GENE THERAPY STRATEGIES FOR TREATMENT OF COOLEY'S ANEMIA

人β-珠蛋白在正常细胞转移后的高水平表达 将人类β珠蛋白基因导入患有 包括β地中海贫血和镰状细胞的人类血红蛋白疾病 贫血可能会导致这些疾病的治愈。 实验中， 将单独的β珠蛋白基因转移到动物的骨髓细胞中 到目前为止还没有导致高水平的感染或这种表达， 基因 这项研究的长期目标是确定 是否使用多药耐药（MDR）基因作为选择性的 与人β珠蛋白基因一起沿着的体内和体外标记 将通过选择的能力来提高基因转移的效率， 含有这两种基因的细胞。 为了做到这一点，MDR和人类测试版 珠蛋白基因将被共转移到受体细胞上， 逆转录病毒载体 本提案的具体目标是：1） 构建能够首先转移MDR的合适的逆转录病毒载体 基因，然后MDR基因与β珠蛋白基因组合， 高效率进入靶向骨髓干细胞 体外和其他药物在体内选择骨髓细胞含有 并表达这些基因; 2）使用小鼠骨髓作为模型系统; 3） 对外周血、北方和 Southern印迹和抗体检测以定量MDR基因转移 4）MDR的进一步增加、转移和表达 和β珠蛋白基因在人类骨髓细胞中的作用。 骨髓培养物; 5）使用免疫缺陷小鼠测试 肝脏中含有MDR和β珠蛋白基因的人骨髓细胞 动物模型;以及6）开发用于评估 - 第一，单独的MDR基因的转移和表达的效率，和 第二，MDR和β珠蛋白基因都进入患者细胞 接受自体移植联合高剂量强化 不涉及骨髓的实体瘤的化疗。 如果 MDR和β珠蛋白基因转移将用于以下患者： 镰状细胞病和β地中海贫血，试图治愈这些 疾病 这些研究应导致对要求的新见解 不仅对库利氏贫血症患者， 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.