The overall goal of this project is to develop techniques that will result in increased gene transfer rates in improve transgene expression hematopoietic stem cells and their progeny to allow the treatment of diseases affect the hematopoietic system. A major objective to successful stem cell gene therapy has been the low gene transfer efficiencies in human gene therapy trials. Highly efficient gene transfer has been achieved into hematopoietic progenitor cells in cultured and in the mouse. Unfortunately, neither in vitro studies nor studies in the mouse have been able to accurately predict gene transfer efficiency into stem cells of large animals or humans. We have therefore developed a competitive repopulation assay which allows efficient evaluation of methods aimed at improving gene transfer into hematopoietic stem cells. We will use the competitive repopulation assay to (1) determine the optimal viral envelope protein (pseudotype) for efficient transduction of hematopoietic stem cells, (2) study whether lentiviral vectors are more efficient in transducing hematopoietic stem cells than Mo-MLV-based vectors, (3) explore whether transgene expression in hematopoietic stem cells and their progeny can be improved using modifier retroviral vectors, and (4) study the effect of insulator elements on sustained promoter activity in repopulating stem cells. Gene transfer into human hematopoietic stem cells will be assessed using the immunodeficient NOD/SCID mouse model, and gene transfer into non-human primate hematopoietic stem cells will be assessed in the baboon. Thus, these studies will also provide information whether gene transfer rates and transgene expression obtained in NOD/SCID repopulating cells can predict stem cell gene transfer efficiencies in the baboon as a surrogate for human stem cell gene transfer.
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