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β珠蛋白基因 β地中海贫血和镰状细胞性贫血的纯合子。这个目标 需要优化转移和表达人珠蛋白的方法 人类骨髓干细胞中的基因。逆转录病毒载体将继续 是基因转入骨髓的主要机制 细胞。这项建议的具体目标是：1)增加 利用较小的新型逆转录病毒载体进行基因转移的效率 含有β珠蛋白基因；2)使用包括IL-3在内的生长因子， IL-6和干细胞因子(SCF)增加骨髓数量 干细胞处于周期，因为最佳逆转录病毒需要周期 感染；3)长期使用骨髓培养，反复 和/或持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)增加 利用DNA序列表达转移的β-珠蛋白基因 人β珠蛋白基因的基因座控制区(LCR) 构建；5)纯化骨髓干细胞；6)展示人β 利用人骨在免疫缺陷小鼠体内表达珠蛋白基因 来自镰状细胞病或贝涛地中海贫血患者的骨髓。这 将作为临床前试验的一个模型系统 人β珠蛋白基因在人骨髓细胞中的活体表达 动物；以及7)如果这些实验成功，就用人类 将逆转录病毒转移到人骨髓的方案 常规接受自体骨髓移植患者的细胞 移植(ABMT)与大剂量化疗和 最终进入镰状细胞患者的骨髓细胞 疾病和贝涛地中海贫血。这些实验有可能 导致包括β地中海贫血在内的血红蛋白疾病的基因治疗 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.