Engineering novel suicide enzymes for improved cancer gene therapy

Suicide gene therapy for cancer offers a selective approach to eliminating tumor cells while leaving normal cells unaffected. One major drawback is the low activity the enzyme displays towards the prodrug. In an effort to overcome this, we sought to optimize two suicide gene therapy systems for enhanced prodrug activity. Herpes Simplex Virus thymidine kinase phosphorylates ganciclovir (GCV) to GCV-monophosphate (GCV-MP). Previously, we characterized two HSVTK-mutants with improved activity towards GCV and generated a fusion protein of HSVTK and mouse guanylate kinase (MGMK) that displayed a 175-fold decrease in IC50 for GCV. By combining these two approaches, we created mutant fusion constructs carrying HSVTK mutants and MGMK (MGMK/30 and MGMK/SR39). Both fusions displayed superior tumor killing in vitro and impressive tumor growth inhibition in an animal tumor model at very low doses of GCV. In addition, we were able to demonstrate that mutations at residue serine 37 in MGMK represent a previously undescribed mechanism for 6-thioguanine resistance, a drug widely used as chemotherapeutic agent to treat leukemia. Bacterial CD (bCD) deaminates 5-fluorocytosine to 5-fluorouracil. From a regio-specific mutagenesis library containing over one million mutants, three bCD variants with improved 5FC activity in E. coli were identified. Kinetic analyses revealed that all variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC50 values and improved bystander effect compared to wild-type bCD in three different cancer cells. In vivo, the best mutant (1525), was shown to have significant tumor growth inhibition at doses that the wild-type enzyme was unresponsive to. When the same mutant is fused with uracil phosphoribosyltransferase, only a modest improvement in tumor killing and surprisingly weaker bystander activity were detected when compared to 1525 alone. We hypothesized that when UPRT was fused to bCD, another rate limiting step in this drug activation pathway developed. From a regio-specific mutagenesis study, three UPRT variants were identified to have enhanced activity towards 5FU in E. coli and kinetic analyses revealed slight shifts in substrate preference towards 5FU. Further studies will be done to generate UPRT mutants with further capability to enhance 5FU sensitization in cancer cells