Stem Cells Models of Familial Combined Hypolipidemia

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，心肌梗死仍然是世界上主要的死亡原因。因此，迫切需要用于预防心肌梗死的新药物。我们已经在人类中使用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，我们将外显子组测序应用于一个家族中的两个健康兄弟姐妹，该家族具有不寻常的脂质模式，我们称之为“家族性组合性低脂血症”-极低的低密度脂蛋白胆固醇（LDL-C），高密度脂蛋白胆固醇（HDL-C）和甘油三酯（TG）水平。我们发现，兄弟姐妹是ANGPTL 3（编码血管生成素样3蛋白）中两个不同无义突变S17 X和E129 X的复合杂合子。ANGPTL 3是一种仅在肝脏中合成并分泌到血流中的蛋白质，据报道可抑制脂蛋白脂肪酶（由LPL编码）和内皮脂肪酶（由LIPG编码），增加啮齿动物的血浆TG和HDL-C水平。我们对ANGPTL 3突变的发现强调了该基因在人类LDL-C代谢中的作用，并暗示该基因是降低LDL-C和预防MI的潜在治疗靶点。在人类中发现ANGPTL 3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ANGPTL 3无义突变携带者极低密度脂蛋白（VLDL）载脂蛋白B（apo B）生成率降低，LDL apo B分解率增加。因此，我们假设ANGPTL 3直接在人肝脏中起作用以调节肝细胞VLDL分泌和LDL清除，从而调节血液中的LDL-C水平。为了验证这一假设，我们试图评估S17 X和E129 X突变对人源性肝细胞中ANGPTL 3功能的影响。我们建议以最严格的可能方式做到这一点：（1）使用人类基因组编辑与尖端TAL效应核酸酶（TALEN）技术，以产生具有或不具有S17 X或E129 X突变的等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加工。我们还提出产生ANGPTL 3报告肝细胞系，用其对降低ANGPTL 3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各种人类遗传疾病。

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.