Oncolytic adenovirus-mediated expression of micro RNA-based short-hairpin RNA under the control of RNA polymerase II promoter induces cancer-specific gene silencing

Abstract

Oncolytic adenovirus (Ad) is a particularly promising vector for cancer gene therapy due to it selectively killing tumor cells while eliciting few side effects. The expression of the therapeutic gene by oncolytic Ad significantly enhances the efficacy of the virus, but overexpression of these genes may lead to adverse side effects. In this study, we tried to minimize the side effects mediated by short hairpin RNA (shRNA) overexpression by controlling the shRNA expression with cancer-specific polymerase (Pol) Ⅱ promoter in a micro RNA-based shRNA expression system (prishRNA). Several different expression constructs were generated to optimize the silencing effect of the prishRNA system with variable loop length, stem length, gap length, terminal signal sequences, and a promoter controlling the expression of shRNA. shRNA constructs with 19 nucleotide (nt) loop show more potent target gene silencing effect than the 9 nt loop and constitutive Pol Ⅱ promoter (such as CMV) mediated gene expression of prishRNA was comparable to those controlled by Pol Ⅲ (such as U6) promoter. prishRNA mediated gene silencing effect was highest when there was a 53 nt gap between the promoter and the hairpin structure while U3B termination sequences showed a highest silencing effect among various termination sequences examined in this study. Importantly, prishRNA under the control of tumor-specific promoters, such as Survivin or modified sex determining region Y-box 2 (SOX2) promoter, induced cancer cell-specific silencing of the target gene, whereas standard Pol Ⅲ-based shRNA expression and prishRNA under the control of constitutive CMV promoter failed to show any cancer specificity. The modified SOX2 promoter was chosen to drive prishRNA targeting hepatocyte growth factor receptor (HGFR; c-Met) in an oncolytic Ad to overcome gefitinib resistance in lung cancer cells due to modified SOX2 promoter showing higher transcriptional activity than Survivin promoter in various cancer cells. Oncolytic Ad mediated cancer-specific silencing of c-Met has sensitized gefitinib-resistant lung cancer cells, leading to synergistic anticancer effect when the oncolytic Ad was used in conjunction with gefitinib.