RECOMBINANT ADENOVIRUSES WITH LARGE DELETIONS GENERATED BY CRE-MEDIATED EXCISION EXHIBIT DIFFERENT BIOLOGICAL PROPERTIES COMPARED WITH FIRST-GENERATION VECTORS IN-VITRO AND IN-VIVO

In vivo gene transfer of recombinant E1-deficient adenoviruses results in early and late viral gene expression that elicits a host immune re sponse, limiting the duration of transgene expression and the use of a denoviruses for gene therapy. The prokaryotic Cre-lox P recombination system was adapted to generate recombinant adenoviruses with extended deletions in the viral genome (referred to here as deleted viruses) in order to minimize expression of immunogenic and/or cytotoxic viral pr oteins. As an example, an adenovirus with a 25-kb deletion that lacked E1, E2, E3, and late gene expression with viral titers similar to tho se achieved with first-generation vectors and less than 0.5% contamina tion with E1-deficient virus was produced. Gene transfer was similar i n HeLa cells, mouse hepatoma cells, and primary mouse hepatocytes in v itro and in vivo as determined by measuring reporter gene expression a nd DNA transfer. However, transgene expression and deleted viral DNA c oncentrations were not stable and declined to undetectable levels much more rapidly than those found for first generation vectors. Intraveno us administration of deleted vectors in mice resulted in no hepatocell ular injury relative to that seen with first-generation vectors. The m echanism for stability of first-generation adenovirus vectors (E1a del eted) appeared to be linked in part to their ability to replicate in t ransduced cells in vivo and in vitro. Furthermore, the deleted vectors were stabilized in the presence of undeleted first-generation adenovi rus vectors. These results have important consequences for the develop ment of these and other nonintegrating vectors for gene therapy.