Replication-defective adenovirus vectors are deficient in early viral gene E1 and are generally grown in cell lines such as HEK-293, which contain and express an integrated copy of E1 for the purpose of complementing the defective adenoviral genome. Although the host cell E1 gene is required for complementation, its presence can also be problematic because recombination between vector and host cell genomes may lead to the reacquisition of E1 by the vector and the formation of replication-competent adenoviruses (RCAs).
This problem has long been recognized, and approaches, such as the minimization of the sequence homology between the adenovirus genes inserted into the host chromosome DNA and viral sequences remaining in the 5' end of the vector genome, have been taken to reduce recombination and RCA formation. However, in the clinical setting, it is important to ensure that the vector dose given to patients contains no more than one RCA infectious particle per patient dose. Emergence of the RCA in the El -deleted replication-defective rAd vector products is a concern for both regulatory agencies and manufacturers.
QVirusTM Platform has developed several methods to determine RCA levels in purified adenovirus batches, including direct PCR analysis for the presence of El sequences and analysis of cytopathic effect (CPE) caused by RCA and infectivity-linked PCR for infectious RCA.
