GENE THERAPY STRATEGIES FOR TREATMENT OF COOLEY'S ANEMIA

正常人移植后人β珠蛋白的高水平表达 人β珠蛋白基因导入慢性粒细胞白血病患者的骨髓细胞 人类血红蛋白疾病，包括β地中海贫血和镰状细胞 贫血可能导致这些疾病的治愈。在这些实验中 单独的β珠蛋白基因被转移到动物的骨髓细胞 到目前为止还没有导致高水平的感染或表达 吉恩。这笔赠款中的研究的长期目标是确定 是否使用多药耐药(MDR)基因作为可选择的 与人β珠蛋白基因在体内和体外的标记 将提高基因转移的效率，因为它能够选择 同时含有这两种基因的细胞。要做到这一点，MDR和人类测试版 珠蛋白基因将被共同转移到相同的 逆转录病毒载体。这项建议的具体目标是：1) 构建可首先转移多药耐药的逆转录病毒载体 基因，然后mdr基因与β珠蛋白基因在 秋水仙碱对靶向骨髓干细胞的高效转化 体外等体内药物筛选为骨髓细胞所含 2)以小鼠骨髓为模型系统；3) 用聚合酶链式反应(PCR)检测外周血、Northern和 Southern印迹和抗体检测定量检测MDR基因转移 和表达；4)MDR的进一步增加和转移表达 和β珠蛋白基因在人骨髓细胞中的长期使用 骨髓培养；5)用免疫缺陷小鼠检测 活体中含有MDR和β珠蛋白基因的人骨髓细胞 动物模型；以及6)制定人类方案以评估 第一，MDR基因单独转移和表达的效率，以及 第二，MDR和β珠蛋白基因都进入了患者的细胞 大剂量强化联合自体移植 不累及骨髓的实体肿瘤的化疗。如果 成功的MDR和β珠蛋白基因转移将用于 镰状细胞病和β地中海贫血试图治愈这些疾病 疾病。这些研究应该会带来对需求的新见解 为成功的基因治疗不仅库利氏贫血患者和 镰状细胞性贫血，但也有其他遗传性疾病。

High-level expression of human beta globin following transfer of a normal human beta globin gene into the bone marrow cells of patients with disorders of human hemoglobin including beta thalassemia and sickle cell anemia could lead to a cure for these diseases. Experiments in which the beta globin gene alone is transferred into the bone marrow cells of animals has thus far not resulted in high level infection or expression of this gene. The long-term goal of the studies in this grant is to determine whether the use of the multiple drug resistance (MDR) gene as a selectable marker both in vivo and in vitro along with the human beta globin gene will increase the efficiency of gene transfer by the ability to select for cells containing both these genes. To do this, both the MDR and human beta globin genes will be co-transferred into recipient cells on the same retroviral vector. The specific goals of this proposal are to: 1) Construct suitable retroviral vectors capable of transferring first the MDR gene, and then the MDR gene in combination with the beta globin gene at high efficiency into target bone marrow stem cells using colchicine in vitro and other drugs in vivo to select for bone marrow cells containing and expressing these genes; 2) Use mouse bone marrow as a model system; 3) Use the polymerase chain reaction (PCR) on peripheral blood, Northern and Southern blotting, and antibody detection to quantitate MDR gene transfer and expression; 4) Further increase and transfer and expression of the MDR and beta globin genes in human bone marrow cells by the use of long-term bone marrow cultures; 5) Use immunodeficient mice to test the expression of human bone marrow cells containing the MDR and beta globin genes in a live animal model; and 6) Develop human protocols for evaluation of the efficiency of transfer and expression of, first, the MDR gene alone, and second, both the MDR and beta globin genes into the cells of patients undergoing autotransplantation in association with high-dose intensive chemotherapy for solid tumors not involving the bone marrow. If successful, MDR and beta globin gene transfer will be used in patients with sickle cell disease and beta thalassemia in an attempt to cure these diseases. These studies should lead to new insights into the requirements for successful gene therapy of patients not only with Cooley's anemia and sickle cell anemia, but with other genetic disorders as well.