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

项目摘要/摘要 美国和世界各地的胰岛素抵抗(IR)患病率预计将在#年大幅上升。 未来几年。由于IR是心血管疾病的重要危险因素，因此发现更有效的治疗方法 预防和治疗这种疾病将对公共健康产生巨大影响。 在过去的几十年里，旨在预防心脏代谢性疾病的新药的开发已经放缓 大幅下降，但人类遗传学的最新进展为药物提供了新的令人兴奋的机会 发展。全基因组关联研究发现了150个与胰岛素抵抗和糖尿病相关的基因座。 在过去的十年里密切相关的特征；但对于这些特征中的绝大多数，因果基因并没有 已明确确定，导致IR的机制尚不清楚。 我们进行了共定位分析，确定了164个GWAS基因座中50个可信的候选基因的优先顺序 与IR相关的特征相关，我们现在的目标是建立和表征与IR相关的基因 IR使用基于CRISPR的基因扰动和单细胞RNA相结合的一系列严格的实验 人类脂肪细胞、骨骼肌细胞和小鼠模型的测序和详细表型。 在目标1中，我们将进行基于CRISPR的作物序列转录干扰(CRISPRi)，然后进行单链干扰。 人脂肪细胞中的细胞RNA-seq(scRNA-seq)-研究差异表达的基因和途径 在基于共定位分析选择的50个基因被敲除之后。 在目标2中，我们将评估代谢表型，如葡萄糖摄取、脂肪分解、胰岛素信号转导、 人脂肪细胞的成脂作用、线粒体功能、脂肪酸氧化和代谢物谱 在目标1的表达谱指导下，CRISPRi敲除25个基因后的骨骼肌细胞。 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、葡萄糖和胰岛素耐量、能量消耗、身体状况 活动，食物摄入量，脂谱，肾脏和肝脏面板，细胞转录组，和组织病理学 给小鼠喂食高脂饲料和喂食后的组织。 通过结合一系列创新方法，包括高通量基因干扰和单一 细胞转录学，体外和体内实验，以确定利用人类遗传学建立的基因座 期望建立参与IR发生发展的新基因的致病基因和作用机制。这 是朝着开发新药迈出的重要的第一步，以解决巨大且日益增长的未得到满足的需求 由IR构成。我们的建议在不同的模型系统中集成了一系列创新方法，提供了 翻译框架可能导致对胰岛素抵抗、2型糖尿病和 心血管疾病，这可能会对公众健康产生巨大影响。

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.