My objective is to transfer genes into human hematopoietic stem cells and attain high expression of the transferred genes. Initially, retrovial gene transfer vectors containing dominantly selectable genes, conferring either methotrexate or neomycin resistance, will be used to define the optimal conditions for transfer and integration of the genes into immortalized human hematopoietic cell lines. Then, retroviral transfer vectors containing the methotrexate or neomycin resistance genes under the transcriptional control of different gene regulatory elements will be constructed. The ability of these vectors to confer drug resistance to hematopoietic cell lines and bone marrow cells will be compared. In this way, gene regulatory sequences with high activity in human hematopoietic cells will be identified. These regulatory sequences will then be incorporated into vectors containing other genes with greater potential clinical usefulness. This strategy is directed toward our long term goals of 1) treating debilitating and lethal genetic disorders of children by inserting normally functioning genes into their hematopoietic stem cells and 2) applying the techniques of gene transfer to the therapy of malignant diseases. I will 1) produce replication-defective helper-free retroviruses containing either the aminoglycoside transferase gene (neomycin resistance gene neo-r) or the mutant dihydrofolate reductase gene (dhfr-r) under the transcriptional control of different regulatory elements; 2) infect human hematopoietic cell lines and normal bone marrow cells with these model vectors and compare the level of gene expression; 3) examine the factors which control the rate of infection by these vectors; and 4) construct helper-free vectors containing normal genes, whose abnormal counterparts result in disease, and transfer these into both normal and abnormal human hematopoietic cells in vitro.
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| Hock, R A; Miller, A D; Osborne, W R (1989) Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines. Blood 74:876-81 |
