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Mechanism by which calcium phosphate coprecipitation enhances adenovirus-mediated gene transfer

Gene Therapy volume 6, pages 1845–1850 (1999)Cite this article

Abstract

Delivery of a normal copy of CFTR cDNA to airway epithelia may provide a novel treatment for cystic fibrosis lung disease. Unfortunately, current vectors are inefficient because of limited binding to the apical surface of airway epithelia. We recently reported that incorporation of adenovirus in a calcium phosphate coprecipitate (Ad:CaPi) improves adenovirus-mediated gene transfer to airway epithelia in vitro and in vivo. To understand better how coprecipitation improves gene transfer, we tested the hypothesis that incorporation in a CaPi coprecipitate increases the binding of adenovirus to the apical surface of differentiated human airway epithelia. When a Cy3-labelled adenovirus was delivered in a coprecipitate, binding increased 54-fold as compared with adenovirus alone. Moreover, infection by Ad:CaPi was independent of fiber knob-CAR and penton base–integrin interactions. After binding to the cell surface, the virus must enter the cell in order to infect. We hypothesized that Ad:CaPi may stimulate fluid phase endocytosis, thereby facilitating entry. However, we found that neither adenovirus nor Ad:CaPi coprecipitates altered fluid phase endocytosis. Nevertheless, Ad:CaPi preferentially infected cells showing endocytosis. Thus, CaPi coprecipitation improves adenovirus-mediated gene transfer by coating the epithelial surface with a layer of virus which enters cells during the normal process of endocytosis.

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Acknowledgements

We thank Pary Weber, Phil Karp, Tom Moninger, Mike Seiler and Theresa Mayhew for excellent assistance. We especially appreciated the generous help of the late Dr Al Fasbender. We thank Dr Sam Wadsworth, Genzyme (Framingham, MA, USA) for the gift of Ad2/GFP and Ad2/βGal. We thank Dr Paul Freimuth for the gift of wild-type adenovirus, and the RAE mutant adenovirus. We thank Dr Jeff Bergelson for the gift of pcDNA1hCAR. We thank the University of Iowa Gene Transfer Vector Core (supported in part by the Roy J Carver Charitable Trust, the National Heart, Lung and Blood Institute, and the Cystic Fibrosis Foundation) for support. This work was supported by the National Heart, Lung and Blood Institute and the Cystic Fibrosis Foundation. MJW is an Investigator of the Howard Hughes Medical Institute.

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  1. Departments of Internal Medicine and Physiology and Biophysics, Howard Hughes Medical Institute, 500 EMRB, Program in Gene Therapy, University of Iowa College of Medicine, Iowa City, 52242, IA, USA

    R W Walters & M J Welsh

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Walters, R., Welsh, M. Mechanism by which calcium phosphate coprecipitation enhances adenovirus-mediated gene transfer. Gene Ther 6, 1845–1850 (1999). https://doi.org/10.1038/sj.gt.3301020

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