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AdEasy-based cloning system to generate tropism expanded replicating adenoviruses expressing transgenes late in the viral life cycle

Gene Therapy volume 12, pages 1347–1352 (2005)Cite this article

Abstract

Replicating adenoviral vectors (RAds) hold great promise for the treatment of cancer. Significant therapeutic effects of these vectors do not only rely on tumor targeting but also on efficient release of viral progeny from host cells. Cytotoxic genes expressed late in the adenoviral life cycle can significantly enhance viral release and spreading. Therefore, an adenoviral cloning system that allows easy integration of established tumor targeting techniques together with late expression of transgenes can be a valuable tool for the development of RAds. We expanded the features of the widely used AdEasy adenoviral cloning system toward the production of tropism modified replicating adenoviral vectors that express transgenes late in the viral life cycle. Three vectors (pIRES, pFIBER and pAdEasy-Sce) that facilitate easy manipulation of the adenoviral fiber region were established. Unique BstBI and I-Sce-1 restriction sites facilitate the introduction of retargeting peptides in the fiber HI-loop and of genes of interest in the fiber transcription unit. We validated the system by constructing an E1-positive adenovirus with an RGD motif in the fiber HI-loop and green fluorescent protein (GFP) expressed from the fiber transcription unit (AdΔ24Fiber-rgd-GFP). Additionally, assessment of E1-negative replication-deficient vectors confirmed strict dependence upon E1 expression for the expression of transgenes inserted into the fiber transcription unit. This flexible cloning system allows for straightforward construction of tropism expanded replicating adenoviral vectors that express transgenes late in the adenoviral life cycle.

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Acknowledgements

We are grateful to D Markusic for critical reading of the manuscript and MA van Geer and JEM Dekker for providing Ad5 Fiber knob. Dr H Mizuguchi (Division of Cellular & Gene Therapy Products, National Institute of Health Sciences, Tokyo, Japan), Dr VW van Beusechem (Department of Medical Oncology, VU University Medical Center, Amsterdam, the Netherlands) AQ Bakker (Department of Immunology, the Netherlands Cancer Institute, Amsterdam, the Netherlands), Dr J Seppen (AMC Liver Center, AMC, Amsterdam, the Netherlands) and Dr AG Jochemsen (Department Molecular and Cell Biology, Leiden University Medical Center, Amsterdam, the Netherlands) are acknowledged for generously providing us with respectively pAdHM15, pShuttleΔ24, pBSK-iresGFP, pMD.G and M37. M Lie-A-Ling was supported by a grant from the Netherlands Organization for Scientific Research (NWO-MW).

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  1. AMC Liver Center, Academic Medical Center/University of Amsterdam, The Netherlands

    M Lie-A-Ling, C T Bakker, J G Wesseling & P J Bosma

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  1. M Lie-A-Ling
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  2. C T Bakker
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  3. J G Wesseling
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Lie-A-Ling, M., Bakker, C., Wesseling, J. et al. AdEasy-based cloning system to generate tropism expanded replicating adenoviruses expressing transgenes late in the viral life cycle. Gene Ther 12, 1347–1352 (2005). https://doi.org/10.1038/sj.gt.3302546

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