A Bicistronic Retroviral Vector for Protecting Hematopoietic Cells Against Antifolates and P-Glycoprotein Effluxed Drugs

Abstract

Chemoresistance gene transfer is an experimental method to protect hematopoietic cells from the toxicity of anticancer drugs. Because multiple drugs are usually given together in cancer therapy, this strategy will ultimately require vectors expressing multiple chemoresistance genes. For this reason, we designed a bicistronic retroviral vector (HaMID) containing a modified human multidrug resistance-1 cDNA and a mutant human dihydrofolate reductase cDNA bearing a leucine to tyrosine substitution at codon 22 (L22Y). To determine if this vector would confer dual drug resistance to hematopoietic cells, recombinant retrovirus was used to transduce the human CEM T lymphoblastic cell line as well as primary murine myeloid progenitors. Growth suppression assays, using polyclonal transduced CEM cells, demonstrated increased resistance to taxol (13-fold), trimetrexate (8.9-fold), vinblastine (5.6-fold), methotrexate (2.5-fold), and etoposide (1.5-fold) when used as single agents. HaMID-transduced cells also grew at a logarithmic rate in the simultaneous presence of 25 nM taxol and 100 nM trimetrexate while control cells were entirely growth inhibited by this drug combination. Similarly, HaMID-transduced murine myeloid progenitors acquired increased resistance to taxol (2.9-fold) and trimetrexate (140-fold), and were able to form colonies in the simultaneous presence of both drugs. Our results suggest that retroviral transfer of HaMID into primary hematopoietic cells should reduce the myelosuppression associated with the combined use of antifolates and P-glycoprotein-effluxed drugs.

Overview summary

Bone marrow toxicity is a major complication of cancer chemotherapy. The introduction of chemoresistance genes into bone marrow cells may decrease the morbidity of therapy and may allow intensification of treatment without associated increased myelotoxicity. We have investigated the ability of a novel bicistronic retroviral vector, expressing both the multidrug resistance gene and a mutated dihydrofolate reductase gene (HaMID), to protect transduced hematopoietic cells from the toxic effects of two classes of chemotherapeutic drugs in vitro. We show that transduced CEM cells acquired a significant increase in resistance to antifolates, P-glycoprotein effluxed drugs, or their combination. Furthermore, similar results were seen in transduced primary murine myeloid progenitor cells. Further studies will determine if the chemoprotection provided by HaMID-transduced marrow will allow repeated high doses of antifolate and taxol combination chemotherapy to be administered in vivo.