Isogenic Human Pluripotent Stem Cell-Based Models of Human Disease Mutations

基于同基因人类多能干细胞的人类疾病突变模型

• 批准号: 8412279
• 负责人: David Altshuler
• 金额: $ 216.69万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-25 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8412279
• 关键词: Adipocytes; Bacteria; Benign; Beta Cell; Biological; Biological Assay; Biological Process; Candidate Disease Gene; Cataloging; Catalogs; Cause of Death; Cell Line; Cell physiology; Cells; Cellular Assay; Cellular biology; Characteristics; Chromosome Mapping; Code; Coronary Arteriosclerosis; DNA; Defect; Derivation procedure; Development; Disease; Disease susceptibility; Dyslipidemias; Engineering; Evaluation; Exons; Fatty acid glycerol esters; Foundations; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Variation; Genome; Genome engineering; Goals; Hepatocyte; Heterogeneity; Human; Human Engineering; Human Genetics; Human Genome; In Vitro; Inherited; Insulin; Knock-out; Knowledge; Laboratories; Left; Lipids; Liver; Measures; Metabolic; Metabolic Diseases; Metabolism; Methodology; Methods; Modeling; Molecular; Morbidity - disease rate; Morphologic artifacts; Mutation; Myocardial Infarction; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Patients; Phenotype; Pluripotent Stem Cells; Population; Prevention; Prevention approach; Process; Protocols documentation; Risk; Series; Source; Stem cells; Structure of beta Cell of islet; System; Technology; Time; Tissues; Variant; Work; base; cell type; cellular engineering; clinical phenotype; disability; disease characteristic; gene discovery; genome sequencing; high throughput screening; human disease; human stem cells; improved; in vitro Assay; in vivo; innovation; insight; insulin secretion; insulin signaling; interest; mortality; next generation; novel strategies; nuclease; programs; recombinase; stem cell biology; stem cell differentiation; therapeutic development

DESCRIPTION (provided by applicant): Type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) are leading causes of morbidity and mortality worldwide. Development of new and more effective approaches to prevention and treatment requires improved understanding of disease mechanisms. Genetic mapping in humans offers an approach to identify genes and DNA variants underlying the inherited contribution to disease susceptibility, unbiased by prior assumptions about the pathophysiological processes responsible. Next-generation sequencing technologies make it possible for the first time to catalog mutations observed in patients and in healthy controls. While human genetics has the potential to dramatically expand our knowledge of biological and disease mechanisms, progress is constrained by two central challenges: (a) determining which DNA changes are functional, and which are benign, and (b) developing cellular assays with which to interrogate the functions of the genes and variants thereby identified. Specifically, the field requires methods to functionally screen large number of mutations in high throughput, using assays that faithfully represent the human cell types of interest. This proposal is built on a foundation of human genetics, genome engineering, and stem cell biology, and is focused on the major metabolic diseases of T2DM, dyslipidemia, and CAD. The approach leverages three recent advances: (a) ongoing next-generation sequencing studies identifying candidate disease mutations, (b) development of methodologies to rapidly alter genes of interest in human pluripotent stem cells (hPSCs) using engineered TAL effector nucleases (TALENs), and (c) protocols to differentiate hPSCs into cell populations with characteristics of hepatocytes, adipocytes, and pancreatic beta cells. Our proposal combines two central innovations. First, we propose to develop two novel approaches to engineer the genomes of hPSCs, rapidly and efficiently knocking out the function of candidate genes, and introducing specific mutations observed in patients. This will generate isogenic human stem cells that differ only at a single mutation of interest. Second, we will develop and improve protocols to differentiate hPSCs into physiologically mature hepatocytes, adipocytes, and beta cells. By engineering stem cells to carry specific mutations, and by differentiating these engineered stem cells into physiologically relevant human metabolic cell types, we will make it possible to study the impact of large numbers of gene variants on human cell biology and function. By relating the functions of gene mutations and cell biological processes with the phenotypes of human patients, we will provide pathophysiological insights and practical in vitro assays to guide development of therapeutics for these challenging diseases. PUBLIC HEALTH RELEVANCE: Next-generation genome sequencing has made it possible to discover gene mutations in patients with type 2 diabetes and heart attack, two of the leading causes of death and disability worldwide. To determine the functional relevance of these gene mutations, we have developed an approach to insert specific mutations into human stem cells, and to grow these engineered cells into tissues that are involved in disease such as liver, fat, and insulin-producing cells of the pancreas. By relating the functional effects of the mutations to the disease characteristics of the patients who carry them, we will provide new insights into the molecular and cellular causes of type 2 diabetes and heart attack, and provide new and more relevant laboratory assays with which to develop new therapies.

描述（由申请人提供）：2 型糖尿病 (T2DM) 和冠状动脉疾病 (CAD) 是全世界发病和死亡的主要原因。开发新的、更有效的预防和治疗方法需要加深对疾病机制的了解。人类基因图谱提供了一种方法来识别疾病易感性遗传因素背后的基因和 DNA 变异，不受先前关于相关病理生理过程的假设的影响。新一代测序技术首次使得对患者和健康对照中观察到的突变进行分类成为可能。虽然人类遗传学有潜力极大地扩展我们对生物和疾病机制的了解，但进展受到两个主要挑战的限制：(a) 确定哪些 DNA 变化是功能性的，哪些是良性的，以及 (b) 开发细胞检测方法询问由此识别的基因和变体的功能。具体来说，该领​​域需要使用忠实代表感兴趣的人类细胞类型的检测方法，以高通量功能性筛选大量突变。该提案建立在人类遗传学、基因组工程和干细胞生物学的基础上，重点关注T2DM、血脂异常和CAD等主要代谢疾病。该方法利用了三项最新进展：(a) 正在进行的下一代测序研究，识别候选疾病突变；(b) 开发使用工程化 TAL 效应核酸酶 (TALEN) 快速改变人​​类多能干细胞 (hPSC) 中感兴趣基因的方法， (c) 将 hPSC 分化为具有肝细胞、脂肪细胞和胰腺 β 细胞特征的细胞群的方案。我们的提案结合了两项核心创新。首先，我们建议开发两种新方法来改造 hPSC 的基因组，快速有效地敲除候选基因的功能，并引入在患者中观察到的特定突变。这将产生仅在单个感兴趣的突变上有所不同的同基因人类干细胞。其次，我们将开发和改进将 hPSC 分化为生理成熟肝细胞、脂肪细胞和 β 细胞的方案。通过工程化干细胞携带特定突变，并将这些工程化干细胞分化为生理相关的人类代谢细胞类型，我们将使研究大量基因变异对人类细胞生物学和功能的影响成为可能。通过将基因突变和细胞生物学过程的功能与人类患者的表型联系起来，我们将提供病理生理学见解和实用的体外测定，以指导这些具有挑战性的疾病的治疗方法的开发。 公共健康相关性：新一代基因组测序使得发现 2 型糖尿病和心脏病患者的基因突变成为可能，这两种疾病是全球死亡和残疾的主要原因。为了确定这些基因突变的功能相关性，我们开发了一种方法，将特定突变插入人类干细胞中，并将这些工程细胞培养成与疾病有关的组织，例如肝脏、脂肪和胰岛素产生细胞。胰腺。通过将突变的功能影响与 根据携带它们的患者的疾病特征，我们将为 2 型糖尿病和心脏病发作的分子和细胞原因提供新的见解，并提供新的、更相关的实验室检测方法来开发新疗法。