Characterization of novel insulin resistance genes by gene editing, high-throughput phenotyping and in vivo studies

项目概要/摘要 胰岛素抵抗（IR）患病率的急剧增加预计在美国和整个世界， 未来几年。由于IR是心血管疾病的重要危险因素，因此发现更有效的方法来治疗心血管疾病是必要的。 预防和治疗这种疾病将对公共卫生产生巨大影响。 在过去的几十年里，旨在预防心脏代谢疾病的新药的开发已经放缓 但人类遗传学的最新进展为药物治疗提供了新的令人兴奋的机会。 发展全基因组关联研究（GWAS）已经发现了>150个与IR相关的基因座， 在过去的十年中，这些基因密切相关的特征;但对于其中的绝大多数，致病基因并没有被发现。 已明确鉴定，导致IR的机制尚不清楚。 我们进行了共定位分析，从164个GWAS位点中优先选择50个可能的候选基因 与IR相关性状相关，我们现在的目标是建立和表征与IR相关的基因。 IR使用一系列严格的实验，将基于CRISPR的基因扰动与单细胞RNA相结合 在人脂肪细胞、骨骼肌细胞和小鼠模型中进行测序和详细的表型分析。 在目标1中，我们将进行基于CROP-seq -CRISPR的转录干扰（CRISPRi），然后进行单克隆测序。 细胞RNA-seq（scRNA-seq）-在人脂肪细胞中表征差异表达的基因和途径 在敲除基于共定位分析选择的50个基因后。 在目标2中，我们将评估代谢表型，如葡萄糖摄取、脂解、胰岛素信号传导， 人脂肪细胞中的脂肪生成、线粒体功能、脂肪酸氧化和代谢产物谱， CRISPRi敲低25个基因后的骨骼肌细胞，由目标1的表达谱指导。 在目标3中，我们将建立和培育三个IR相关基因的敲除小鼠模型，然后比较野生型 和基因敲除小鼠的脂肪分布，葡萄糖和胰岛素耐受性，能量消耗，身体状况， 活动，食物摄入，脂质谱，肾脏和肝脏面板，细胞转录组和组织病理学， 组织中的脂肪含量。 通过结合一系列创新方法，包括高通量基因扰动，然后单克隆抗体， 细胞转录组学，体外和体内实验，以表征基因座建立使用人类遗传学，我们 期望建立致病基因和参与IR发展的新基因的作用机制。 是开发新药以解决巨大且日益增长的未满足需求的第一个重要步骤 我们的建议在不同的模型系统中集成了一系列创新方法， 翻译框架，这可能会导致新的重要见解胰岛素抵抗，2型糖尿病和 心血管疾病，这可能会对公共卫生产生巨大影响。

PROJECT SUMMARY/ABSTRACT Dramatic increases in insulin resistance (IR) prevalence are expected in the U.S. and throughout the world in coming years. Since IR is an important risk factor for cardiovascular disease, discovery of more efficient ways of preventing and treating this condition would have a huge public health impact. Over the past decades, development of new drugs aimed at preventing cardiometabolic disease has slowed down substantially, but recent advances in human genetics offer new exciting opportunities for drug development. Genome-wide associations studies (GWAS) have discovered >150 loci associated with IR and closely related traits over the past decade; but for the vast majority of these, the causal gene has not been definitely identified and the mechanisms leading to IR are unknown. We have performed colocalization analyses to prioritize 50 plausible candidate genes from 164 GWAS loci associated with IR-related traits, and we now aim to establish and characterize genes causally associated with IR using a rigorous series of experiments combining CRISPR-based gene perturbation with single-cell RNA sequencing and detailed phenotyping in human adipocytes, skeletal myocytes and mouse models. In Aim 1, we will perform CROP-seq – CRISPR-based transcriptional interference (CRISPRi) followed by single- cell RNA-seq (scRNA-seq) – in human adipocytes to characterize differentially expressed genes and pathways after knockdown of 50 genes selected based on colocalization analyses. In Aim 2, we will evaluate metabolic phenotypes, such as glucose uptake, lipolysis, insulin signaling, adipogenesis, mitochondrial function, fatty acid oxidation, and metabolite profiles in human adipocytes and skeletal myocytes after CRISPRi knockdown of 25 genes, guided by expression profiles from aim 1. In Aim 3, we will create and breed knockout mouse models for three IR-related genes, and then compare wildtype and knockout mice with regards to fat distribution, glucose and insulin tolerance, energy expenditure, physical activity, food intake, lipid profiles, kidney and liver panels, cellular transcriptome, and histopathology of different tissues in mice on chow and after high-fat feeding. By combining a range of innovative methods including high-throughput gene perturbations followed by single cell transcriptomics, in vitro and in vivo experiments to characterize loci established using human genetics, we expect to establish causal genes and mechanisms of action for novel genes involved in development of IR. This is a first important step towards development of new drugs to address the huge and increasing unmet need posed by IR. Our proposal integrates a range of innovative approaches in different model systems providing a translational framework that is likely to lead to new important insights into insulin resistance, type 2 diabetes and cardiovascular disease which could have a huge public health impact.