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原位正常的珠蛋白基因的功能， 珠蛋白基因的组织特异性和效率 转录。 DNA介导的基因转移实验 结果显示，人类β-珠蛋白基因的5'端和 3'侧翼序列可以在组织中表达， 发展阶段的具体方式。 然而高水平 这种基因的转录可能需要不 存在于珠蛋白结构基因及其直接侧翼 序列的 比较DNA缺失的程度， 荷兰人和英国人的伽马δ β地中海贫血表明 可能存在的调控序列上游的 胚胎ε-珠蛋白基因，它可以调节表达 β-珠蛋白基因的下游。 一段DNA， ε-珠蛋白基因5'端的10 Kb已显示出 红细胞特异性增强子活性。 因此， β-珠蛋白基因的转录激活可能涉及 至少两个协同活化步骤，分别由 这个远距离的增强子序列， β-珠蛋白基因 DNA介导的基因转移 在实验中，这种增强子元件的存在，顺式于 β-珠蛋白或测试基因，因此可以显著增强 在红系宿主中这种基因的转录效率。 这 该提案试图解决这种可能性。 增强子 与其他可能的调控序列的元件将被剪接 将β-珠蛋白或测试基因插入无增强子的质粒中， 插入到无增强子的逆转录病毒载体中， 瞬时或稳定地导入适当的细胞系或导入 小鼠骨髓细胞 的转录效率。 测试基因将与内源性α-or基因进行比较 红系宿主细胞的β-珠蛋白基因。 红细胞特异性珠蛋白基因增强子的鉴定， 了解其作用于β- 如珠蛋白基因，以及它在协调 激活α-和β-样珠蛋白基因， 分离的染色体，都可能有助于阐明调节 人类β-珠蛋白基因的机制， β-地中海贫血症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.