Enhancing Oncolytic Virotherapy Using Functional Genomic Screening

Abstract

The treatment of cancer patients with oncolytic vaccinia virotherapy has yielded encouraging results in early phase clinical trials, but there are yet unexplored opportunities for improving this therapy that could lead to even better outcomes for patients. To this end, we harnessed the power of functional genomic screening to identify both host and vaccinia virus (VV) genes that could be manipulated to enhance the efficacy of VV. We hypothesized that one approach to enhancing the efficacy of VV was to increase its ability to spread in tumours by manipulating host genes. We conducted human genome-wide RNAi screens in three tumour cell lines and identified 237 genes that upon silencing enhanced spread in two out of the three cells lines tested and 23 across all three cell lines. Muscle RAS oncogene homolog (MRAS) emerged as a promising candidate following in vitro validation studies. To test the impact of inhibiting this gene in vivo, we engineered an oncolytic VV encoding a dominant negative of MRAS. The treatment of tumour-bearing mice with this virus significantly delayed tumour progression, prolonged survival and resulted in durable responses. As a parallel approach to enhancing the therapy, we also sought to discover alternative means of conferring VV tumour specificity that would lead to a larger therapeutic index than that of the current clinical candidates that rely on deletions of thymidine kinase and/or vaccinia growth factor. We conducted RNAi VV genome-wide screens in two tumour cell lines to identify VV genes that could be deleted without diminishing its potency in tumour cells but would be concomitantly attenuating in normal cells. We identified 35 VV gene candidates and engineered deletion mutants of 13 of these. We found a VV with the DNA ligase gene deleted to be as attenuating as the current clinical iii candidate vvDD in normal cells, but more potent in multiple tumour cell lines and provided a therapeutic advantage in vivo. This work serves as a valuable resource for designing the next generation of oncolytic VVs and showcases functional genomic screening as a powerful tool for identifying genes that can be manipulated to potentially improve clinical outcomes.