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Oncolytic adenoviruses are being developed as new anti-cancer agents. Their efficacy can be improved by incorporating RNA interference (RNAi) molecules. RNAi molecules can be expressed in various precursor formats. The aim of this study was to determine the most effective format. To this end, we constructed three Δ24-type oncolytic adenoviruses, with human microRNA-1 (miR-1) expression cassettes in short hairpin RNA (shRNA), precursor microRNA (pre-miRNA), and primary miRNA (pri-miRNA) format, respectively. The viruses were compared for virus replication, mature miR-1 expression, and target gene silencing in cancer cells. Incorporation of the cassettes had only minor effects on virus replication. Mature miR-1 expression from the pri-miRNA format reached on average 100-fold higher levels than from the other two formats. This expression remained stable upon long-term virus propagation. Infection with the pri-miR-1-expressing virus silenced the validated miR-1 targets FOXP1 and MET. Drosha knockout almost completely abrogated mature miR-1 expression, confirming that processing of adenovirus-encoded pri-miR-1 was dependent on the host cell miRNA machinery. Using simple in vitro recombination cloning, a similar virus expressing miR-26b was made and shown to silence the validated miR-26b target PTGS2. We thus provide a platform for construction of oncolytic adenoviruses with high expression of RNAi molecules of choice.

Oncolytic virus therapy of cancer is an actively pursued field of research. Viruses that were once considered as pathogens threatening the wellbeing of humans and animals alike are with every passing decade more prominently regarded as vehicles for genetic and oncolytic therapies. Oncolytic viruses kill cancer cells, sparing healthy tissues, and provoke an anticancer immune response. Among these viruses, recombinant adenoviruses are particularly attractive agents for oncolytic immunotherapy of cancer. Different approaches are currently examined to maximize their therapeutic effect. Here, knowledge of virus⁻host interactions may lead the way. In this regard, viral and host microRNAs are of particular interest. In addition, cellular factors inhibiting viral replication or dampening immune responses are being discovered. Therefore, applying RNA interference is an attractive approach to strengthen the anticancer efficacy of oncolytic viruses gaining attention in recent years. RNA interference can be used to fortify the virus’ cancer cell-killing and immune-stimulating properties and to suppress cellular pathways to cripple the tumor. In this review, we discuss different ways of how RNA interference may be utilized to increase the efficacy of oncolytic adenoviruses, to reveal their full potential.