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Introduction and Objectives: Oncolytic viruses preferentially replicate in and lyse cancer cells while sparing normal cells. Virus replication requires an abundant supply of deoxyribonucleotide triphosphates (dNTPs), but because cellular enzymes that synthesize dNTPs are degraded at the end of S­phase, VAC expresses its own biosynthetic enzymes including both I4L (large, R1) and F4L (small, R2) subunits of the heterodimeric ribonucleotide reductase (RR). We have shown that deleting the F4L gene inhibits virus replication and reduces virulence in a mouse model. Most importantly for oncolytics, ΔF4L mutations render virus growth sensitive to low levels of cellular RR.
Methods: VACs are tagged with the gene encoding mCherry fluorescent protein and carrying deletions in F4L, J2R (viral thymidine kinase, TK) or both. Oncolytic activity of these VACs was evaluated in a panel of human and rodent bladder cancer or non­tumorigenic bladder cell lines in vitro. The orthotopic KU7­luc bladder cancer xenograft model in nude mice was used to assess the mutant VACs in vivo.
Results: Cytotoxicity assays showed a high degree of cell killing in infections with ΔF4L, ΔJ2R or the double mutant VACs. Highly efficient VAC replication was observed in our panel of bladder cancer cell lines and particularly in the human urothelial cell carcinoma (UCC) cell line KU7­luc as well as the rat UCC cell line AY­27. Preliminary data indicate that pre­treatment of bladder cancer cells with gemcitabine sensitized them to oncolytic VAC killing. We show that our mutant VAC selectively replicated in KU7­luc bladder cancer xenografts in nude mice. Significant tumor regression in mice treated with the mutant VAC was seen.
Conclusions: These data indicate that the VACs have retained much of their replication proficiency and cytotoxicity in bladder cancer and in vivo replication is selective for the cancerous cells and avoids transmission despite deletions of these essential viral genes.