QUANTITATIVE AND IN-VIVO ACTIVITY OF ADENOVIRAL-PRODUCING CELLS MADE BY COTRANSDUCTION OF A REPLICATION-DEFECTIVE ADENOVIRUS AND A REPLICATION-ENABLING PLASMID

Achieving limited recombinant viral replication may provide a means of amplifying viral-mediated gene transfer in vivo. We have previously s hown that cotransduction of an El-defective adenovirus with a plasmid containing the deleted El functions into prostate carcinoma cells resu lted in El-defective virus production by those cells. The studies desc ribed here have extended these findings to more firmly establish the c apacity of the trans complementation approach to achieve amplification of recombinant viral transgene expression. The recombinant virus used for all the studies was AdCMV-luc which contained a luciferase expres sion cassette; the replication-enabling plasmid, pE1, encoded the El f unctions deleted from AdCMV-luc. Quantitative in vitro studies with th e HeLa cell line showed that for each plaque forming unit of AdCMV-luc originally exposed to the cells, 0.54 x 10(3) new replication-defecti ve viruses were detected in supernatants and lysates over the followin g 3 days. Multiple cell lines were shown to support new virus producti on following cotransduction of AdCMV-luc and pE1. Small numbers of rep lication-competent viruses were detected in the lysates and supernatan ts from the cotransduced cells such that for every 10(5) replication-d efective viruses approximately two replication-competent viruses were produced. Tumor nodules produced by engrafting a mixture of AdCMV-luc/ pE1-cotransduced HeLa cells with uninfected HeLa cells yielded much hi gher levels of luciferase expression than control rumors containing mi xtures of cells infected with AdCMV-luc alone. In total, these results demonstrate new virus production by cells receiving a replication-def ective adenovirus and a replication-enabling plasmid are capable of am plifying recombinant viral transgene expression in vivo.