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The adenovirus (Ad) genome encodes one or two non-coding small RNAs called virus-associated (VA)-RNAs, that are transcribed by polymerase III and support Ad replication. A replication-incompetent Ad vector, which is widely used in not only gene therapy studies, including clinical trials, but also basic researches as a gene delivery vehicle, as well as wild-type Ad (WT-Ad) express VA-RNAs, and VA-RNAs activate innate immunity, including the production of type I interferons. In addition, VA-RNAs perturb cellular microRNA (miRNA) expression profiles via competitive inhibition of key components involved in the miRNA maturation pathway.