Abstract
Viral replication of thymidine kinase deleted (tk−) vaccinia virus (VV) is attenuated in resting normal cells, enabling cancer selectivity, however, replication potency of VV-tk− appears to be diminished in cancer cells. Previously, we found that wild-type herpes simplex virus (HSV)-tk (HSV-tk) disappeared in most of the recombinant VV after multiple screenings, and only a few recombinant VV containing naturally mutated HSV-tk remained stable. In this study, VV-tk of western reserve (WR) VV was replaced by A167Y mutated HSV-tk (HSV-tk418m), to alter nucleoside selectivity from broad spectrum to purine exclusive selectivity. WOTS-418 remained stable after numerous passages. WOTS-418 replication was significantly attenuated in normal cells, but cytotoxicity was almost similar to that of wild type WR VV in cancer cells. WOTS-418 showed no lethality following a 5 × 108 PFU intranasal injection, contrasting WR VV, which showed 100% lethality at 1 × 105 PFU. Additionally, ganciclovir (GCV) but not BvdU inhibited WOTS-418 replication, confirming specificity to purine nucleoside analogs. The potency of WOTS-418 replication inhibition by GCV was > 10-fold higher than that of our previous truncated HSV-tk recombinant OTS-412. Overall, WOTS-418 demonstrated robust oncolytic efficacy and pharmacological safety which may delegate it as a candidate for future clinical use in OV therapy.
Highlights
Vaccinia virus (VV) is one of the more preferred backbones for oncolytic virus (OV) engineering based on its long history of use as a vaccine for smallpox
The nucleoside binding site (NBS) coding regions of vaccinia virus (VV)-tk and human TK1 (H-tk) share significant homology, which may provide a basis for the cancer selectivity of tk deleted VV (VV-tk− ), but there is no significant homology shared with the NBS of herpes simplex virus tk (HSV-tk) [8]
Wild type HSV-tk was modified for purine selectivity and gene stability and incorporated in western reserve (WR) VV via deletion of native tk gene using homologous recombination
Summary
Vaccinia virus (VV) is one of the more preferred backbones for oncolytic virus (OV) engineering based on its long history of use as a vaccine for smallpox. Tk (human cytosolic tk (TK1) or viral tk) uses ATP as a phosphate donor to deoxyribonucleosides during DNA synthesis [6] Nucleoside selectivity of these different TKs is determined by the nucleoside binding site (NBS) encoded in the tk gene [7]. Efficient DNA synthesis is determined by nucleoside selectivity as well as the coordinated increase (10–20 fold) of other kinases—such as thymidylate synthase [9], ribonucleotide diphosphate reductase [10], and dihydrofolate reductase [11]—in the G1/S phase of cell cycle This orchestrated kinase activity plays slightly different roles in each of the following: viral DNA replication, cancer cell proliferation, and proliferation in normal cells, which may be further subdivided by cell type, where activity may vary between cell types such as bone marrow cells and gastrointestinal epithelial cells
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