Development and application of the technologies for manipulationg hematopoietic stem cells using cell-regulatory genes

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1) For ex vivo hematopoietic stem cell (HSC) expansion, the transient block of differentiation would be required during the ex vivo culture and we utilized a dominant negative form of the retinoic acid receptor α (RARE). The 32D cells transduced with a retroviral vector containing the RARE gene flanked by the loxP sites at the both ends remained immature and continued to proliferate without showing differentiation even in the presence of G-CSF.When the 32D cells expressing the RARE gene were further transduced with the Cre recombinase gene, the cells differentiated into granulocytes in the presence of G-CSF, indicating that the differentiation block was abolished. The reversible integration of the RARE gene using the Cre/loxP system may be applicable to ex vivo HSC expansion.2) To overcome the low efficiency of gene transfer into HSCs, we have developed a novel strategy for selective expansion of transduced hematopoietic cells. This system involves "selective amplifier genes (SAGs)" which encode fusion proteins between cytokine receptors and the steroid receptor hormone-binding domains (HBDs). The prototype SAG encoded a fusion molecule (GCRER) between the granulocyte colony-stimulating factor receptor (GCR) and the estrogen-binding domain (ER). We have evaluated the SAG system using primary murine bone marrow cells in vivo. The SAG-transduced bone marrow cells were transplanted into lethally irradiated mice, and the reconstituted animals were challenged with Tm. The results suggested that SAG conferred a steroid-responsive growth ability on the transduced hematopoietic cells in vivo, and that selective expansion of these cells is feasible.