Open AccessLong-Term Transgene Expression in Proliferating Cells Mediated by Episomally Maintained High-Capacity Adenovirus Vectors
Author(s) -
Florian Kreppel,
Stefan Kochanek
Publication year - 2004
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.78.1.9-22.2004
Subject(s) - biology , transgene , transduction (biophysics) , cre recombinase , genome , microbiology and biotechnology , recombinase , viral replication , scaffold/matrix attachment region , dna replication , gene , cell culture , virology , gene expression , genetics , genetically modified mouse , biochemistry , recombination , chromatin remodeling
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.