Insulation from viral transcriptional regulatory elements enables improvement to hepatoma-specific gene expression from adenovirus vectors

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Abstract

We previously reported that the HS-4 insulator, derived from the chicken β-globin locus, was able to shield a downstream inducible promoter from viral enhancers or silencers present in the genome of adenovirus vectors. In this study, we constructed two recombinant adenoviruses (Ad) that express an alkaline phosphatase (AP) reporter gene driven by an α-fetoprotein (AFP) enhancer/promoter with and without HS-4 insulator (Ad.HS4.AFP-AP and Ad.AFP-AP). The insulated vector, Ad.HS4.AFP-AP, conferred significantly higher AP expression than Ad.AFP-AP in all AFP-producing hepatocellular carcinoma cell lines (HepG2, Hep3B, and HuH7) examined. AP expression from Ad.HS4.AFP-AP was specific to hepatoma cells and barely detectable in AFP-negative tumor cell lines and normal human cells, including human hepatocytes. Intravenous infusion of viral vectors into mice with liver metastasis derived from Hep3B hepatoma cells resulted in AP expression exclusively localized to tumor cells. The number of tumor cells with detectable AP expression was significantly higher in mice infused with Ad.HS4.AFP-AP than in mice that received the non-insulated vector. This study demonstrates that the HS-4 insulator in the context of an Ad vector can increase the activity of the AFP promoter, while maintaining its tumor-specificity in vitro and in vivo. Considering that the anti-tumor activity of oncolytic vectors often depends on the level of pro-apoptotic or suicide gene expression, insulators might be a useful tool to improve the efficacy and specificity of these vectors.

Section snippets

Materials and methods

Construction of adenovirus. All adenoviruses used were created in 293 cells by using pHVad2 [8] as the left-end shuttle plasmid with pBHG10 (Microbix, Toronto, Canada) as the right end. Briefly, the SV40 polyadenylation signal was removed from pRep4 (Invitrogen, Carlsbad, CA) by XhoI and SalI digestion and cloned into the SalI site pHVad2, generating pHV.SV40pA. We created pHV.AP.SV40pA by removing the AP gene from pLAPSN [9] by digestion with EcoRI and Bpu10I, blunting, and inserting it into

Results and discussion

Transcriptional targeting is a means to restrict gene expression from Ad vectors to a particular cell type [15]. In our studies, we used a modified version of the AFP promoter to achieve transgene expression specifically in HCC cells. The AFP promoter contains two enhancer elements, which have been previously demonstrated to be activated specifically in AFP-expressing HCC cell lines [16]. The promoter also includes six copies of a silencer element, responsible for silencing the promoter in

Acknowledgements

Xun Ye and Min Liang contributed equally to this work. We thank Kathrin Bernt for help with the in vitro transduction studies; A. Taketa and J. Sato for the modified version of the AFP promoter. This work was supported in part by national 863 Grant 2001AA217131 (LM) and NIH Grant R01 CA 80192 (AL).

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