Retroviral expression of Escherichia coli thymidylate synthase cDNA confers high-level antifolate resistance to hematopoietic cells

Drug resistance gene therapy has the potential to protect against the myelo suppressive side effects of chemotherapy or to be used as a dominant in viv o selectable marker of genetically modified cells. Steady state kinetic stu dies have indicated the Escherichia coli thymidylate synthase (ecTS) is int rinsically more resistant to several TS-directed inhibitors as compared wit h the human enzyme, suggesting that ecTS is suitable for use as a drug-resi stant marker. However, we found a disparity between the kinetic properties of ecTS and the degree of resistance conferred to cells transfected with th e cDNA encoding this enzyme. It was determined that although ecTS is as sta ble as human TS (hTS) in transfected mammalian cells, ecTS is produced at o nly 40% the level of hTS, indicating poor translation of ecTS in eukaryotic cells. To circumvent this problem, the entire cDNA sequence of ecTS was sy nthesized by using codons optimized for expression in mammalian cells. In t ransfected Chinese hamster lung cells, expression of ecTS from the optimize d construct, termed OPTecTS, is as efficient as hTS. Furthermore, cells tra nsfected with the OPTecTS cDNA are significantly more resistant to the TS i nhibitor raltitrexed as compared with transfected cells expressing similar levels of hTS. High-titer retroviral packaging cells were generated with OP TecTS and >80% of transduced mouse hematopoietic progenitor cells are resis tant to raltitrexed, Thymitaq, and U89 at concentrations that eliminated co lony growth of mock-transduced cells. The transgene was detectable by PCR i n transduced bone marrow selected in U89 or raltitrexed, and expression of ecTS from the OPTecTS cDNA in bone marrow exhibited a catalytic rate consta nt comparable to that of purified recombinant ecTS. These data indicate tha t OPTecTS is a viable dominant selectable marker that can confer resistance to antifolates when introduced into cells.