A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-competent virus

Production of E1-deleted adenovirus vectors for gene therapy has been plagued by the emergence of replication-competent adenovirus. A number of investigators have minimized homologous sequences between the vector and transfected E1 DNA in an attempt to avoid replication-competent adenovirus. We describe a HeLa-based cell line called GH329 that stably expresses the E1 locus from a promoter derived from the phosphoglycerate kinase gene. Overlap sequences with a standard E1-deleted vector that retains a full pIX transcriptional unit have been eliminated at the 5' end and minimized at the 3' end. The GH329 cell line plaques and produces E1-deleted adenovirus as well as 293 cells. Replication-competent virus has emerged after 5 passages of vector on 293 cells but was not detected after 20 passages on GH329 cells.