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Journal of Virology, January 2004, p. 9-22, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.9-22.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Long-Term Transgene Expression in Proliferating Cells Mediated by Episomally Maintained High-Capacity Adenovirus Vectors

Florian Kreppel1,2 and Stefan Kochanek1,2*

Center for Molecular Medicine Cologne, University of Cologne, D-50931 Cologne,1 Division of Gene Therapy, University of Ulm, D-89081 Ulm, Germany2

Received 28 May 2003/ Accepted 19 September 2003

High-capacity "gutless" adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

* Corresponding author. Mailing address: Division of Gene Therapy, University of Ulm, Helmholtzstrasse 8/1, D-89081 Ulm, Germany. Phone: 49-731-50033601. Fax: 49-731-50033664. E-mail: stefan.kochanek{at}medizin.uni-ulm.de.

Journal of Virology, January 2004, p. 9-22, Vol. 78, No. 1
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.1.9-22.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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