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  • Viral Transfer Technology
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Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo

Gene Therapy volume 7, pages 556–567 (2000)Cite this article

Abstract

Recombinant adenoviruses (Ad) are attractive vectors for gene transfer in vitro and in vivo. However, the widely used E1-deleted vectors as well as newer generation vectors contain viral sequences, including transcriptional elements for viral gene expression. These viral regulatory elements can interfere with heterologous promoters used to drive transgene expression and may impair tissue-specific or inducible transgene expression. This study demonstrates that the activity of a metal-inducible promoter is affected by Ad sequences both upstream and downstream of the transgene cassette in both orientations. Interference with expression from the heterologous promoter was particularly strong by viral regulatory elements located within Ad sequences nucleotides 1–341. This region is present in all recombinant Ad vectors, including helper-dependent vectors. An insulator element derived from the chicken γ-globin locus (HS-4) was employed to shield the inducible promoter from viral enhancers as tested after gene transfer with first-generation Ad vectors in vitro and in vivo. Optimal shielding was obtained when the transgene expression cassette was flanked on both sides by HS-4 elements, except for when the HS-4 element was placed in 3′→5′ orientation in front of the promoter. The insulators reduced basal expression to barely detectable levels in the non-induced stage, and allowed for induction factors of approximately 40 and approximately 230 in vitro and in vivo, respectively. Induction ratios from Ad vectors without insulators were approximately 40-fold lower in vitro and approximately 15-fold lower in vivo. This study proves the potential of insulators to improve inducible or tissue-specific gene expression from adenovirus vectors, which is important for studying gene functions as well as for gene therapy approaches. Furthermore, our data show that insulators exert enhancer-blocking effects in episomal DNA.

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Acknowledgements

We thank Zong-Yi Li and Greg Priestley for technical assistance. We are grateful to Cheryl Carlson, Dmitry Shayakhmetov, and David Russell for critical discussion. We thank David Emery for providing the HS-4 insulator element and Richard Palmiter for the MRE promoter fragment. This work was supported by the Cystic Fibrosis Foundation, and NIH grants R01 CA80192–01, R21 DK55590–01. DS is a recipient of a predoctoral DAAD fellowship.

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    D S Steinwaerder & A Lieber

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Steinwaerder, D., Lieber, A. Insulation from viral transcriptional regulatory elements improves inducible transgene expression from adenovirus vectors in vitro and in vivo. Gene Ther 7, 556–567 (2000). https://doi.org/10.1038/sj.gt.3301139

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