Mechanisms of cis-acting HbF regulation in sickle cell anemia

摘要 镰状细胞贫血是人类最常见的遗传性单基因疾病之一，是一种全球性的健康问题， 负担一个治疗目标是增加胎儿血红蛋白浓度，以保护红细胞免受镰状 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 反式作用基因座已被深入研究，BCL 11 A正被遗传靶向， 在某些情况下，这可能至关重要。在此我们 我们将重点放在新的顺式作用位点上，我们发现这些位点与阿拉伯-印度人胎儿血红蛋白升高有关。 镰状细胞性贫血的单倍型我们的中心假设是：β-珠蛋白基因簇位点控制的变体 阿拉伯-印度单倍型镰状细胞性贫血中的一个区域通过调节胎儿血红蛋白基因表达 基因座控制区与珠蛋白基因启动子之间的差异性成环，部分解释了基因座控制区与珠蛋白基因启动子之间的差异。 胎儿血红蛋白水平与不同HBB单倍型相关。我们将使用一种诱导多能干细胞 基于iPSC的系统能够重现胎儿和成人型血细胞产生， 灵活的机制研究模型，以验证这一假设。我们提出三个具体目标： 目的1：遗传修饰镰状细胞病单倍型特异性iPSC，以评估推定的 胎儿血红蛋白表达的顺式作用调节剂。我们将使用基因编辑来靶向选定的顺式作用 调节镰状细胞来源的成人型成红细胞中胎儿血红蛋白基因表达的元件 贫血患者具有高阿拉伯-印度和低贝宁胎儿血红蛋白HBB基因簇单倍型。 目的2：使用定向的顺式作用的胎儿血红蛋白调节剂在功能上验证新的顺式作用的胎儿血红蛋白调节剂。 镰状细胞病特异性iPSC向胎儿和成人型血细胞的分化。引导 将镰状iPSC分化为表达血红蛋白的胎儿和成人成红细胞将使我们能够探索 修饰的调控区的细胞内在效应。 目的3：剖析顺式激活胎儿血红蛋白调节剂在iPSC衍生的红系细胞中的作用 使用染色体构象捕获、染色质免疫沉淀和全局转录 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： 珠蛋白基因表达、胎儿血红蛋白合成和染色体成环的工程改变。 拟议的研究使用既有和新的系统和策略：1）评估的作用， iPSC衍生的红系细胞中的新型顺式作用胎儿血红蛋白调节剂，2）揭示了基础生物学 在血细胞发育过程中球蛋白转换的背后，以及3）为发展奠定基础， 测试新的、针对患者的治疗方法。这些跨学科研究将在 补充实验室的三名研究人员与专业知识的干细胞生物学（墨菲），镰状细胞 疾病和球蛋白基因调控（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).