Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector

Replication-deficient adenoviral vectors have been developed for the delive ry of DNA sequences encoding a variety of proteins intended for the managem ent of disease through gene therapy. One concern is the occurrence of repli cation-competent adenovirus (RCA) in the population of replication-deficien t adenoviral vectors as a result of recombination or contamination, To addr ess this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivit y of RCA, rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppresser gene p53 f or treatment of various cancers. Multiple preparations of the replication-d eficient adenoviral vector rAd/p53 were tested for the presence of RCA, emp loying a sensitive biological assay. Single plaques from RCA-positive prepa rations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complet e El sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host a denoviral sequence. Results from viral infectivity analysis by flow cytomet ry demonstrate no substantial difference in infectivity of RCA, rAd/p53, an d wild-type adenovirus type 5 in 293 cells.