A new kind of versatile adenoviral vector (AdV) has been constructed, one that 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 was made possible by deletion of the Ad protease gene (PS), which is essential for many steps of the Ad life cycle. The PS-deleted virus can be propagated in 293-derived cell lines engineered to express PS. In these new complementing cells, the PS gene was expressed from a tetracycline-inducible promoter in a dicistronic vector coexpressing the green fluorescent protein (GFP). When induced, the best 293-PS stable clones produced the PS in amounts greater than the level reached after Ad infection. Biological activity was first demonstrated by the ability of 293-PS cells to support the replication of Ad2ts1, a mutant expressing a functionally defective PS. While overexpression of the Ad PS slightly affected cell growth, moderate expression at levels sufficient to fully complement Ad2ts1 was well tolerated in 293 cells. Two PS-deleted mutants, deleted or not deleted for E1/E3, were then generated and characterized. Despite their complete loss of infectivity after a single round of replication in permissive cells, the PS-deleted mutants produced as much viral protein as wildtype Ad. These new vectors should thus be both safer and more efficient for applications in which enhancement of transgene expression is desirable, as in the case of vaccination, in situ therapy for tumors, protein production, or the large-scale production of other viral vectors such as adeno-associated virus (AAV).

Adenoviruses (Ad) deleted in the protease (PS)

As a first step in the evaluation of the safety of replication-defective adenoviruses used as gene transfer vectors, the dissemination of wild-type (wt) adenovirus (Ad) type 5, Ad-d1327 (deleted for the E3 gene) and Adgp50 (deleted for E3 and E1A and therefore replication defective) was studied in mice and cotton rats. Of each virus, 108 p.f.u, was inoculated, either by the intranasal or the intramuscular route, and virus isolation was undertaken after sacrifice of the animals 3 or 31 days after inoculation. E3 deletion had no significant effect on viral spread, whereas E1A deletion reduced it significantly. After intramuscular inoculation of the E3-/E1A-virus, it could be isolated only from the local lymph node. Intranasal inoculation led to a wider distribution including lungs, liver, kidneys and lymph nodes. The pattern of dissemination of the E3 -/E1A-virus was the same in mice and cotton rats, providing indirect evidence of the lack of replication of this virus in this species permissive for the wt virus. However, in the case of infection with a wt strain in E3-/E1A-adenovirus-inoculated recipients, both viruses were found in lymph nodes. In this situation the risk of phenotypic complementation of the E1A gene remains to be studied further.Adenovirus vectors are being used more and more widely for gene transfer, especially in non-dividing cells (Stratford-Perricaudet et al

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