Highlights ► SiRNAs inhibit adenovirus multiplication. ► Adenoviral DNA replication is a key target for siRNA-mediated inhibition. ► SiRNAs can delay the death of adenovirus-infected cells.

Highlights► AmiRNAs can be utilized to inhibit adenovirus replication in vitro. ► Adenoviral vectors are an effective expression vehicle for anti-adenoviral amiRNAs. ► A combination of amiRNA and cidofovir results in additive effects.

We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.

BackgroundHuman adenoviruses are a frequent threat to immunocompromised patients, and disseminated disease is associated with severe morbidity and mortality. Current drugs are not capable of preventing all fatalities, thus indicating the need for alternative treatment strategies. Adenoviruses can be rendered susceptible to antiherpetic prodrugs such as ganciclovir (GCV), upon expression of the herpes simplex virus thymidine kinase (HSV-TK) gene in adenovirus-infected cells. Furthermore, adenoviruses are amenable to post-transcriptional gene silencing via small interfering RNAs (siRNAs) or artificial micro RNAs (amiRNAs).ResultsIn this study, we combined these 2 approaches by constructing a combinatorial gene expression cassette that comprises the HSV-TK gene and multiple copies of an amiRNA directed against the mRNA encoding the adenoviral preterminal protein (pTP). HSV-TK gene expression was controlled by the adenoviral E4 promoter, which is activated in the presence of the adenoviral E1 gene products (i.e., when adenovirus is present in the cell). When inserted into a replication-deficient (E1-, E3-deleted) adenoviral vector, this cassette effectively inhibited the replication of wild-type adenovirus in vitro. The reduction rate mediated by the combinatorial approach was higher compared to that achieved by either of the 2 approaches alone, and these obvious additive effects became most pronounced when the GCV concentration was low.ConclusionsThe concept presented here has the potential to aid in the inhibition of wild-type adenovirus replication. Furthermore, the combinatorial expression cassette may constitute a safeguard to potentially control unintended replication of adenoviral vectors and to prevent immune responses provoked by them.