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  • Viral Transfer Technology
  • Published:

Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells

Abstract

Gene therapy of cancer requires high-level expression of therapeutic transgenes in the target cells. Poor gene transfer is an important limitation to adenovector-mediated cancer gene therapy. We investigated two fundamentally different approaches to improve transgene expression in poorly permissive cancer cells. First, overexpression of the adenovirus attachment receptor CAR to facilitate receptor-mediated adenovector (AdV) uptake into the target cells; second, co-infection of this vector together with traces of replication competent adenovirus (RCA) accidentally arising by back-recombination during large-scale vector preparation. Among eight gastrointestinal cancer cell lines, the colorectal cancer lines showed particularly poor vector-mediated transgene expression (down to 67-fold lower than in HeLa cells). Expression of the adenovirus receptors CAR, αvβ5- and αvβ3-integrin were highly variable between cell lines. AdV uptake was significantly associated with CAR levels on the cell surface, but not with those of the integrins. AdV-mediated CAR overexpression increased CAR density on the surface of all investigated tumor cells and led to enhancement of transgene expression by 1.8- to 6.7-fold. The other principle to enhance transgene expression was ‘trans-complementation’ of the therapeutic vector, ie induction of its replication within the target cells. Traces of RCA in a vector preparation, as well as purified RCA were found to provide sufficient E1-region transcripts to induce replication of the therapeutic vector genome. The number of adenovector-based transgene expression cassettes was greatly amplified by this principle, notably without any influence on the rate of vector entry. Co-infection of four colorectal cancer cell lines with marker vector plus RCA (at around 240:1 particle ratio) resulted in far stronger enhancement of transgene expression (up to 46-fold) as compared with CAR overexpression, even in cancers almost refractory to standard adenovector-mediated gene transfer. Whereas RCAs need to be strictly avoided in gene therapy of non-malignant diseases for safety reasons, the magnitude of helper virus-induced therapeutic transgene expression could possibly warrant application of this principle to overcome the resistance of highly malignant cancers against gene therapy.

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Acknowledgements

We thank Kerstin Hinze for assistance with the cell culture experiments. KH was supported by a grant from the Freie Universität Berlin and will incorporate part of the data presented here in her doctoral thesis. This work was supported by Deutsche Forschungsgemeinschaft through a Heisenberg Fellowship to WP (WP378/2–1 and 378/2–2), by a grant from the Centeon Pharmaceutical Company, and by the Cardiovascular Research Center at the University Hospital Benjamin Franklin, Freie Universität Berlin. This work was also supported by grants from the NIH (AI35667 and HL54734), and by an Established Investigator Award from the American Heart Association to JB.

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Fechner, H., Wang, X., Wang, H. et al. Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells. Gene Ther 7, 1954–1968 (2000). https://doi.org/10.1038/sj.gt.3301321

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