Fast Track Adaptation of Oncolytic Coxsackie B3 Virus to Resistant Colorectal Cancer Cells - a Method to Personalize Virotherapy

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Leslie Elsner - , Technical University of Berlin (Author)
  • Lisanne Heimann - , Technical University of Berlin (Author)
  • Anja Geisler - , Technical University of Berlin (Author)
  • Babette Dieringer - , Technical University of Berlin (Author)
  • Klaus Peter Knoch - , Molecular Diabetology, German Center for Diabetes Research (DZD e.V.) (Author)
  • Luisa Hinze - , Technical University of Berlin (Author)
  • Karin Klingel - , University Hospital Tübingen (Author)
  • Michel Solimena - , Molecular Diabetology, German Center for Diabetes Research (DZD e.V.) (Author)
  • Jens Kurreck - , Technical University of Berlin (Author)
  • Henry Fechner - , Technical University of Berlin (Author)

Abstract

Background: The efficacy of oncolytic viruses (OV) in cancer treatment depends on their ability to successfully infect and destroy tumor cells. However, patients’ tumors vary, and in the case of individual insensitivity to an OV, therapeutic efficacy is limited. Here, we present a protocol for rapid generation of tumor cell-specific adapted oncolytic coxsackievirus B3 (CVB3) with enhanced oncolytic potential and a satisfactory safety profile. This is achieved by combining directed viral evolution (DVE) with genetic modification of the viral genome and the use of a microRNA-dependent regulatory tool. Methods: The oncolytic CVB3 variant PD-H was adapted to the refractory colorectal carcinoma cell line Colo320 through serial passaging. XTT assays and virus plaque assays were used to determine virus cytotoxicity and virus replication in vitro. Recombinant PD-H variants were generated through virus mutagenesis. Apoptosis was detected by Western blots, Caspase 3/7 assays, and DAPI staining. The therapeutic efficacy and safety of the adapted recombinant OV PD-SK-375TS were assessed in vivo using a subcutaneous Colo320 xenograft mouse model. Results: PD-H was adapted to the colorectal cancer cell line Colo320 within 10 passages. Sequencing of passage 10 virus P-10 revealed a heterogenous virus population with five nucleotide mutations resulting in amino acid substitutions. The genotypically homogeneous OV PD-SK was generated by inserting the five detected mutations of P-10 into the genome of PD-H. PD-SK showed significantly stronger replication and cytotoxicity than PD-H in Colo320 cells, but not in other colorectal carcinoma cell lines. Increase of apoptosis induction was detected as key mechanisms of Colo320 cell-specific adaptation of PD-SK. For in vivo safety PD-SK was engineered with target sites of the miR-375 (miR-375TS) to exclude virus replication in normal tissues. PD-SK-375TS, unlike the PD-H-375TS not adapted homolog suppressed the growth of subcutaneous Colo320 tumors in nude mice without causing any side effects. Conclusion: Taken together, here we present an optimized protocol for the rapid generation of tumor cell-specific adapted oncolytic CVB3 based on the oncolytic CVB3 strain PD-H. The protocol is promising for the generation of personalized OV for tumor therapy and has the potential to be applied to other OV.

Details

Original languageEnglish
Article number11
Number of pages16
Journal Biological procedures online : BPO
Volume26 (2024)
Issue number1
Publication statusPublished - 25 Apr 2024
Peer-reviewedYes

Keywords

Sustainable Development Goals

Keywords

  • Cancer Therapy, Colorectal Carcinoma, Coxsackievirus B3, Direct Virus Evolution, Oncolytic Virus, Personalized Therapy