Protease-deleted adenovirus vectors and complementing cell lines: Potential applications of single-round replication mutants for vaccination and genetherapy

A new kind of versatile adenoviral vector (AdV) has been constructed, one t hat is completely replication disabled in the absence of Ad-E1 proteins but is capable of a single round of replication when Ad-E1 is present. This wa s made possible by deletion of the Ad protease gene (PS), which is essentia l for many steps of the Ad life cycle. The PS-deleted virus can be propagat ed in 293-derived cell lines engineered to express PS. In these new complem enting cells, the PS gene was expressed from a tetracycline-inducible promo ter in a dicistronic vector coexpressing the green fluorescent protein (GFP ). When induced, the best 293-PS stable clones produced the PS in amounts g reater than the level reached after Ad infection. Biological activity was f irst demonstrated by the ability of 293-PS cells to support the replication of Ad2ts1, a mutant expressing a functionally defective PS. While overexpr ession of the Ad PS slightly affected cell growth, moderate expression at l evels sufficient to fully complement Ad2ts1 was well tolerated in 293 cells . Two PS-deleted mutants, deleted or not deleted for E1/E3, were then gener ated and characterized. Despite their complete loss of infectivity after a single round of replication in permissive cells, the PS-deleted mutants pro duced as much viral protein as wildtype Ad. These new vectors should thus b e both safer and more efficient for applications in which enhancement of tr ansgene expression is desirable, as in the case of vaccination, in situ the rapy for tumors, protein production, or the large-scale production of other viral vectors such as adeno-associated virus (AAV).