Genetic modification of hematopoietic progenitor cells for combined resistance to 4-hydroperoxycyclophosphamide, vincristine, and daunorubicin

Aim: To investigate whether human peripheral blood hematopoietic progenitor cells (PBPC) modified with human aldehyde, dehydrogenase, class-3 gene (AL DH-3) and multidrug resistance gene 1 (MDR1) would increase chemotherapy re sistance to 4-hydroperoxycyclophosphamide (4-HC) and P-glycoprotein effluxe d drugs. Methods: A bicistronic retroviral vector GlNa-ALDH3-IRES-MDR1 cDNA was constructed and used to tranfect the packaging cell lines PA317 by ele ctroporation. CD34(+) PBPC were isolated with a high-gradient magnetic cell sorting system (MACS), and then were transfected with supernatant of retro virus containing human ALDH-3 and MDR1 cDNA. PCR, RT-PCR, Southern blot, No rthern blot, FACS, and MTT assay were used to evaluate the transfection and expression of the transgene in target cells. Results. The bicistronic retr oviral vector construction was verified by PCR and restriction endonuclease analysis. Dual drug resistance genes were integrated into the genomic DNA of CD34(+) PBPC and expressed efficiently. The efficiency of gene transfect ion in CD34(+) PBPC was tested to be 18% on colonies. Nested PCR and Neo(r) rescue assay indicated that no helper virus was present in this system. Co mpared with the untransduced cells, transgene recipient cells conferred 4.5 -fold resistance to 4-14C, 6.6-fold and 7.8-fold resistance to P-glycoprote in effluxed drug, vincristine and daunorubicin, respectively. Conclusion: E fficient transduction of two different types of drug resistance genes into human peripheral blood hematopoietic progenitor cells and the co-expression may decrease cumulative myelosuppression of combination chemotherapy.