A mammalianized synthetic nitroreductase gene for high-level expression

We performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays.In our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect.Our results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans.Cancer is the second most frequent cause of death in developed countries and a leading cause of death in the world. Fifty-eight million people died worldwide in 2005 and cancer accounted for 7.6 million (13%) of these deaths [1]. Twelve million people were newly diagnosed with cancer last year and the number is prognosed to continue rising with estimated up to 26 million new cases in 2030 [2]. Thus, novel and effective anti-cancer therapies are needed to encounter this extending disease.Conventional cancer treatment involves chemotherapy, which is able to cure a certain number of cancers, such as leukemia. However, the chemotherapeutic effect on solid tumors is often only transient [3]. Chemotherapeutic drugs interfere with a broad spectrum of intracellular