Mechanisms of cis-acting HbF regulation in sickle cell anemia

摘要 镰状细胞性贫血是人类最常见的遗传性单基因疾病之一，是一种全球性的疾病 负担。治疗目标是提高胎儿血红蛋白浓度以保护红细胞免受镰刀的伤害。 聚合物引起的损伤。胎儿血红蛋白基因表达受顺式和反式作用基因座调控。虽然 已经对反式作用基因座进行了深入的研究，而BCL11A正在成为基因靶标，但人们对此的关注较少 支付给独联体代理监管机构，在某些情况下，这可能是最重要的。在此，我们 关注新的顺式作用基因，我们发现这些基因与阿拉伯裔印度人胎儿血红蛋白升高有关 单倍型镰状细胞性贫血。我们的中心假设是：β-珠蛋白基因簇位置的变异控制 阿拉伯-印度单倍型镰状细胞贫血区通过以下途径调节胎儿血红蛋白基因的表达 位点控制区对珠蛋白基因启动子的差异环，并部分解释了 不同HBb单倍型与胎儿血红蛋白水平的关系我们将使用一种诱导的多能干细胞 基于IPSC的系统，能够概括胎儿和成人型血细胞的产生，提供 用于机制研究的灵活模型来验证这一假设。我们提出三个具体目标： 目的1：对镰状细胞病单倍型特异的IPSCs进行基因修饰，以评估推测的 胎儿血红蛋白表达的顺式作用调节因子。我们将使用基因编辑来针对选定的顺式作用 镰状细胞来源的成人型红细胞中调节胎儿血红蛋白基因表达的因素 贫血患者具有高阿拉伯-印度人和低贝宁胎儿血红蛋白HBB基因簇单倍型。 目的2：从功能上验证新型顺式作用的胎儿血红蛋白调节剂 镰状细胞疾病特异性ipscs分化为胎儿和成人型血细胞。定向 将镰刀状IPSCs分化为胎儿和成人表达血红蛋白的红细胞将使我们能够探索 修饰的调节区对细胞的内在影响。 目的3：分析顺式激活胎儿血红蛋白调节剂对IPSC来源的红系细胞的影响 利用染色体构象捕获、染色质免疫沉淀和整体转录 分析。我们将通过检测以下因素的影响来确定顺式作用调节的机制 基因工程改变珠蛋白基因的表达、胎儿血红蛋白合成和染色体环路。 拟议的研究使用既有的和新的系统和战略来：1)评估 IPSC来源的红系细胞中新型顺式作用的胎儿血红蛋白调节剂，2)揭示基础生物学 在血细胞发育过程中珠蛋白转换的背后，以及3)为发育和 测试针对患者的新型治疗方法。这些跨学科研究将在 补充三名具有干细胞生物学专业知识的研究人员的实验室(墨菲)、镰刀细胞 疾病和珠蛋白基因调控(Steinberg)和染色质动力学(DAI)。

ABSTRACT Sickle cell anemia is one of mankind's most common hereditary monogenic diseases and a global health burden. A therapeutic goal is to increase fetal hemoglobin concentration to protect erythrocytes from sickle polymer-induced injury. Fetal hemoglobin gene expression is regulated by cis-and trans-acting loci. Although the trans-acting loci have been intensively studied and BCL11A is being targeted genetically, less attention has been paid to cis-acting regulators, which in some instances might be of paramount importance. Herein we focus on novel cis-acting loci that we found to be associated with elevated fetal hemoglobin in the Arab-Indian haplotype of sickle cell anemia. Our central hypothesis is: Variants in the β-globin gene cluster locus control region in sickle cell anemia with the Arab-Indian haplotype modulate fetal hemoglobin gene expression by differential looping of the locus control region to globin gene promoters and account, in part, for the variance in fetal hemoglobin levels associated with different HBB haplotypes. We will use an induced pluripotent stem cell (iPSC)-based system capable of recapitulating both fetal and adult-type blood cell production that provides a flexible model for mechanistic studies to validate this hypothesis. We propose three specific aims: Aim 1: Genetically modify sickle cell disease haplotype-specific iPSCs to assess the effects of putative cis-acting regulators of fetal hemoglobin expression. We will use gene editing to target selected cis-acting elements to modulate fetal hemoglobin gene expression in adult-type erythroblasts derived from sickle cell anemia patients with the high Arab-Indian and low Benin fetal hemoglobin HBB gene cluster haplotypes. Aim 2: Functionally validate novel cis-acting fetal hemoglobin modulators using the directed differentiation of sickle cell disease-specific iPSCs into fetal and adult-type blood cells. Directed differentiation of sickle iPSCs into fetal and adult hemoglobin-expressing erythroblasts will allow us to explore the cell intrinsic effects of modified regulatory regions. Aim 3: Dissect the effects of cis-activating fetal hemoglobin modulators in iPSC-derived erythroid cells using chromosome conformation capture, chromatin immunoprecipitation, and global transcriptional analyses. We will define the mechanism of cis-acting regulation through assays examining the effects of engineered changes on the expression of globin genes, fetal hemoglobin synthesis and chromosomal looping. The proposed studies use both established and novel systems and strategies to: 1) evaluate the role of novel, cis-acting fetal hemoglobin modulators in iPSC-derived erythroid cells, 2) reveal the basic biology behind globin switching during blood cell development, and 3) lay the groundwork for the development and testing of novel, patient-specific therapeutics. These transdisciplinary studies will be carried out in the complementing laboratories of three investigators with expertise in stem cell biology (Murphy), sickle cell disease and globin gene regulation (Steinberg), and chromatin dynamics (Dai).