Improvement of the amiRNA expression cassette by concatemerization of amiRNA hairpins and inhibition of adenoviral DNA replication at decreased GCV concentrations. A549 cells were transduced with the adenoviral HSV-TK/pTP-mi5 expression vector, AdTO-TK-pTP-mi5×6 (carrying 6 copies of pTP-mi5), or the respective control vector, AdTO-TK-mi-×6 (carrying 6 copies of a negative control amiRNA) at an MOI of 100 TCID50/cell. At 24 h post-transduction, cells were infected with wt Ad5 at an MOI of 0.01 TCID50/cell, and GCV was or was not added at concentrations of 0.15 μM (A), 0.3 μM (B), 0.6 μM (C), or 1.2 μM (D). Wt Ad5 genome copy numbers were determined at 48 h post-infection with wt Ad5 by qPCR using a primer/probe set specific for the E1A gene present only on wt Ad5. Data represent the means ± SD of 3 independent transduction/infection experiments, each performed in triplicate. In each panel, data points for cells that were not treated with GCV are given as a reference. **p < 0.01, ***p < 0.001.