A novel oncolytic vaccinia virus with multiple gene modifications involved in viral replication and maturation increases safety for intravenous administration while maintaining proliferative
Go Okita, Kiyotaka Suenaga, Masashi Sakaguchi, Toshio Murakami
Abstract
To generate a novel oncolytic vaccinia virus with improved safety and productivity, the genome of smallpox vaccine strain LC16m8 was modified by a bacterial artificial chromosome system. By using LC16m8, a replicating virus homologous to the target virus, as a helper virus for the bacterial artificial chromosome system, we successfully recovered genome-edited infectious viruses. Oncolytic viruses with limited growth in normal cells were obtained by deleting the genes for vaccinia virus growth factor (VGF), extracellular signal-regulated kinase-activating protein (O1L), and ribonucleotide reductase (RNR) present in the viral genome.
Introduction
Oncolytic virotherapy with various viruses represents a promising new therapeutic modality for cancer. Clinical trial data on the intravenous administration of oncolytic viruses demonstrated that they may be delivered safely and systemically with reduced toxicity; however, efficacy varied among individuals. This is primarily because at doses and regimens that are known to be safe, the virus is rapidly cleared from the circulation before it reaches its target. This phenomenon is mainly caused by neutralizing antibodies, complement activation, antiviral cytokines, and endogenous tissue macrophages, and is accompanied by non-specific uptake by other tissues, such as the lung, liver, and spleen [1].
Materials and method
Cells
The immortalized cell line HeLa derived from human cervical cancer cells and the cell line RK13 exhibiting an epithelial morphology that was isolated from the rabbit kidney were purchased from the American Type Culture Collection. Primary normal human dermal fibroblasts (NHDF) isolated from the dermis of adult skin were purchased from Lonza. These cells were generally handled according to the supplier’s recommendations.
Results
Decrease in cytotoxicity in normal cells by the RNR gene deficiency
Cancer cells (HeLa) or normal cells (NHDF) were infected with LC16m8-B5RmO, in which the B5R gene of LC16m8 was reverted to the sequence of the parent strain (LC16mO), and its modified viruses under serum-free culture conditions, and the cytotoxicity of these viruses was compared. The modified viruses used were MD-RVV lacking the C11R and O1L genes of the LC16m8-B5RmO virus genome, MD-RVV-ΔRR lacking the F4L gene encoding the RNR small subunit of MD-RVV, and viruses that modified the EEV-related gene of MD-RVV-ΔRR (MD-RVV-ΔRR-A34R, MD-RVV-ΔRR-EEV6, and MD-RVV-ΔRR-EEV7).Citation: Okita G, Suenaga K, Sakaguchi M, Murakami T (2025) A novel oncolytic vaccinia virus with multiple gene modifications involved in viral replication and maturation increases safety for intravenous administration while maintaining proliferative potential in cancer cells. PLoS ONE 20(3): e0312205. https://doi.org/10.1371/journal.pone.0312205
Discussion
Novel oncolytic vaccinia viruses derived from the vaccine strain LC16m8 virus genome were examined in the present study. The virus genome was modified using a BAC system. BAC technology has made it possible to exploit the genetics of E. coli to precisely introduce multiple genetic mutations into the viral genome [17]. The rescue of infectious genome-edited vaccinia viruses has so far been consistently achieved by transfecting mammalian cells infected with a helper non-replicating fowlpox virus with a BAC plasmid with a vaccinia virus insert. The risks associated with using fowlpox as a helper virus include the possibility of contamination with viruses other than the vaccinia virus of interest and unintended recombination between the virus of interest and the helper virus.
Acknowledgments
The authors are grateful to Dr. Yasushi Kawaguchi from Tokyo University for providing expertise to construct recombinant viruses using BAC technology and sharing pEPkan-S and GS1783. We also thank Y. Kamizuru, S. Etou, K. Gotou, A. Tsune, M. Shinmura, Y. Kanadome, and S. Kuriyama for constructing the recombinant viruses and obtaining evaluation data as lab technicians.
Citation: Okita G, Suenaga K, Sakaguchi M, Murakami T (2025) A novel
oncolytic vaccinia virus with multiple gene modifications involved in viral replication and maturation increases safety for intravenous administration while maintaining proliferative potential in cancer cells. PLoS ONE 20(3): e0312205. https://doi.org/10.1371/journal.pone.0312205
Editor: Brian M. Ward, University of Rochester School of Medicine and Dentistry, UNITED STATES OF AMERICA
Received: October 12, 2023; Accepted: September 6, 2024; Published: March 6, 2025
Copyright: © 2025 Okita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript (DOCX) and figures (TIFF) files.
Funding: This work was fully supported by KM Biologics Co., Ltd. All authors were employees of KM Biologics Co., Ltd. at the time of this study. The founder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: GO and TM are employees of KM Biologics Co., Ltd. KS and MS are employees of Meiji Animal Health Co., Ltd. In connection with this research, KS, GO, and MS are the inventors of a registered patent (JP7034818B2), TM and GO are the inventors of an applied patent (WO/2021/029385). These does not alter our adherence to PLOS ONE policies on sharing data and materials.
