CONSTRUCTION OF ADENOVIRUS VECTORS THROUGH CRE-LOX RECOMBINATION

Two barriers prevent adenovirus-based vectors from having wide applica tion One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here He descri be uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simple method for constructing E1-substituted adenoviru ses. Second, we demonstrate a method to construct adenovirus vectors c arrying recombinant genes in place of all of the viral genes, so-calle d gutless adenovirus vectors. The pivotal feature in each method is th e use of a negatively selected adenovirus named Psi 5. We engineered a cis-acting selection into Psi 5 by flanking its packaging site with l oxP sites. When Psi 5 was grown in cells making a high level of Cre re combinase, the packaging site was deleted by recombination and the yie ld of Psi 5 was reduced to 5% of the wild-type level. To make a new E1 -substituted virus, we used Psi 5 as a donor virus and recombined it w ith a shuttle vector via a loxP site. The resulting recombinant virus has a single loxP site next to the packaging site and therefore outgro ws Psi 5 in the presence of Cre recombinase. To make a gutless virus, we used Psi 5 as a helper virus. The only viral sequences included in the gutless vector are those needed in cis for its replication and pac kaging. We found that a loxP site next to the packaging site of the gu tless virus was necessary to neutralize homologous recombination betwe en Psi 5 and the gutless viruses within their packaging domains.