Selection of drug-resistant transduced cells with cytosine nucleoside analogs using the human cytidine deaminase gene

Hematopoietic toxicity produced by most anticancer drugs limits their poten tial for curative therapy. We have shown previously that the human cytidine deaminase (CID) gene can confer drug resistance in murine bone marrow cell s (BMCs) to the nucleoside analog, cytosine arabinoside (ARA-C). In the pre sent study, as the first objective we showed that the CID gene can also ren der-drug resistance in BMCs to related analogs, 2',2'-difluorodeoxycytidine (dFdC) and 5-azadeoxycytidine (5-AZA-CdR). As a second objective, we inves tigated the potential of ex vivo selection with cytosine nucleoside analogs of CD-transduced BMC. The goal of this approach was to enrich the fraction of CD-transduced BMCs so as to increase the transgene expression and [eve[ of drug resistance before transplantation. This strategy may have the pote ntial to circumvent the problem in clinical gene therapy of low level of ge ne transfer and adequate long-term gene expression. Using a bicistronic ret roviral vector containing the CD and the green fluorescent protein (CDiGFP) , we transduced murine L1210 leukemic cells. All three analogs, ARA-C, dFdC , and 5-AZA-CdR were demonstrated in vitro to enrich ( >95%) the population of leukemic cells expressing the GFP transgene. However, with CD-transduce d primary murine BMCs cultivated at high cell density we observed that in v itro selection with ARA-C was riot possible due to release of CID into the culture medium at amounts that were sufficient to inactivate the analog. Th e CD-containing medium produced a chemoprotective effect on mock BMCs as sh own by lack of significant growth inhibition in the presence of ARA-C. Howe ver, at low cell density in a cell mixture containing CD-transduced cells, the mock BMCs showed marked drug sensitivity to ARA-C as determined by clon ogenic assay. Selection with ARA-C was shown to significantly increase the CID enzyme activity in transduced BMC. These results suggest that CD gene h as the potential to be a good selectable marker and a possible tool for che moprotection in cancer gene therapy.