Retroviral Expression ofEscherichia coliThymidylate Synthase cDNA Confers High-Level Antifolate Resistance to Hematopoietic Cells

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Abstract
Drug resistance gene therapy has the potential to protect against the myelosuppressive side effects of chemotherapy or to be used as a dominant in vivo selectable marker of genetically modified cells. Steady state kinetic studies have indicated the Escherichia coli thymidylate synthase (ecTS) is intrinsically more resistant to several TS-directed inhibitors as compared with the human enzyme, suggesting that ecTS is suitable for use as a drug-resistant marker. However, we found a disparity between the kinetic properties of ecTS and the degree of resistance conferred to cells transfected with the cDNA encoding this enzyme. It was determined that although ecTS is as stable as human TS (hTS) in transfected mammalian cells, ecTS is produced at only 40% the level of hTS, indicating poor translation of ecTS in eukaryotic cells. To circumvent this problem, the entire cDNA sequence of ecTS was synthesized by using codons optimized for expression in mammalian cells. In transfected Chinese hamster lung cells, expression of ecTS from the optimized construct, termed OPTecTS, is as efficient as hTS. Furthermore, cells transfected with the OPTecTS cDNA are significantly more resistant to the TS inhibitor raltitrexed as compared with transfected cells expressing similar levels of hTS. High-titer retroviral packaging cells were generated with OPTecTS and >80% of transduced mouse hematopoietic progenitor cells are resistant to raltitrexed, Thymitaq, and U89 at concentrations that eliminated colony growth of mock-transduced cells. The transgene was detectable by PCR in transduced bone marrow selected in U89 or raltitrexed, and expression of ecTS from the OPTecTS cDNA in bone marrow exhibited a catalytic rate constant comparable to that of purified recombinant ecTS. These data indicate that OPTecTS is a viable dominant selectable marker that can confer resistance to antifolates when introduced into cells.

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