TRANFERRED GLOBIN REGULATION IN ERYTHROBLASTS

镰状细胞病和β地中海贫血的严重程度是 在个体产生高水平的 胎儿血红蛋白 因此，合理治疗这些疾病， 将是增加胎儿珠蛋白水平的患者， 否则发生。 为此，研究的直接目标是 更好地了解调节胎儿和成人珠蛋白的机制 成年人的水平。 至少有三个区域的DNA序列以外的 已经鉴定出珠蛋白基因转录单位，其具有重要的 在调节胎儿和成人β-珠蛋白基因表达中的作用。 这些是侧翼于DNA序列的基因座激活区（LAR）序列。 β-珠蛋白基因簇（包含两个γ-珠蛋白基因）， γ-和β-珠蛋白基因启动子元件，和增强子元件3 ′ 和β-珠蛋白基因之间的联系 我们计划研究 这些序列对转移的γ-和β-珠蛋白的表达的影响 基因. 特别是，我们将确定是否个别伽玛和 β-珠蛋白基因可以通过LARs和增强子竞争激活 从而影响它们的相对表达。 一组完整的 实验将确定自然发生的因果关系 与其他正常胎儿中胎儿珠蛋白产生升高有关的突变 成人（胎儿遗传持续性的非缺失型） 血红蛋白）。 我们将通过逆转录病毒转移来实现这些目标， 珠蛋白调节序列和珠蛋白基因进入早期红系 分离自人的祖细胞（爆发形成单位-红系或BFU-E） 外周血 将培养携带转移基因的BFU-E 在无血清条件下产生表达 生理水平的γ-珠蛋白。 转移的表达 将在从成红细胞分离的RNA中测量珠蛋白基因， 与内源性珠蛋白基因水平相比。 珠蛋白的逆转录病毒转移 人类成纤维细胞的基因将提供人类珠蛋白的信息， 基因调控在其他系统中无法获得，也将直接帮助 发展基因治疗。

The severity of sickle cell disease and beta-thalassemia is ameliorated under conditions where individuals produce high levels of fetal hemoglobin. It follows that a rational therapy for these diseases would be to increase fetal globin levels in patients where this does not otherwise occur. To this end, the immediate goal of the research is a better understanding of the mechanisms regulating fetal and adult globin levels in adults. At least three regions of DNA sequences outside of the globin gene transcription unit have been identified which have important roles in regulating fetal gamma- and adult beta-globin gene expression. These are locus activation region (LAR) sequences flanking the beta-globin gene cluster (which contains the two gamma-globin genes), gamma- and beta-globin gene promoter elements, and enhancer elements 3' to the gamma- and beta-globin genes. We plan to study the effects of these sequences on the expression of transferred gamma- and beta-globin genes. In particular, we will determine whether individual gamma- and beta-globin genes can compete for activation by LARs and enhancers thereby influencing their relative expression. An integral set of experiments will be to determine causality of naturally-occurring mutations linked to elevated fetal globin production in otherwise normal adults (the nondeletion types of hereditary persistence of fetal hemoglobin). We will accomplish these aims by retroviral transfer of globin regulatory sequences and globin genes into early erythroid progenitors (burst forming units-erythroid or BFU-E) isolated from human peripheral blood. BFU-Es carrying the transferred gene will be cultured under serum-free conditions to produce mature erythroblasts expressing physiological levels of gamma-globin. Expression of the transferred globin genes will be measured in RNA isolated from the erythroblasts and compared to endogenous globin gene levels. Retroviral transfer of globin genes into human erythoblasts will provide information on human globin gene regulation unobtainable in other systems and will also directly help develop gene therapy.