In vivo methotrexate selection of murine hemopoietic cells transduced with a retroviral vector for Gaucher disease

The studies described were performed to investigate whether in vivo selection of retrovirus-transduced hemopoietic cells is feasible starting from a low percentage of transduced hemopoietic stem cells (PHSCs). The vector used is an amphotropic bicistronic retroviral vector carrying a cDNA for human lysosomal glucocerebrosidase (hGC) for treatment of Gaucher disease and a methotrexate (MTX) resistant mutant cDNA encoding human dihydrofolate reductase (DHFR). We tested the effect of MTX selection in mice that were either myeloablated or not before infusion of transduced cells. In addition, we determined whether repeated administration of transduced bone marrow cells has an additional effect on the percentage of hGC expressing cells. The results obtained have shown that, in myeloablated mice transplanted once with transduced bone marrow and treated twice weekly with 10 mg/kg of MTX for a total of 6 months, a two- to three-fold increased numbers of hGC expressing cells could be detected in both peripheral blood and bone marrow as compared with non-MTX treated mice. In mice transplanted with transduced bone marrow once every 2 weeks for a total of four times, percentages of hGC expressing cells were not significantly increased as compared with mice transplanted once. In non-ablated mice neither MTX selection nor multiple infusions of transduced bone marrow resulted in detection of hGC expressing cells 6 months after transplantation, indicating that the success of in vivo selection using MTX is highly dependent on the ratio of transduced hemopoietic stem cells transplanted versus residing and untransduced stem cells.