Development and validation of a real-time PCR assay for the detection and quantitation of p53 recombinant adenovirus in clinical samples from patients treated with Ad5CMV-p53 (INGN 201).

The purpose of this study was to assess the usefulness of real-time PCR as a quantitative, highly reproducible, and sensitive method, for detecting and quantifying p53 recombinant adenovirus in biological samples from cancer patients receiving injections of Ad5CMV-p53. The dynamic range of this real-time PCR-based assay was wide (at least five orders of magnitude). Our assay used an internal positive control in the same PCR tube that is capable of detecting residual PCR inhibitors. Serial spiked samples in plasma with known quantities of Ad5CMV-p53 were evaluated. The minimum detection limit was 2pfu per PCR (∼50pfu per ml of plasma) and the quantification values were reproducible. A total of 2069 controls tested with 1780 plasma samples from 286 patients enrolled in gene therapy trials using Ad5CMV-p53 were investigated. Using calibrators to adjust the quantitation value, the results confirmed the good performance of the assay. In conclusion, the high sensitivity, simplicity and reproducibility of the real-time Ad5CMV-p53 assay, allowing screening of large numbers of samples, combined with its wide dynamic range, make this method particularly suitable for monitoring gene therapy trials.