mus; Mutagenesis; Mutate; Mutation; Natural regeneration; Nature; Neuroglia; Neurons; Nitrosourea Compounds; Optical Coherence Tomography; Pathway interactions; Phenotype; Photoreceptors; Physiology; Play; Predisposition; Process; Proteomics; Protocols documentation; Publishing; Recovery; Reporting; Research; Retina; Retinal Defect; Retinal Diseases; Role; Science; Spottings; Streptozocin; Suggestion; System; Techniques; Technology; Testing; Thick; Thinness; Vision; base; causal variant; cell type; experimental study; exposure pathway; forward genetics; gene discovery; genetic approach; genetic pedigree; immunoregulation; improved; insight; interest; mouse genome; mutant; new therapeutic target; novel; novel therapeutics; outer plexiform layer; retinal imaging; retinal regeneration; ribbon synapse; screening; single-cell RNA sequencing; success; trafficking; treatment strategy

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.

项目摘要 视网膜发育、稳态和视网膜免疫的精心协调过程中的缺陷 监测导致或促成了多种疾病。现在很清楚，遗传学不仅在 这些过程，但也可以调节糖尿病视网膜病变。我们的短期目标是识别和描述 基因/蛋白质缺陷和分子途径，导致异常视网膜发育/稳态，改变 糖尿病视网膜病变的视网膜免疫监视和调节。长期目标是利用我们的 研究发现，以了解视网膜疾病的过程，并确定新的治疗机会。 我们建议，一个高通量和公正的战略提供了一个理想的方法来发现 基因/表型相关性。在与诺贝尔奖获得者布鲁斯Beutler合作，我们将聘请 一个强大的国家的科学和公正的向前遗传学的方法，其中数千个新的随机 产生突变，并通过使用眼底扫描筛选来鉴定显示视网膜异常的小鼠， 照片和OCT。 与其他现有协议相比，我们的方法具有显着的优势。最重要的是， 这是第一个也是唯一一个在所有突变位点对所有小鼠进行预基因分型的方案。此外，大 我们系统的规模和大的谱系规模也将增加发现能力。这些优势加在一起 将使我们能够识别和追求与视网膜发育相关的新基因/表型关联， 稳态和疾病。我们已经确定了超过43个基因-表型协会后，覆盖只有8%的， 小鼠基因组。其中，12个基因在文献中与视网膜有弱关联，另外20个基因在文献中与视网膜有弱关联。 尚未报道与视网膜相关。这是一个强有力的证据， 包括其余92%的小鼠基因组将产生更多的基因-表型关联， 视网膜发育、体内平衡和免疫监视。值得注意的是，我们的建议首先选择了一些 我们已经确定的最有希望的基因进行进一步的研究。我们将利用 CRISPR/Cas9基因编辑、单细胞RNA测序、免疫共沉淀实验， 灵敏的质谱和蛋白质组学分析、我们最近发表的光损伤模型和其他 技术来探索这些协会的机制。我们还将应用链脲佐菌素模型， 糖尿病视网膜病变的OCT视网膜成像管道，以确定基因，可以调节早期糖尿病 视网膜病变这项拟议的研究将推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。