AN EFFICIENT AND FLEXIBLE SYSTEM FOR CONSTRUCTION OF ADENOVIRUS VECTORS WITH INSERTIONS OR DELETIONS IN EARLY REGION-1 AND REGION-3

Human adenoviruses (Ads) are attracting considerable attention because of their potential utility for gene transfer and gene therapy, for de velopment of live viral vectored vaccines, and for protein expression in mammalian cells. Engineering Ad vectors for these applications requ ires a variety of reagents in the form of Ads and bacterial plasmids c ontaining viral DNA sequences and requires different strategies for co nstruction of vectors for different purposes. To simplify Ad vector co nstruction and develop a procedure with maximum flexibility, efficienc y, and cloning capacity, we have developed a vector system based on us e of Ad5 DNA sequences cloned in bacterial plasmids. Expanded deletion s in early region 1 (3180 bp) and early region 3 (2690 or 3132 bp) can be combined in a single vector that should have a capacity for insert s of up to 8.3 kb, enough to accommodate the majority of cDNAs encodin g proteins with regulatory elements. Genes can be inserted into either early region 1 or 3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. To illustrate the flexibili ty of the system, we have introduced a wild-type early region 3 into t he vectors, and to illustrate the high capacity for inserts, we have i solated a vector with two genes totaling 7.8 kb.