Adenovirus-Delivered Antisense RNA and shRNA Exhibit Different Silencing Efficiencies for the Endogenous Transforming Growth Factor- β (TGF- β ) Type II Receptor

Abstract

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-β type II receptor (TGFβRII). We used a dicistronic reporter, consisting of the coding sequences for TGFβRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFβRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFβRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFβRII. In contrast, the shRNAs were able to silence endogenous TGFβRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFβRII/GFP reporter reduced endogenous TGFβRII protein expression by 70% in A549 cells and reduced TGFβ signaling by >80% in HeLa cells.