Stem Cells Models of Familial Combined Hypolipidemia

家族性混合性低脂血症的干细胞模型

• 批准号: 9212742
• 负责人: Kiran Musunuru
• 金额: $ 40.25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9212742
• 关键词: ANGPTL3 gene; Affect; Apolipoproteins B; Biological Models; Blood; Blood Circulation; Cause of Death; Cell Line; Cell model; Cells; Cholesterol; Cholesterol Homeostasis; DNA; Disease; Drug Targeting; Family; Genes; Genetic study; Genomics; Goals; Hepatocyte; Hereditary Disease; Heterozygote; High Density Lipoprotein Cholesterol; Human; Human Genetics; Human Genome; Hypertriglyceridemia; Knowledge; LDL Cholesterol Lipoproteins; LIPG gene; Libraries; Link; Lipids; Lipoproteins; Liver; Low-Density Lipoproteins; Mutation; Myocardial Infarction; Nonsense Mutation; Pattern; Pharmaceutical Preparations; Phenotype; Plasma; Prevention; Production; Proteins; Protocols documentation; Reporter; Reporter Genes; Reporting; Risk Factors; Rodent; Role; Siblings; Site; Stem cells; Technology; Testing; Therapeutic; Triglycerides; Variant; Very low density lipoprotein; base; effective therapy; exome sequencing; genome editing; genome wide association study; human pluripotent stem cell; hypolipidemia; lipid disorder; lipoprotein lipase; lipoprotein triglyceride; mutant; next generation; novel; novel therapeutics; nuclease; prevent; public health relevance; screening; small molecule; success; therapeutic target; uptake

DESCRIPTION (provided by applicant): Despite the widespread use of cholesterol-lowering medications, principally the statin drugs, myocardial infarction remains the leading cause of death in the world. There is therefore a critical need for new medications for the prevention of myocardial infarction. We have used genome-wide association studies and exome sequencing studies in humans to identify a number of novel genes related to cholesterol metabolism. Recently, we applied exome sequencing to two healthy siblings in a family with an unusual lipid pattern that we have termed "familial combined hypolipidemia"-extremely low low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels. We discovered that the siblings were compound heterozygotes for two distinct nonsense mutations-S17X and E129X-in ANGPTL3 (encoding the angiopoietin-like 3 protein). ANGPTL3, a protein exclusively synthesized in liver and secreted into the bloodstream, has been reported to inhibit lipoprotein lipase (encoded by LPL) and endothelial lipase (encoded by LIPG), increasing plasma TG and HDL-C levels in rodents. Our finding of ANGPTL3 mutations highlights a role for the gene in LDL-C metabolism in humans, as well as implicating the gene as a potential therapeutic target for LDL-C reduction and prevention of MI. Having discovered a novel link between ANGPTL3 and LDL-C in humans, we now seek to define the mechanism by which the gene alters LDL-C in the blood. We found that carriers of ANGPTL3 nonsense mutations had decreased rates of very-low-density lipoprotein (VLDL) apolipoprotein B (apoB) production and increased fractional catabolic rates for LDL apoB. Thus, we hypothesize that ANGPTL3 acts directly in the human liver to regulate hepatocellular VLDL secretion and LDL clearance, thereby modulating LDL-C levels in the blood. To test this hypothesis, we seek to evaluate the effects of the S17X and E129X mutations on ANGPTL3 function in human-derived hepatocytes. We propose to do this in the most rigorous possible way by (1) using human genome editing with cutting-edge TAL effector nuclease (TALEN) technology to generate isogenic human pluripotent stem cell (hPSC) lines with or without the S17X or E129X mutations; (2) differentiating the hPSC lines into hepatocytes; and (3) assessing VLDL/LDL processing in the hepatocytes. We also propose to generate an ANGPTL3 reporter hepatocyte cell line with which to perform a small molecule screen for compounds that reduce ANGPTL3 expression and thereby reduce blood cholesterol levels. If successful, our approach of using genome-edited, hPSC-derived cells to study the effects of disease-associated mutations could be applied widely to a large variety of human genetic disorders.

描述（由申请人提供）： 尽管广泛使用降胆固醇药物，主要是他汀类药物，但心肌梗塞仍然是世界上死亡的主要原因。因此，迫切需要新的药物来预防心肌梗塞。我们利用人类全基因组关联研究和外显子组测序研究来鉴定一些与胆固醇代谢相关的新基因。最近，我们对一个家庭中的两个健康兄弟姐妹进行了外显子组测序，他们的血脂模式异常，我们称之为“家族性混合性低脂血症”——低密度脂蛋白胆固醇（LDL-C）极低，高密度脂蛋白胆固醇（HDL-C）极低。 ）和甘油三酯（TG）水平。我们发现这对兄弟姐妹是 ANGPTL3（编码血管生成素样 3 蛋白）中两个不同的无义突变（S17X 和 E129X）的复合杂合子。 ANGPTL3 是一种仅在肝脏中合成并分泌到血液中的蛋白质，据报道可以抑制脂蛋白脂肪酶（由 LPL 编码）和内皮脂肪酶（由 LIPG 编码），从而增加啮齿动物的血浆 TG 和 HDL-C 水平。我们对 ANGPTL3 突变的发现凸显了该基因在人类 LDL-C 代谢中的作用，并表明该基因是降低 LDL-C 和预防 MI 的潜在治疗靶点。在人类中发现了 ANGPTL3 和 LDL-C 之间的新联系后，我们现在试图确定该基因改变血液中 LDL-C 的机制。我们发现 ANGPTL3 无义突变的携带者极低密度脂蛋白 (VLDL) 载脂蛋白 B (apoB) 的产生率降低，而 LDL apoB 的分解代谢率增加。因此，我们假设 ANGPTL3 直接作用于人肝脏，调节肝细胞 VLDL 分泌和 LDL 清除，从而调节血液中 LDL-C 水平。为了检验这一假设，我们试图评估 S17X 和 E129X 突变对人源肝细胞中 ANGPTL3 功能的影响。我们建议以最严格的方式做到这一点，（1）使用尖端TAL效应核酸酶（TALEN）技术的人类基因组编辑来生成具有或不具有S17X或E129X突变的同基因人类多能干细胞（hPSC）系； (2)将hPSC系分化为肝细胞； (3) 评估肝细胞中的 VLDL/LDL 处理。我们还建议生成 ANGPTL3 报告肝细胞系，用其对减少 ANGPTL3 表达从而降低血液胆固醇水平的化合物进行小分子筛选。如果成功，我们使用基因组编辑的 hPSC 衍生细胞来研究疾病相关突变的影响的方法可以广泛应用于多种人类遗传疾病。