Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication

Background A major limitation of adenovirus-mediated gene therapy for metab olic and inherited diseases is the instability of transgene expression in v ivo. This instability results, at least in part, from the inability of the vector genome to maintain the transgene through replication or integration. In this study we evaluated the possibility of stabilization of an adenovir us-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 it s 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-delet ed 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 ep isome can occur in a cell-free system as well as in cultured cells transfec ted with plasmid DNA or transduced by a virus vector carrying the episome-e xcising cassette. PCR studies have shown that in proliferating, nonpermissi ve, 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 appro ach for the stabilization of transgenes delivered to dividing cells by aden ovirus vectors. Copyright (C) 2000 John Wiley & Sons, Ltd.