Gutted adenoviral vector growth using E1/E2b/E3‐deleted helper viruses

Abstract

Background Helper-dependent, or gutted, adenoviruses (Ad) lack viral coding sequences, resulting in reduced immunotoxicity compared with conventional Ad vectors. Gutted Ad growth requires a conventional Ad to supply replication and packaging functions in trans. Methods that allow high-titer growth of gutted vectors while reducing helper contamination, and which use safer helper viruses, will facilitate the use of gutted Ad vectors in vivo. Methods Replication-defective helper viruses were generated that are deleted for Ad E1, E2b and E3 genes, but which contain loxP sites flanking the packaging signal. Complementing Ad packaging cell lines (C7-cre cells) were also generated by transfecting 293 cells with the Ad E2b genes encoding DNA polymerase and pre-terminal protein, and with a cre-recombinase plasmid. Results We show that C7-cre cells allow efficient production of gutted Ad using ΔE1 + ΔE2b + ΔE3 helper viruses whose growth can be limited by cre-loxP-mediated excision of the packaging signal. Gutted Ad vectors carrying ∼28 kb cassettes expressing full-length dystrophin were prepared at high titers, similar to those obtained with E2b+ helpers, with a resulting helper contamination of <1%. Conclusions These new packaging cell lines and helper viruses offer several significant advantages for gutted Ad vector production. They allow gutted virus amplification using a reduced number of passages, which should reduce the chances of selecting rearranged products. Furthermore, the residual helper contamination in gutted vector preparations should be less able to elicit immunological reactions upon delivery to tissues, since E2b-deleted vectors display a profound reduction in viral gene expression. Copyright © 2002 John Wiley & Sons, Ltd.