New Helper Cells and Matched Early Region 1-Deleted Adenovirus Vectors Prevent Generation of Replication-Competent Adenoviruses

Abstract

The presence of replication-competent adenoviruses (RCAs) in batches of replication-defective adenovirus (Ad) vectors is a major problem for the application of these vectors in gene therapy. RCAs are generated by recombination between sequences in the Ad vector and homologous Ad sequences in the helper cells, resulting in the acquisition by the vector of early region 1. To prevent the formation of RCAs, we have developed helper cell lines, which we named PER, and matched Ad vectors that do not have sequence overlap. PER cells contain the Ad serotype 5 (Ad5) E1A- and E1B-encoding sequences (Ad5 nucleotides 459–3510) under the control of the human phosphoglycerate kinase (PGK) promoter. We demonstrate that PER cells synthesize high levels of the Ad5 E1A and E1B proteins. The yields from PER cells of E1-deleted Ads are similar to those obtained from earlier helper cells, such as 911 and 293 cells. Propagation of matched Ad vectors, which lack Ad5 nucleotides 459–3510, in one of the PER clones, PER.C6, does not result in the generation of RCAs, in contrast to propagation in 293 cells. We conclude that the combination of PER.C6 cells and nonoverlapping El-deleted adenoviral vectors eliminates the problem of RCA generation by homologous recombination, and allows cost-effective production of safe, clinical-grade batches of recombinant Ad vectors. The authors describe a new helper cell line and matched, nonhomologous adenoviral vectors. The absence of homologous sequences prevents the formation of replication-competent adenoviruses as a result of homologous recombination between the vector and the adenovirus sequences in the helper cells. With these new cells, the yields of E1-deleted adenoviral vectors are similar to the yields obtained with the conventional 293 and 911 cell lines. In none of nine batches of adenoviral vectors produced in these new cells could replication-dependent adenovirus be detected. The authors conclude that this new cell–vector combination allows cost-effective production of safe, clinical-grade batches of recombinant adenovirus vectors.