*6.5. Experimental Evolution of VSV*

Replication-competent OVs can evolve under natural selection, and reversion to virulence or loss of oncolytic potential can threaten the safety and efficacy of virotherapeutic treatments. Evolutionary risk assessment studies are required to confirm safety, and can also serve the benefit of producing more potent OV, however, the most effective strategy is to combine rational design with evolution, allowing each engineered virus to mutate and fully adapt to its intended target cells. In our recent study, two VSV-∆M51 variants containing human p53 were serially passaged on Suit-2 cells, resulting in mutations that

adapted the viruses to better replicate in multiple PDAC cell lines without developing mutations in the p53 gene or losing oncoselectivity. The mutations of note that were acquired by the viruses include two separate mutations within the G protein sequence; both of these mutations achieved fixation within the span of the experiment in either virus and were identical. It was determined that the acquired G mutations stimulate VSV replication at least in part due to improved virus attachment to PDAC cells [62,194].
