REGULATION OF HUMAN B-GLOBIN GENE IN ERYTHROID CELLS

地中海贫血综合征的基因治疗需要 引入的珠蛋白基因模拟了原位正常的功能 珠蛋白基因在组织特异性和效率上的研究 抄写。DNA介导的基因转移实验已经 研究表明，人类的β-珠蛋白基因及其直接的5‘和 3‘侧翼序列可以在组织中表达，并且 发展阶段特有的方式。但仍处于较高水平 这种基因的转录可能需要序列元素而不是 驻留在珠蛋白结构基因及其邻近侧翼 序列。人类基因组DNA缺失程度的比较 荷兰和英国的伽马三角洲β-地中海贫血提示 可能存在调控序列的上游 调控基因表达的胚胎表型珠蛋白基因 远下游的β-珠蛋白基因。一段DNA位于 已经显示了10kb 5‘的epsilon-珠蛋白基因 红系特异性增强子活性。因此，有可能 β-珠蛋白基因的转录激活可能涉及到 至少两个协同激活步骤，分别由 这个遥远的增强子序列，通过更接近的序列 β-珠蛋白基因。在DNA介导的基因转移中 实验证明，这种增强子元件在顺式基因中的存在 β-珠蛋白或测试基因，可能因此显著增强 这种基因在红系宿主中的转录效率。这 该提案试图解决这种可能性。增强剂 具有其他可能调控序列的元件将被拼接 将β-珠蛋白或测试基因转化为无增强子的质粒或 转化为无增强型逆转录病毒载体，随后将 瞬时或稳定地导入适当的细胞系或导入 小鼠骨髓细胞。该基因的转录效率 测试基因将与内源性α-or的基因进行比较 红系宿主细胞的β-珠蛋白基因。 鉴定一种红系特异的珠蛋白基因增强子， 了解其对β-内酰胺酶作用的分子机制 就像珠蛋白基因，以及它在协调 α和β样珠蛋白基因的激活 分离的染色体，可能都有助于阐明调控 人类β-珠蛋白基因的机制以及由此带来的 β-地中海贫血的基因治疗更接近于 现实。

Gene therapy for the thalessemia syndromes requires that the introduced globin gene mimic the function of an in situ normal globin gene both in tissue specificity and efficiency of transcription. DNA mediated gene transfer experiments have shown that the human beta-globin gene with its immediate 5' and 3' flanking sequences can be expressed in a tissue and developmental-stage specific manner. Yet high level transcription of such a gene may require sequence elements not residing in the globin structural gene and its immediate flanking sequences. Comparison of the extents of DNA deletions in the Dutch and English gamma delta beta-thalassemias suggests the possible existence of regulatory sequences upstream of the embryonic epsilon-globin gene, which can regulate the expression of the far downstream beta-globin gene. A segment of DNA at 10 Kb 5' of the epsilon-globin gene has been shown to display erythroid specific enhancer activity. It is therefore possible that transcriptional activation of the beta-globin gene may involve at least two synergistic activation steps, mediated respectively by this distant enhancer sequence, and by sequences much closer to the beta-globin gene. In DNA mediated gene transfer experiments, the presence of this enhancer element, in cis to the beta-globin or a test gene, may thus significantly enhance the transcriptional efficiency of such a gene in erythroid hosts. This proposal attempts to address this possibility. The enhancer element with other probable regulatory sequences will be spliced with a beta-globin or a test gene into enhancerless plasmids or into an enhancerless retroviral vector, which will subsequently be transiently or stably introduced into appropriate cell lines or into mouse bone marrow cells. The transcriptional efficiency of the test gene will be compared to that of the endogenous alpha-or beta-globin gene of the erythroid host cells. Identification of an erythroid specific, globin gene enhancer, understanding the molecular mechanism of its action on the beta- like globin genes, and its possible role in the coordinated activation of the alpha- and beta-like globin genes located on separate chromosomes, may all aid in elucidating the regulatory mechanism of the human beta-globin gene and thus in bringing gene therapy for the beta-thalassemias significant steps closer to reality.