'Autoreplication' of the vector genome in recombinant adenoviral vectors with different E1 region deletions and transgenes

High transgene stabilities of 1 year and more have been reported in immunod eficient hosts after adenovirus-mediated gene transfer Transgene persistenc e of this duration could be due to inherently high stability of the episoma l viral vector DNA. An alternative explanation would be limited 'autoreplic ation' of transgenic vector DNA, just sufficient to counteract slow but con tinuous degradation within the host cells. Autoreplication could occur in t he absence of any production of infectious virus particles, based on residu al activity of the adenoviral DNA replication system only. To test this hyp othesis, a series of DNA metabolic labeling studies in non-permissive cells cultures transfected with different vectors was conducted. Due to extensiv e E1 region deletions none of the vectors was able to produce viral progeny in non-permissive cells. Vectors fell into two categories, however, with r espect to their autoreplication potential. Neosynthesis of vector DNA in no npermissive vector-transfected cells was readily detectable in 'type A,' bu t not in 'type B' vectors. In addition to their different transgene express ion cassettes, vector DNA sequencing showed a less extensive E1 deletion in type A (nucleotides 453-3333 of wild-type virus) as compared to type a vec tors (nucleotides 325-3523). Autoreplication was also associated with high transcriptional activity of several viral genes (E1B-14k, adenoviral DNA po lymerase, single-strand DNA-binding protein, E4-25k), in contrast to type B vectors. In addition to these 'wild-type' transcripts, 'irregular' recombi nant transcripts were detected in autoreplication vectors which contained t he transgenic cDNA in conjunction with adenoviral vector sequences. Exogeno us or cryptic promotors may (under certain conditions) enhance the transcri ptional activity of a vector in such a way that autoreplication occurs. Con ditions determining the level of transcriptional enhancement (extent of E1 deletion, type of promoter and transgene, etc) need to be further defined b efore rational design of adenovectors with high autoreplication capacity be comes possible. In summary, we have shown autoreplication to be a novel fea ture of certain E1-deleted adenovectors with likely relevance for their sta bility in vivo, but also with possibly adverse consequences for target cell function or vector immunogenicity. Full characterization of adenoviral vec tor systems should therefore include a description of their autoreplication capacity.