Abstract
Optimization of oncolytic viruses for therapeutic applications requires the strategic removal or mutagenesis of virulence genes alongside the insertion of transgenes that enhance viral replication, spread and immunogenicity. However, the complexity of many viral genomes and the labour-intensive nature of methods for the generation and isolation of recombinant viruses have hindered the development of therapeutic oncolytic viruses. Here we report an iterative strategy that exploits the preferential susceptibility of viruses to certain antibiotics to accelerate the engineering of the genomes of oncolytic viruses for the insertion of immunomodulatory cytokine transgenes, and the identification of dispensable genes with regard to replication of the recombinant oncolytic viruses in tumour cells. We applied the strategy by leveraging insertional mutagenesis via the Sleeping Beauty transposon system, combined with long-read nanopore sequencing, to generate libraries of herpes simplex virus type 1 and vaccinia virus, identifying stable transgene insertion sites and gene deletions that enhance the safety and efficacy of the viruses.
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Data availability
The data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed long-read sequencing datasets generated during the study are too large to be publicly shared, but are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank other members of the J.C.B. and T. Azad laboratories for contributing to this work. We also acknowledge the support of the personnel from the Flow Cytometry Core Facility, Histology Core Facility and Animal Care and Veterinary Services of the Faculty of Medicine at the University of Ottawa. J.C.B. discloses support from the Canadian Institutes of Health Research (CIHR; grant number 448323), Canadian Cancer Society Research Institute, Prostate Cancer Canada and BioCanRx. T. Azad discloses support from the CIHR (grant number 49634), Terry Fox New Investigator Awards and Cancer Research Society Next Generation of Scientists Award. R.R. discloses support from a Vanier Canada Graduate Scholarship from CIHR. S.B. discloses support from a CIHR Postdoctoral Fellowship (grant number 187898).
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J.C.B. and T. Azad proposed and supervised the project. R.R., S.B., M.A., J. Petryk, N.K.Z., R.S., G.S.-L., L.D., A.P., J. Poutou, A.I.M.Z., C.C., V.H.G., Z.K., B.A., K.A.O., R.M., Z.A., F.M., V.M., E.W., A.N.-A., A.A. and F.P.A. performed the in vitro and in vivo experiments. R.R., T. Azad, S.B. and M.D.S. engineered the viruses. A.S., R.R. and T. Azad designed the figures and illustrations. R.R., T. Azad, R.S., A.S., F.M. and S.B. performed the data analyses. R.R., R.S., T. Azad, A.S. and J.C.B. wrote the manuscript. S.S.G.F., T.C.H., T. Alain, L.-H.T., C.S.I. and J.-S.D. contributed to study design.
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Rezaei, R., Boulton, S., Ahmadi, M. et al. Antibiotic-mediated selection of randomly mutagenized and cytokine-expressing oncolytic viruses. Nat. Biomed. Eng 9, 822–835 (2025). https://doi.org/10.1038/s41551-024-01259-7
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DOI: https://doi.org/10.1038/s41551-024-01259-7
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