RETROVIRUS-MEDIATED TRANSFER OF THE MULTIDRUG-RESISTANCE GENE INTO HUMAN HEMATOPOIETIC PROGENITOR CELLS

We report the utilization of cord blood (CB) or bone marrow (BM) deriv ed low density or purified CD34(+) cells as a target for human multidr ug resistance (MDR1) gene transfer. Cells were cocultivated for 48 h w ith an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progen itor cells and in peripheral blood cells, efficiency of MDR1 gene tran sfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 mu g/ml taxol. In uni nfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-res istant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progen itors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA th rough polymerase chain reaction. MDR1 expression was very low in cultu res of uninfected controls, whereas, after drug selection, MDR1 mRNA l evels in transduced cells was as high as in the MDR1 retroviral produc er line (positive controls). (3) Flow cytometric analysis of the expre ssion of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-gly coprotein was found on 17-25% of viable CD34(+) cells. (4) Cytochemica l localization by APAAP staining of P-glycoprotein. No specific locali zation was found in untransduced controls, whereas transduced and cult ured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor c ells seems a feasible and attractive approach to generate a drug-resis tant haemopoiesis.