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Journal of Virology, February 2002, p. 1600-1609, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1600-1609.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Variables Affecting In Vivo Performance of High-Capacity Adenovirus Vectors

Gudrun Schiedner,1* Sabine Hertel,1 Marion Johnston,1 Volker Biermann,1 Volker Dries,2 and Stefan Kochanek1

Center for Molecular Medicine (ZMMK),1 Institute for Pathology, University of Cologne, D-50931 Cologne, Germany2

Received 2 July 2001/ Accepted 15 October 2001

In high-capacity adenovirus (HC-Ad) vectors the size and/or composition of the vector genome influences vector stability during production and the expression profile following gene transfer. Typically, an HC-Ad vector will contain both a gene or an expression cassette and stuffer DNA that is required to balance the final vector genome to a size of between 27 and 36 kb. To gain an improved understanding of factors that may influence gene expression from HC-Ad vectors, we have generated a series of vectors that carry different combinations of human alpha-1 antitrypsin (hAAT) expression constructs and stuffer DNAs. Expression in vitro did not predict in vivo performance: all vectors expressed hAAT at similar levels when tested in cell culture. Hepatic expression was evaluated following in vivo gene transfer in C57BL/6J mice. hAAT levels obtained from genomic DNA were significantly higher than levels achieved with small cDNA expression cassettes. Expression was independent of the orientation and only marginally influenced by the location of the expression cassette within the vector genome. The use of lambda stuffer DNA resulted in low-level but stable expression for at least 3 months when higher doses were applied. A potential matrix attachment region element was identified within the hAAT gene and caused a 10-fold increase in expression when introduced in an HC-Ad vector genome carrying a phosphoglycerate kinase (pgk) hAAT cDNA construct. We also illustrate the influence of the promoter on anti-hAAT antibody formation in C57BL/6J mice: a human cytomegalovirus but not a pgk promoter resulted in an anti-hAAT antibody response. Thus, the overall design of HC-Ad vectors may significantly influence amounts and duration of gene expression at different levels.

* Corresponding author. Mailing address: Center for Molecular Medicine (ZMMK), University of Cologne, Kerpenerstr. 34, D-50931 Cologne, Germany. Phone: 49 221 478 3536. Fax: 49 221 478 3510. E-mail: Gudrun.Schiedner{at}medizin.uni-koeln.de.

Journal of Virology, February 2002, p. 1600-1609, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1600-1609.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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Copyright © 2002 by the American Society for Microbiology. All rights reserved.