Decreased homing of transduced human bone marrow CD34+ cells in NOD/SCID mice
Many clinical gene therapy trials have described poor engraftment of CD34+ cells transduced with retroviral vectors. Since engraftment is dependent on successful homing of graft cells to the bone marrow (BM), we examined whether retroviral-mediated gene transfer (RMGT) induces a homing defect in CD34+ cells. Homing of fluorescently labeled human BM CD34+ cells transduced with three separate retroviral vectors (MFG-eGFP, LNC-eGFP and LXSN) was assessed in NOD/SCID mice. Homing of transduced CD34+ cells was significantly decreased 20h after transplantation compared to freshly isolated control and cultured untransduced control cells. Specifically, homing of GFP+ cells in the graft was preferentially decreased thus skewing the contribution of transduced cells to engraftment. Transduced cells were not selectively trapped in other organs and BM-homed transduced cells did not undergo apoptosis at a higher rate than untransduced cells. Furthermore, adhesion molecule expression and binding activity was not altered by RMGT. To determine if soluble factors present in vector supernatant mediated the homing defect, homing of cells cultured with supernatant from empty AM12 packaging cells was assessed and a consistent (though not significant) decrease in homing was observed. Therefore, we analyzed homing following exposure to viral particles alone after AM12-LNCeGFP vector supernatant was pre-loaded onto CH-296-coated plates, and the loaded supernatant was pooled as the remainder fraction. However, culture on pre-loaded plates and with the remainder fraction both lead to decreased homing, suggesting that the defect is caused by an interaction with factors present in both vector-containing supernatant and/or viral particles or proteins. This homing defect was reversed when transduced cells were cultured over CH-296 for two additional days with SCF only. These data suggest that RMGT of hematopoietic cells may compromise their homing potential and implicate transduction-induced defective homing in the observed low engraftment of retrovirally-transduced CD34+ cells. These results may have a direct clinical application for the improvement of current gene therapy protocols.