DNA Methylation, Chromatin, and Globin Gene Silencing

描述(申请人提供)：胎儿血红蛋白(HBF)水平升高对镰状细胞性贫血患者的临床有益。在狒狒模型中进行的实验表明，使用5-氮杂-2‘-脱氧胞苷(地他滨)、丁酸盐和羟基尿素等药物可以提高HBF水平。这些药物对镰状细胞病患者的有效性已在一些临床试验中得到证实。MSH的研究表明，羟基尿素疗法减少了疼痛危象的数量、急性胸部综合征的发生率和患者的输血需求。相当多的人(10-40%)对治疗无效，HBF水平的微小变化证明了这一点。此外，由于增加的HbF在红细胞中分布不均，很大比例的红细胞仍未受到脱氧HBS分子在细胞内聚合的保护。因此，必须开发新的和改进的药物和疗法，在更大比例的患者中将HBF提高到更高的水平，并最大限度地增加F细胞的产生数量。我们的目标是在使用去甲基化药物地他滨、组蛋白脱乙酰酶抑制剂和生长因子的基础上，为镰状细胞病患者开发一种更好的治疗方案。我们打算通过确定DNA甲基化和组蛋白乙酰化在成人珠蛋白基因表达的发育调节和HBF表达的重新激活中的作用来研究这些药物的作用机制。利用流式细胞仪、亚硫酸氢盐测序和免疫沉淀甲醛固定染色质片段(CHIP)结合聚合酶链式反应分析少量高纯度造血祖细胞中基因甲基化和组蛋白乙酰化状态是可能的。我们建议在胎儿发育和正常的红系分化过程中观察伽玛珠蛋白基因表达、DNA甲基化和组蛋白乙酰化的变化，以及在给予地他滨和组蛋白去乙酰酶抑制剂诱导的HbF产生增加之后。我们将使用过去20年来一直使用的体外培养系统和体内狒狒模型系统来研究这些机制。这些研究将确定伽马珠蛋白基因沉默的机制，并将有助于开发新的程序，以增加镰状细胞疾病患者的HBF产生。

DESCRIPTION (provided by applicant): Increased level of fetal hemoglobin (HbF) is clinically beneficial ill patients with sickle cell anemia. Experiments performed in the baboon model demonstrated that HbF levels could be elevated using pharmacologic agents such as 5-aza-2'-deoxycytidine (decitabine), butyrates, and hydroxyurea. The usefulness of these drugs in patients with sickle cell disease was confirmed in a number of clinical trials. The MSH study demonstrated that hydroxyurea therapy reduced the number of pain crises, incidence of acute chest syndrome, and transfusion requirements in patients. A significant number (10-40%) are refractory to treatment as evidenced by minimal changes in HbF levels. Furthermore, because the increased HbF is distributed heterogeneously among red cells, a large percentage of erythrocytes remain unprotected from intracellular polymerization of deoxy-HbS molecules. New and improved agents and therapies must therefore be developed which increase HbF to higher levels in a greater proportion of patients and maximize the number of F cells produced. It is our goal to develop a better therapeutic regimen for patients with sickle cell disease based upon the use of the demethylating drug decitabine, histone deacetylase inhibitors, and growth factors. We intend to investigate the mechanism of action of these agents by determining the role of DNA methylation and histone acetylation in both the development regulation of globin gene expression and the reactivation of HbF expression in the adult. Analysis of the methylation and histone acetylation status of genes in small numbers of highly purified hematopoietic progenitor cells is now possible using FACS, bisulfite sequencing and immunoprecipitation of formaldehyde-fixed chromatin fragments (CHIP) in combination with PCR. We propose to follow changes in gamma-globin gene expression, DNA methylation, and histone acetylation during fetal development and normal erythroid differentiation, and following augmentation of HbF production induced by administration of decitabine and histone deactylase inhibitors. We will use an in vitro culture system and an in vivo baboon model system that we have used for the past 20 years to study these mechanisms. These studies will define the mechanisms of gamma-globin gene silencing, and will aid in the development of new procedures to augment HbF production in patients with sickle cell disease.