TRANSFER AND EXPRESSION OF HUMAN BETA GLOBIN GENES

这些实验的长期目标是转移和表达 患者骨髓干细胞中的人类β珠蛋白基因 β地中海贫血和镰状细胞贫血的纯合子。 这一目标 需要人类球蛋白最佳转移和表达的方法 人类骨髓干细胞中的基因。 逆转录病毒载体将继续 是基因转移到骨髓中的主要机制 细胞 该提案的具体目标是：（1）增加 使用更小和新的逆转录病毒载体的基因转移效率 含有β珠蛋白基因; 2）使用包括IL-3的生长因子， IL-6和干细胞因子（SCF），以增加骨髓数量 干细胞在循环中，因为循环是最佳逆转录病毒所必需的。 感染; 3）利用长期骨髓培养反复 和/或连续感染小鼠和人骨髓干细胞， 含有人β珠蛋白基因的逆转录病毒; 4）为了增加 使用来自以下的DNA序列表达转移的β珠蛋白基因： 添加到人β珠蛋白基因的基因座控制区（LCR） 构建体; 5）纯化骨髓干细胞; 6）证明人β 使用人骨在免疫缺陷小鼠中体内珠蛋白基因表达 镰状细胞病或β地中海贫血患者的骨髓。 这 将作为临床前试验的模型系统， 人β珠蛋白基因在人骨髓细胞中表达 动物; 7）如果这些实验成功，使用人类 将逆转录病毒转移到人骨髓中的方案 常规接受自体骨髓移植的患者 与高剂量化疗相关的ABMT， 最终进入镰状细胞病患者的骨髓细胞 疾病和β地中海贫血。 这些实验有可能 导致对血红蛋白病包括β地中海贫血的基因治疗 和镰状细胞性贫血

The long-term goal of these experiments is to transfer and express the human beta globin gene in the bone marrow stem cells of patients homozygous for beta thalassemia and sickle cell anemia. this goal requires methods for the optimal transfer and expression of human globin genes in human marrow stem cells. Retroviral vectors will continue to be the major mechanism by which genes are transferred into bone marrow cells. The specific goals of this proposal are: 1) To increase the efficiency of gene transfer using smaller and novel retroviral vectors containing the beta globin gene; 2) To use growth factors including IL-3, IL-6, and stem cell factor (SCF) to increase the number of bone marrow stem cells in cycle since cycling is required for optimal retroviral infection; 3) to utilize long-term bone marrow cultures to repeatedly and/or continuously infect mouse and human bone marrow stem cells with retroviruses containing the human beta globin gene; 4) To increase the expression of the transferred beta globin genes using DNA sequences from the locus controlling region (LCR) added to the human beta globin gene constructs; 5) To purify marrow stem cells; 6) To demonstrate human beta globin gene expression in vivo in immunodeficient mice using human bone marrow from patients with sickle cell disease or betao thalassemia. This will serve as a model system for preclinical trials of the efficiency of human beta globin gene expression in human bone marrow cells in a live animal; and 7) If these experiments are successful, to use human protocols in which retroviruses are transferred into human bone marrow cells in patients routinely undergoing autologous bone marrow transplantation (ABMT) associated with high-dose chemotherapy and eventually into the bone marrow cells of patients with sickle cell disease and betao thalassemia. These experiments have the potential of leading to gene therapy for hemoglobinopathies including beta thalassemia and sickle cell anemia.