GENE THERAPY IN HEMATOPOIETIC CELLS

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The goal of this project is to develop hematopoietic stem cell gene therapy for diseases affecting the hematopoietic system. A recent study in children with X-linked serve combined immunodeficiency has shown the successful application of hematopoietic stem cell gene therapy. While these results were very encouraging for the gene therapy field, most genetic diseases do not have selective advantages for gene-corrected cells, and are therefore not likely to be cured by currently available techniques. Further improvements in gene transfer efficiency and in the engraftment of transduced cells using less toxic non-myeloablative conditioning regimens will be required. We have used the dog model to study gene transfer into hematopoietic repopulating cells because of our long-standing experience with hematopoietic stem cell transplantation in this model and because of the availability of disease models. During the previous funding period, we have improved gene transfer into hematopoietic repopulating cells by (1) using a gibbon ape leukemia virus (GALV) envelope, (2) transducing CD34-enriched cells in flasks coated with the human fibronectin fragments CH-296, and (3) using a growth factor combination which included canine stem cell factor (cSCF), canine granulocyte-colony stimulating factor (cG-CSF) and human FLT3-L. Although gene marking, especially in a non-myeloablative setting. Thus our objectives for this project are threefold. First, in Specific Aims 1-3, we propose to study techniques to further improve gene transfer rates in hematopoietic stem cells. Second, Specific Aims 4 and 5 will explore less toxic conditioning regimens in combination with immunosuppression and also investigate a novel in vivo selection system. Third, Specific Aims 6 and 7 will apply gene transfer techniques to disease models. Fanconi anemia has been chosen as the first target for hematopoietic stem cell gene therapy since gene-corrected stem cells in this disorder are thought to have a selective advantage over uncorrected stem cells.