TRANSFER AND EXPRESSION OF HUMAN BETA GLOBIN GENES

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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.