Stem Cell Models of Familial Combined Hypolipidemia

家族性混合性低脂血症的干细胞模型

• 批准号: 8792243
• 负责人: Kiran Musunuru
• 金额: $ 41.21万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8792243
• 关键词: Affect; Angiopoietins; Apolipoproteins B; Biological Models; Blood; Blood Circulation; Cause of Death; Cell Line; Cell model; Cells; Cholesterol; Cholesterol Homeostasis; DNA; Disease; Drug Targeting; Family; Gene Proteins; Genes; Genetic study; Genomics; Goals; Health; Hepatocyte; Hereditary Disease; Heterozygote; High Density Lipoprotein Cholesterol; Human; Human Genetics; Human Genome; Hypertriglyceridemia; Knowledge; LDL Cholesterol Lipoproteins; Libraries; Link; Lipase; Lipids; Lipoproteins; Liver; Low-Density Lipoproteins; Mutation; Myocardial Infarction; Nonsense Mutation; Pattern; Pharmaceutical Preparations; Phenotype; Plasma; Pluripotent Stem Cells; Prevention; Process; 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; hypolipidemia; link protein; lipoprotein lipase; mutant; next generation; novel; nuclease; prevent; protein expression; protein function; 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）： 尽管降低胆固醇的药物（主要是他汀类药物），但心肌梗死仍然是世界上死亡的主要原因。因此，对于预防心肌梗死的新药物迫切需要。我们已经在人类中使用了全基因组关联研究和外显子组测序研究，以鉴定许多与胆固醇代谢有关的新基因。最近，我们将外显子组测序应用于一个具有不寻常脂质模式的家庭中的两个健康兄弟姐妹，我们称其为“家族性降低低脂蛋白血症” - 高密度低密度脂蛋白胆固醇（LDL-C），高密度脂蛋白胆固醇（HDL-C）和Triglycerider（TG）和TG）级别（TG）级别。我们发现，兄弟姐妹是两个不同的无意义突变S17X和E129X-IN ANGPTL3（编码Angiopoietin样3蛋白）的复合杂合子。据报道，一种仅在肝脏中合成并分泌到血液中的蛋白质，据报道抑制脂蛋白脂肪酶（由LPL编码）和内皮脂肪酶（由LIPG编码）抑制脂肪蛋白（由LIPG编码），从而增加了Rodents的血浆TG和HDL-C水平。我们对ANGPTL3突变的发现突出了该基因在人类LDL-C代谢中的作用，并将基因牵涉到LDL-C减少和预防MI的潜在治疗靶标。在人类中发现了Angptl3和LDL-C之间的新型联系后，我们现在试图定义基因在血液中改变LDL-C的机制。我们发现，Angptl3废话突变的载体降低了非常低密度的脂蛋白（VLDL）载脂蛋白B（APOB）的产生，而LDL APOB的分数分解代谢率提高。因此，我们假设ANGPTL3直接在人肝脏中起作用，以调节肝细胞VLDL分泌和LDL清除率，从而调节血液中的LDL-C水平。为了检验这一假设，我们试图评估S17X和E129X突变对人体衍生肝细胞中ANGPTL3功能的影响。我们建议以最严格的方式来实现（1）使用人类基因组编辑使用尖端的TAL效应核酸酶（TALEN）技术来生成具有或没有S17X或E129X突变的同基因人类多能干细胞（HPSC）线条； （2）将HPSC线区分为肝细胞； （3）评估肝细胞中的VLDL/LDL处理。我们还建议生成一个ANGPTL3报告基因肝细胞细胞系，以对降低ANGPTL3表达的化合物进行小分子筛选，从而降低血液胆固醇水平。如果成功，我们使用基因组编辑的HPSC衍生细胞来研究与疾病相关突变的作用的方法可以广泛应用于各种各样的人遗传疾病。