TRANSFERRED GLOBIN REGULATION IN HUMAN 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，针对这些疾病的合理治疗方法 将是提高患者的胎儿珠蛋白水平，而这不会 否则就会发生。为此，这项研究的直接目标是 更好地了解胎儿和成人珠蛋白的调节机制 成人体内的水平。至少有三个DNA序列区域不在 珠蛋白基因转录单位已被鉴定，它们具有重要的 在调节胎儿和成人β-珠蛋白基因表达中的作用。 这些是基因座激活区(LAR)序列 β-珠蛋白基因簇(包含两个伽马珠蛋白基因)， 伽马和β-珠蛋白基因启动子元件和增强子元件3‘ 与伽马和β-珠蛋白基因有关。我们计划研究 这些关于转移的γ-和β-珠蛋白表达的序列 基因。特别是，我们将确定个人伽马和 β-珠蛋白基因可以竞争LARs和增强剂的激活 从而影响它们的相对表达。一套完整的 实验将确定自然发生的因果关系 在其他正常情况下，与胎儿珠蛋白产生增加有关的突变 成人(胎儿遗传性持续性的非缺失型 血红蛋白)。我们将通过逆转录病毒转移来实现这些目标 珠蛋白调控序列和珠蛋白基因进入早期红系 从人类分离的祖细胞(爆裂形成单位-红系或BFU-E) 外周血液。携带转移基因的BFU-ES将被培养 在无血清条件下产生成熟的红细胞，表达 生理水平的丙种球蛋白。被转移者的表达 珠蛋白基因将在从红细胞中分离的RNA中进行测量 与内源性珠蛋白基因水平进行比较。珠蛋白的逆转录病毒转移 进入人类红细胞的基因将提供有关人类珠蛋白的信息 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.