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Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication
Author(s) -
Krougliak Valeri A.,
Krougliak Natalia,
Eisensmith Randy C.
Publication year - 2000
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/1521-2254(2000)9999:9999<::aid-jgm150>3.0.co;2-#
Subject(s) - extrachromosomal dna , biology , transgene , vector (molecular biology) , genetic enhancement , plasmid , vectors in gene therapy , genome instability , virology , viral vector , dna replication , microbiology and biotechnology , gene , genetics , dna , recombinant dna , dna damage
Background A major limitation of adenovirus‐mediated gene therapy for metabolic and inherited diseases is the instability of transgene expression in vivo . This instability results, at least in part, from the inability of the vector genome to maintain the transgene through the replication or integration. In this study we evaluated the possibility of stabilization of an adenovirus‐delivered transgene by non‐adenovirus replicative elements. Methods We have developed a novel system for the maintenance of transgenes delivered by adenovirus vectors through extrachromosomal replication. In its initial configuration, this system combines the Epstein‐Barr virus (EBV) replicative elements, a tetracycline (Tc)‐inducible expression system, and the Cre‐lox recombination system in the context of a single E1/E3/E4‐deleted adenovirus vector. Induction of Cre expression initiates a Cre‐mediated recombination, resulting in the excision of a fragment of the vector genome and its circularization into an EBV‐based episome. Results In vitro studies have demonstrated that excision of the circular episome can occur in a cell‐free system as well as in cultured cells transfected with plasmid DNA or transduced by a virus vector carrying the episome‐excising cassette. PCR studies have shown that in proliferating, non‐permissive, cultured primate cells, the episome generated from the adenovirus vector is maintained much more stably than the genome of the parent vector. This episome was also able to replicate in mammalian cells. Conclusion Together these studies demonstrate the feasibility of this approach for the stabilization of transgenes delivered to dividing cells by adenovirus vectors. Copyright © 2000 John Wiley & Sons, Ltd.