Although hematopoietic stem cell gene therapy offers a potential means to treat a wide variety of genetic diseases that affect blood cells, its clinical utility has been hampered by inefficient gene transfer into repopulating stem cells. To address this important limitation, we have developed a system for the in vivo selection of genetically modified hematopoietic stem cells. This strategy entails using a mutated dihydrofolate reductase (DHFR) gene as a dominant selectable marker. Stem cells containing a retroviral vector expressing the DHFR gene are then selected by administered a novel anti-folate-based drug combination to animals after transplant. We have shown that this system enables significant enrichment of transduced stem cells in transplanted mice. We now propose to further develop this system for clinical gene therapy by pursuing two specific aims. In the first specific aim, we will determine if transduced stem cells undergo self-renewal divisions after selection, and if enforced self-renewal will increase the efficiency of selection. The effects of induced stem cell cycling on selection efficiency will also be analyzed. This new information will then be tested in a potentially improved selection protocol to determine if transduced stem cells can then be amplified in non-myeloablated hosts. The second specific aim will develop this system for use in non-human primates. An effective protocol for transducing stem cells will be systematically derived in transplanted Rhesus monkeys. A separate set of experiments will be undertaken to define an appropriate drug selection scheme. These results will then be incorporated into experiments that will test if stem cell selection can be obtained in this preclinical model. We anticipate that these studies will fully determine the clinical feasibility of this approach, and will yield data broadly relevant to stem cell gene therapy.
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