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

描述（由申请方提供）：2型糖尿病（T2DM）和冠状动脉疾病（CAD）是全球发病率和死亡率的主要原因。开发新的和更有效的预防和治疗方法需要更好地了解疾病机制。人类遗传图谱提供了一种方法来确定基因和DNA变异的遗传贡献的疾病易感性，无偏见的事先假设的病理生理过程负责。下一代测序技术首次使在患者和健康对照中观察到的突变编目成为可能。虽然人类遗传学有可能极大地扩展我们对生物学和疾病机制的知识，但进展受到两个核心挑战的限制：（a）确定哪些DNA变化是功能性的，哪些是良性的，以及（B）开发细胞测定法，以询问由此鉴定的基因和变体的功能。具体地，该领域需要使用忠实地代表感兴趣的人细胞类型的测定以高通量功能性地筛选大量突变的方法。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等主要代谢性疾病。该方法利用了三个最新进展：（a）正在进行的下一代测序研究，鉴定候选疾病突变，（B）开发使用工程化TAL效应核酸酶（TALEN）快速改变人多能干细胞（hPSC）中感兴趣基因的方法，以及（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。我们的建议结合了两个核心创新。首先，我们建议开发两种新的方法来工程化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处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。通过工程干细胞携带特定的突变，并将这些工程干细胞分化为生理相关的人类代谢细胞类型，我们将有可能研究大量基因变异对人类细胞生物学和功能的影响。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相关联，我们将提供病理生理学见解和实用的体外试验，以指导这些具有挑战性的疾病的治疗方法的开发。

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.