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Abstract

Immuno-virotherapy (IVT) of cancer employs oncolytic viruses (OVs) that selectively infect and lyse malignant cells, thereby establishing a systemic anti-tumor immune response. Arming of OVs with therapeutic payloads can potentiate this immune activation. However, the clinical translation of armed OVs is limited by toxicities associated with uncontrolled expression of immunostimulatory proteins, as well as adverse anti-viral immune responses that lead to premature termination of viral oncolysis. Therefore, reliable strategies enabling external control over the timing and dosage of therapeutic protein expression from armed OVs are needed to improve the safety and efficacy of IVT. In this regard, tetracycline (Tet)-responsive RNA-switches represent an alternative to conventional gene regulatory mechanisms as they function without the expression of auxiliary, potentially immunogenic proteins, require minimal genomic capacity, and are activated by an approved drug. Nevertheless, the engineering of potent RNA-switch systems for therapeutic applications presents a bottleneck, and Tet-inducible RNA-switches have not yet been validated in OVs. In this work, I hypothesized that Tet-inducible RNA-switches can be exploited for controllable expression of therapeutic transgenes from oncolytic adenoviruses (oAds) to generate potent oncolytic agents. First, I identified candidate RNA-switches in plasmid-based vectors by comparative reporter assays, including a splicing-based RNA-switch (S) and an aptazyme (A). Thereby, I developed the RNA-switch combination S+A with superior performance. Next, I generated a versatile toolbox of regulated oAds by adapting selected single RNA-switches and the S+A RNA-switch combination for controllable firefly luciferase (Luc) expression from oAds. In this context, I employed distinct transgene cassette expression strategies and defined a functional insertion site into the oAd genome. Consequently, I demonstrated Tet-inducible Luc expression from these regulated oAds and validated oAd TK-Luc-S+A as the lead candidate. I further optimized the S+A RNA-switch combination by mutagenesis, resulting in oAd TK-Luc￾S m+Am, which exhibited enhanced induction of Luc activity and sensitivity towards Tet. Importantly, I showed that this oAd retained inducibility during ongoing viral replication, whereas a corresponding oAd based on a Tet-inducible promoter did not. Subsequently, I validated the functionality of oAd TK-Luc-S m+Am for Tet inducible Luc expression in tumor￾bearing mice, providing proof-of-principle evidence that RNA-switches can be harnessed for ligand-dependent transgene expression from OVs in vivo. Finally, I demonstrated S m+Am RNA￾switch-dependent expression of the murine cytokines interleukin-2 (mIL2), mIL12 and interferon-β (mIFNβ) from oAds, while preserving their oncolytic properties. Further characterization of these regulated armed oAds revealed rapid induction and reversibility of cytokine expression, sustained expression upon repeated Tet administration, and robust induction at pharmaceutically achievable Tet concentrations. Notably, the expressed cytokines from oAds showed comparable bioactivity to recombinant proteins. Hence, these regulated armed oAds represent optimal candidates to demonstrate safer and more effective cancer treatment through controllable expression of therapeutic payloads. In conclusion, my findings establish optimized Tet-inducible RNA-switches as a reliable strategy for regulated transgene expression from oAds. The potent and sensitive RNA-switch system may overcome limitations of IVT and encourage applications in various immunotherapeutic, or gene-therapy approaches that would benefit from drug-inducible gene expression.