Improving adenovirus vector-mediated RNAi efficiency by lacking the expression of virus-associated RNAs.
Several studies have reported that short hairpin RNA (shRNA)-mediated RNA interference (RNAi) was competitively inhibited by the expression of adenovirus (Ad)-encoded small RNAs (VA-RNAs), which are expressed from a replication-incompetent Ad vector, as well as a wild-type Ad; however, it remained to be clarified whether an shRNA-expressing Ad vector-mediated knockdown was inhibited by VA-RNAs transcribed from the same Ad vector genome. In this study, we demonstrated that a lack of VA-RNA expression from the Ad vector leads to an increase in knockdown efficiencies of Ad vector-mediated RNAi. In the cells transduced with a first-generation Ad vector (FG-Ad) expressing shRNA (FG-Ad-shRNA), the copy numbers of shRNA and VA-RNAs incorporated into the RNA-induced silencing complex (RISC) was comparable. In contrast, higher amounts of shRNA were found in the RISC when the cells were transduced with an shRNA-expressing helper-dependent Ad (HD-Ad) vector, in which all viral genes, including VA-RNAs, were deleted (HD-Ad-shRNA), compared with FG-Ad-shRNA. HD-Ad vectors expressing shRNA against luciferase and p53 showed 7.4% and 37.3% increases in the knockdown efficiencies compared to the corresponding FG-Ad-shRNA, respectively, following in vitro transduction. Furthermore, higher levels of knockdown efficiencies were also found by the transduction with shRNA-expressing Ad vectors lacking VA-RNA expression (AdΔVR-shRNA) than by transduction with FG-Ad-shRNA. These results indicate that VA-RNAs expressed from an Ad vector inhibit knockdown by the shRNA-expressing Ad vector and that HD-Ad-shRNA and AdΔVR-shRNA are a powerful framework for shRNA-mediated knockdown.
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
M
M
Machitani
F
F
Sakurai
K
K
Katayama
M
M
Tachibana
T
T
Suzuki
H
H
Matsui
T
T
Yamaguchi
H
H
Mizuguchi
D000256:Adenoviridae / Q000235:genetics*; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014773:Virology / Q000379:methods
D034741:RNA, Small Interfering; D012367:RNA, Viral
Adenovirus vector; Helper-dependent adenovirus vector; RISC; RNAi; VA-RNA