Characterization of Replication-Competent Adenovirus Isolates from Large-Scale Production of a Recombinant Adenoviral Vector

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Abstract
Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occur- rence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the fre- quency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to de- liver the tumor suppresser gene p53 for treatment of various cancers. Multiple preparations of the replica- tion-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biologi- cal assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA sug- gests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no sub- stantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.