Improved production of adenovirus vectors expressing apoptotic transgenes

Abstract

Adenovirus vectors expressing gene products that can induce apoptosis have potential utility in gene therapy applications ranging from the treatment of proliferative diseases to transplantation. However, adenovirus vectors carrying proapoptotic gene products are difficult to produce, as the apoptotic environment is not conducive to adenovirus gene expression and replication. Production of AdFasL/G, an adenovirus vector that expresses high levels of Fas ligand, was severely reduced in the 293 packaging cell line. Increased yields of AdFasL/G were achieved by inclusion of peptide-based caspase inhibitors in the growth medium. However, use of these inhibitors for large-scale production would be difficult and expensive. A screen for gene products that increase the yield of AdFasL/G in 293 cells revealed that the poxvirus serpin CrmA and the adenovirus 14.7K product were able to increase virus yields significantly. Apoptosis induced by AdFasL/G was attenuated in 293CrmA cell lines and virus titers were increased dramatically. However, serial passage of AdFasL/G on 293CrmA cells resulted in the generation of replication-competent adenovirus. To resolve this problem, the CrmA gene was introduced into AE25 cells, an E1-complementing cell line that has limited sequence identity with the vectors. AdFasL/G titers were increased 100-fold on AE25CrmA cells relative to the AE25 cells and RCA contamination was not detectable. In addition, adenovirus vectors that express FADD, caspase 8, and Fas/APO1 were produced efficiently in AE25CrmA and 293CrmA.