Medicina molecular y genética

Quantitative Analysis of Virus-associated RNAI Expression following Transduction with a Replication-incompetent Adenovirus Vector In Vitro and In Vivo

Abstract

Keisaku Wakabayashi, Mitsuhiro Machitani, Kahori Shimizu, Masashi Tachibana, Fuminori Sakurai and Hiroyuki Mizuguchi

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. As previously reported, 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. Although these characteristics of VA-RNAs might negatively affect the application of Ad vectors, VA-RNA expression profiles following transduction with an Ad vector have been not fully examined. In this study, we quantitatively analyzed the expression profiles of VA-RNAI, which is a major species of VA-RNAs, following transduction with Ad vectors in vitro and in vivo using real-time RT-PCR. The VA-RNAI expression levels in the cells transduced with a conventional Ad vector expressing luciferase (Ad-CAL2) at a multiplicity of infection (MOI) of 100 were approximately 2000- to 3000-fold lower than those infected with WT-Ad at the same MOI at 48 h after treatment. The expression levels of VA-RNAI in the mouse liver following administration with Ad-CAL2 were approximately 600-fold lower than those following administration with WT-Ad at 48 h post-administration. miRNA-mediated suppression of leaky expression of the Ad E4 genes resulted in about five-fold reduction in the VA-RNAI copy numbers in the liver following systemic administration in mice. These data provide informative clues for the development of novel safer Ad vectors.