Recombinant adenoviral vectors expressing Ad5-directed amiRNAs were amplified in T-REx-293 cells. All other adenoviral vectors and wt Ad5 (ATCC VR-5) were amplified in HEK 293 cells. Titers of infectious adenoviruses expressing amiRNAs were determined on T-REx-293 cells by 50% tissue culture infective dose (TCID50) assays. Titers of wt Ad5 present in mixed virus suspensions containing both wt and recombinant virus as obtained in combined transduction/infection experiments were determined on A549 cells using

the same method. All other TCID50 assays were performed with HEK 293 cells. The vectors employed in dual-luciferase assays for the screening of Ad5-directed amiRNAs have been described elsewhere (Kneidinger et al., 2012). The dual-luciferase target vector used for the MK-2206 clinical trial determination of Renilla luciferase gene silencing in Ad5-infected cells was constructed as follows: a part of the modified coding region of the firefly (Photinus pyralis) luciferase open reading frame (ORF) representing the target sequence for the corresponding amiRNA was amplified Screening Library by PCR with primers Fluc-f2 (5′-ATAAGGCTATCTCGAGATACGCCCTGGTTCC-3′) and Fluc-r2 (5′-AATGTCGTTCGCGGCCGCAACTGCAACTCCGAT-3′) from vector pGL3 (Promega, Mannheim, Germany). This fragment was restricted with XhoI and NotI and inserted into the corresponding sites located

within the 3′UTR of the Renilla luciferase gene present on plasmid psiCHECK-2 (Promega, Mannheim, Germany). From the resulting vector (psiCHECK-FLuc2), a BglII-BamHI fragment comprising both the firefly and Renilla luciferase expression cassettes was transferred into pENTR4 (Life Technologies Austria, Vienna, Austria) that had been restricted with XmnI and EcoRV. From the resulting vector (pENTR-Luc), the entire expression GABA Receptor cassette was eventually moved into the deleted E1 region of the adenoviral vector pAd/PL-DEST (Life Technologies Austria, Vienna, Austria) giving rise to vector Ad-Luc-as ( Fig. 1). This final transfer was mediated

by employing Life Technologies’ Gateway technology, i.e., by site-specific recombination between sequences flanking the expression cassette on pENTR-Luc and the corresponding sequences located on the adenoviral vector. The recombination reaction was performed according to the instructions of the manufacturer (Life Technologies Austria, Vienna, Austria). The adenoviral vector expressing the amiRNA directed against the target sequence present in the 3′UTR of the Renilla gene on Ad-Luc-as was constructed in a similar way by transferring the enhanced green fluorescence protein (EGFP)/amiRNA expression cassette of plasmid pcDNA6.2-GW/EmGFP-miR-luc (Life Technologies Austria, Vienna, Austria) into pAd/CMV/V5-DEST™ via site-specific recombination as before.