PERSISTENCE OF RECOMBINANT ADENOVIRUS IN-VIVO IS NOT DEPENDENT ON VECTOR DNA-REPLICATION

Recombinant adenovirus vectors represent an efficient means of transfe rring genes into many different organs, The first-generation E1 delete d vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver, The mechan ism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-spec ific methylation strategy to monitor the replicative fate of E1-delete d adenovirus vectors in vitro and in vivo. Methylation-marked adenovir us vectors were produced by the addition of a methyl group onto the N- 6 position of the adenine base of XhoI sites, CTCGAG, by propagation o f vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltr ansferase, The methylation did not affect vector production or transge ne expression but did prevent cleavage by XhoI. Loss of methylation th rough viral replication restores XhoI cleavage and was observed by Sou thern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures, In contrast, following liver-directed gene transfer of methy lated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show th at stabilization of the vector within the target tissue prior to clear ance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persiste nt molecules. This information will be useful for the design of optima l adenovirus vectors and perhaps nonviral episomal vectors for clinica l gene therapy.