137. pFex: A Novel Fiber Exchange System for the Creation of Modified-Fiber Gene Therapy Vectors

Abstract

The application of conditionally replicating adenoviruses to cancer gene therapy has shown much promise. However, it has become apparent that these vectors must be more specifically re-targeted to increase therapeutic effect. Although the number of re-targeted vectors is growing, the total number is small, mostly due to cloning difficulties associated with the large viral genome. Here, we have developed a unidirectional shuttle exchange system, called pFex, where one can easily transfer modified fiber genes from a smaller shuttle vector directly into the fiber portion of the Adenovirus genome. The adenoviral acceptor vector, termed pFex, was derived from the AdEasy1 vector where the entire fiber gene has been replaced by the sucrose lethal gene, SacB, and two flanking non-compatible half-mutant lox sites. Several smaller shuttle vectors contain the modified fiber gene, which is also flanked by two non-compatible half mutant lox sites. In the presence of Cre recombinase, the modified fiber gene is shuttled into pFex, replacing SacB. The resulting lox sites are fully mutated and no longer recognized by Cre recombinase, producing a unidirectional exchange. The combination of several selection markers allows one to exchange fiber either before or after the traditional RecA mediated E1 region recombination. The pFex system is highly efficient and has been used to produce eight recombinant viral vectors in less than two weeks. This exchange system should dramatically increase the output of re-targeting adenoviral vectors.