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Engineering Rapalog-Inducible Genetic Switches Based on Split-T7 Polymerase to Regulate Oncolytic Virus-Driven
Nikolas T Martin1,2, Mathieu J F Crupi1,2, Zaid Taha1,2
1Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.
Abstract:
The approval of different cytokines as anti-neoplastic agents has been challenged by dose-limiting toxicities. Although reducing dose levels affords improved tolerability, efficacy is precluded at these suboptimal doses. Strategies combining cytokines with oncolytic viruses have proven to elicit potent survival benefits in vivo, despite promoting rapid clearance of the oncolytic virus itself. Herein, we developed an inducible expression system based on a Split-T7 RNA polymerase for oncolytic poxviruses to regulate the spatial and temporal expression of a beneficial transgene. This expression system utilizes approved anti-neoplastic rapamycin analogues for transgene induction. This treatment regimen thus offers a triple anti-tumour effect through the oncolytic virus, the induced transgene, and the pharmacologic inducer itself. More specifically, we designed our therapeutic transgene by fusing a tumour-targeting chlorotoxin (CLTX) peptide to interleukin-12 (IL-12), and demonstrated that the constructs were functional and cancer-selective. We next encoded this construct into the oncolytic vaccinia virus strain Copenhagen (VV-iIL-12mCLTX), and were able to demonstrate significantly improved survival in multiple syngeneic murine tumour models through both localized and systemic virus administration, in combination with rapalogs. In summary, our findings demonstrate that rapalog-inducible genetic switches based on Split-T7 polymerase allow for regulation of the oncolytic virus-driven production of tumour-localized IL-12 for improved anti-cancer immunotherapy.
Insights
We developed an inducible oncolytic virus system using Split-T7 polymerase and rapamycin to control transgene expression. This approach enhances cancer immunotherapy by delivering targeted interleukin-12 (IL-12) and improving survival rates.
Area of Science:
- Oncolytic virotherapy
- Gene therapy
- Cancer immunotherapy
Background:
- Cytokine therapy for cancer is limited by dose-limiting toxicities.
- Oncolytic viruses combined with cytokines show survival benefits but face rapid viral clearance.
- Controlling transgene expression is crucial for effective oncolytic virus therapy.
Purpose of the Study:
- To develop an inducible expression system for oncolytic poxviruses to regulate transgene expression spatially and temporally.
- To create a therapeutic transgene by fusing chlorotoxin (CLTX) to interleukin-12 (IL-12) for tumor targeting.
- To evaluate the efficacy of a novel oncolytic vaccinia virus encoding the CLTX-IL-12 construct in preclinical cancer models.
Main Methods:
- Engineered an inducible expression system using Split-T7 RNA polymerase for oncolytic poxviruses.
- Utilized rapamycin analogues to induce transgene expression.
- Fused chlorotoxin (CLTX) to interleukin-12 (IL-12) to create a tumor-targeting therapeutic transgene.
- Constructed and tested the vaccinia virus strain Copenhagen (VV-iIL-12mCLTX) in syngeneic murine tumor models.
Main Results:
- Demonstrated functional and cancer-selective CLTX-IL-12 fusion constructs.
- Achieved significantly improved survival in murine tumor models using localized and systemic administration of VV-iIL-12mCLTX with rapalogs.
- Showcased the ability of the Split-T7 system to regulate tumor-localized IL-12 production.
Conclusions:
- Rapalog-inducible Split-T7 polymerase systems enable controlled production of therapeutic transgenes in oncolytic viruses.
- This strategy offers a triple anti-tumor effect: oncolytic virus, induced transgene, and pharmacologic inducer.
- The developed system holds promise for enhanced anti-cancer immunotherapy through targeted IL-12 delivery.
