title: Long-term transgene expression in proliferating cells mediated by episomally maintained high-capacity adenovirus vectors.
abstract: 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.
authors.affiliation: Center for Molecular Medicine Cologne, University of Cologne, D-50931 Cologne, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Kreppel
authors.forename: Stefan
authors.initials: S
authors.lastname: Kochanek
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D002455:Cell Division; D045744:Cell Line, Tumor; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D034882:Lamins / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D018741:Replication Origin / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes / Q000502:physiology*; D014779:Virus Replication
chemical_list: D019309:Epstein-Barr Virus Nuclear Antigens; D034882:Lamins; C417029:EBV-encoded nuclear antigen 1