Stem Cells Models of Familial Combined Hypolipidemia

描述(由申请人提供)： 尽管降胆固醇药物的广泛使用，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，迫切需要新的药物来预防心肌梗死。我们在人类中使用了全基因组关联研究和外显子组测序研究来识别一些与胆固醇代谢相关的新基因。最近，我们对一个家族中的两个健康的兄弟姐妹进行了外显子组测序，该家族有一种不寻常的血脂模式，我们将其称为“家族性混合性低脂血症”--极低的低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDLC)和甘油三酯(TG)水平。我们发现，这两个兄弟姐妹是Angptl3(编码血管生成素样3蛋白)中两个明显的无义突变-S17X和E129X-的复合杂合子。Angptl3是一种在肝脏中特异合成并分泌到血液中的蛋白质，已有报道抑制脂蛋白脂酶(LPL编码)和内皮脂肪酶(LIPG编码)，升高啮齿类动物的血浆甘油三酯和高密度脂蛋白胆固醇水平。我们对Angptl3突变的发现突出了该基因在人类低密度脂蛋白-C代谢中的作用，并暗示该基因可能成为降低低密度脂蛋白和预防心肌梗死的治疗靶点。在人类中发现了Angptl3和低密度脂蛋白-C之间的一种新的联系后，我们现在试图定义该基因改变血液中低密度脂蛋白-C的机制。我们发现，Angptl3无义突变携带者降低了极低密度脂蛋白(VLDL)的生成率，增加了载脂蛋白B(ApoB)的部分分解代谢率。因此，我们假设Angptl3直接作用于人的肝脏，调节肝细胞VLDL的分泌和低密度脂蛋白的清除，从而调节血液中的低密度脂蛋白水平。为了验证这一假设，我们试图评估S17X和E129X突变对人类来源的肝细胞中Angptl3功能的影响。我们建议以最严格的方式完成这项工作：(1)使用具有尖端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突变的等基因人类多能干细胞(HPSC)株；(2)将hPSC株分化为肝细胞；以及(3)评估肝细胞中VLDL/LDL的处理。我们还建议建立一个Angptl3报告肝细胞系，用来进行小分子筛选，以寻找降低Angptl3表达从而降低血液胆固醇水平的化合物。如果成功，我们使用基因组编辑的hPSC来源的细胞来研究疾病相关突变的影响的方法可能会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.