Abstract
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.
Highlights
Many advances were made in the treatment of cancer in recent years, cancer still remains one of the leading causes of death worldwide
We set out to compare different precursor formats expressed from the genome of oncolytic adenoviruses during their replication in cancer cells
In contrast to what was previously reported for transfected replication-defective adenovirus shuttle plasmids,[30] we found that expression from a conditionally replicating adenovirus genome in pri-miRNA format yielded 2 orders of magnitude higher production of mature silencing molecules than expression in short hairpin RNA (shRNA) or pre-miRNA format and that this increased production resulted in the most efficient and rapid knockdown of endogenous target genes
Summary
Many advances were made in the treatment of cancer in recent years, cancer still remains one of the leading causes of death worldwide. There is, a great need for new treatments. One promising approach is oncolytic virus therapy, using viruses that selectively replicate in and kill cancer cells, evoking an antitumor immune response.[1,2] Several different oncolytic viruses are evaluated for this purpose, adenoviruses being among the most extensively studied. While preclinical results with these viruses are very prom-.
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