GENE THERAPY STRATEGIES FOR TREATMENT OF COOLEY'S ANEMIA

人β -珠蛋白的高水平表达后转移正常

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.