Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Detection Assay for Replication-Competent Adenovirus by Concentration Passage and Real-Time qPCR

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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 (Louis et al. 1997) 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 result in 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 (Fallaux et al. 1998). Nevertheless, 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 (NIH 2001). Historically, this has been done by plating the vector stock on a cell line that does not contain the adenovirus E1 gene and then visually scoring the RCA cytopathic effect (CPE) (Hehir et al. 1996). The sensitivity of the assay is established by spiking known amounts of an RCA surrogate (e.g., wild-type human adenovirus type 5 [HuAd5] for vectors based on this serotype) into the vector stock and assaying the spiked samples alongside the test vector. Reliance on the detection of CPE results in low sensitivity and is generally only sufficient to screen for relatively high amounts of RCAs. More recently, qPCR directed at the adenovirus E1 gene has been used to enhance sensitivity and also shorten the assay time (Schalk et al. 2007). The sensitivity of the assay can also be affected by a phenomenon known as interference, whereby a low level of RCAs is outcompeted by large excesses of vector for occupancy of available cell-surface viral receptors and subsequent replication pathways. An excess of vector can also lead to toxicity and cell death. Thus, the initial ratio of virus to cells (or MOI) must be kept to levels at which toxicity and interference are minimized but are still practical in terms of the tissue culture burden incurred.

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