Oncolytic viruses (OVs) represent a promising class of cancer therapies, capable of replicating within and destroying tumor cells while delivering therapeutic payloads. However, the heterogeneity and adaptability of tumor ecosystems pose significant challenges to OV efficacy. Conventional cancer treatments often struggle against metastatic disease due to tumor evolution and heterogeneity. Replicating cancer therapeutics, such as engineered viruses, offer a potential solution by adapting and responding to the evolving tumor environment. OVs, like vaccinia virus (VV), offer a large coding potential for delivering therapeutic transgenes, but controlling viral replication and transgene expression remains a major hurdle for safe and effective treatment. Limited regulatory switches exist to control the timing and dosing of transgene expression. This research aims to address this limitation by developing and integrating drug-controlled gene switches into VV vectors. The goal is to create a toolbox of precisely controllable synthetic promoters and regulatory networks to finely tune OV replication and therapeutic payload delivery, thereby enhancing the safety and efficacy of oncolytic virotherapy.

Existing literature highlights the potential of OVs as replicating cancer gene therapy vectors. Studies demonstrate the ability of engineered bacteria, immune cells, and viruses to elicit robust, systemic anti-tumor immune responses. However, the balance between optimal virus spread and the generation of immune responses needs careful management. The incorporation of genetic circuits into OVs to regulate virulence genes and therapeutic payloads is crucial for improving therapeutic outcomes. Previous research has explored various inducible systems for gene expression, but their optimization for use with viral vectors like VV is an ongoing challenge. Specifically, the lack of easily controllable, FDA-approved systems for regulating viral replication and therapeutic transgene expression within the context of an oncolytic virus limits the clinical translation of this technology. This study leverages the power of synthetic biology to address these limitations.

The researchers adapted several drug-controlled gene switches to regulate VV-encoded transgene expression. They focused on systems controlled by FDA-approved reagents such as rapamycin and doxycycline, along with the less explored cumate-inducible system. The study involved the design of synthetic promoters by rationally fusing operator elements of different drug-inducible systems with VV promoters. They aimed for robust inducible expression with undetectable baseline levels. The generation of chimeric synthetic promoters further enhanced the regulatory layers for VV-encoded synthetic transgene networks. These inducible systems were then applied to control the expression of various transgenes: fusogenic proteins, toxic cytokines, and elements directly regulating VV replication. Multiple cell lines (U205 osteosarcoma, HeLa, HT29 colorectal adenocarcinoma, A549 lung carcinoma) were used for in vitro testing, and in vivo studies utilized HT-29 xenograft and syngeneic mouse tumor models. The efficacy of rapamycin, its analogs, and doxycycline was evaluated in controlling viral gene expression. Furthermore, the cumate-inducible system was investigated for its efficacy and safety profile in vivo. Combinatorial application of the developed chemogenetic switches (e.g., combining rapamycin and doxycycline inducible systems) was also explored to improve selectivity. The researchers characterized the systems’ kinetics and dose-responsiveness and assessed their impact on viral replication in both in vitro and in vivo settings. Toxicity studies were conducted to evaluate the safety profile of the cumate-inducible system in mice. Finally, the application of the TetR/TetO system as a safety switch for conditionally replicating VV was investigated, assessing both its in vitro and in vivo effects on viral replication and its potential as a vaccine vector. Bioluminescence imaging was employed to monitor transgene expression and viral replication in vivo. Various assays, including luciferase assays, cell viability assays, and ELISA were used to quantify the effects of the systems.

The researchers successfully generated a rapamycin-inducible expression system in a VV vector, showing robust induction of transgene expression in various cell lines and in vivo in the presence of rapamycin or its analogs. They also developed a doxycycline-inducible system, identifying optimal VV promoters for TetO-controlled expression and demonstrating its effectiveness in controlling transgene expression both in vitro and in vivo. A cumate-inducible system was developed and characterized; showing high inducibility and minimal basal expression, along with a favorable safety profile in mice, even with prolonged cumate administration. Combinatorial application of the rapamycin- and doxycycline-inducible systems demonstrated synergistic effects on transgene expression. The use of a Dox-inducible system as a safety switch to control VV replication was demonstrated in vivo, achieving robust tumor-selective viral replication in the presence of doxycycline and effectively extinguishing replication upon doxycycline removal. The conditionally replicating VV was shown to be safer than a control virus in an in vivo model, indicating reduced adverse events. The impact of expressing fusogenic proteins on viral spread and therapeutic efficacy was also investigated. The researchers showed that by temporally controlling the expression of a fusion protein, they could improve the therapeutic effect of the oncolytic virus.

The findings demonstrate the successful development and application of multiple chemogenetic switches for precise control of oncolytic virus replication and transgene expression. These systems provide a significant advancement in the field, addressing crucial limitations in the development of safe and effective oncolytic virus therapies. The combinatorial approach of using multiple switches further refines control, reducing off-target effects and enhancing the therapeutic window. The successful application of these systems in both in vitro and in vivo models strongly supports their potential for clinical translation. The study's results have implications for developing safer and more potent oncolytic virus therapies, potentially expanding the clinical applications of this technology. The successful use of conditionally replicating vaccinia virus as a potential vaccine vector is a particularly promising finding. The ability to control both viral replication and therapeutic transgene expression opens doors for developing highly effective oncolytic viruses with minimal side effects.

This study presents a significant advancement in oncolytic virus therapy by developing and validating novel chemogenetic switches for precise control of viral replication and transgene expression. The integration of these systems enhances the safety and efficacy of oncolytic viruses, paving the way for more effective and less toxic cancer treatments. Future research could focus on optimizing these systems for use with other viral vectors and exploring their applications in various cancer types and treatment paradigms. Further investigation into the long-term safety and efficacy of these systems in larger animal models and ultimately in clinical trials is crucial for translating this promising technology into clinical practice.

While the study demonstrates promising results, some limitations should be noted. The in vivo studies were conducted primarily in xenograft and syngeneic mouse models, which may not fully capture the complexity of human tumors. The long-term effects of cumate administration in mice warrant further investigation, despite the observed short-term safety profile. The scope of the study focused on a limited number of inducible systems and transgenes; further investigation is needed to expand the range of applicable systems and therapeutic payloads. The generalizability of the findings to other oncolytic viral vectors beyond VV and HSV needs further investigation.