Packaging cell line DNA contamination of vector supernatants: implication for laboratory and clinical research.

Investigators conducting retroviral gene therapy trials are required to monitor for the presence of replication-competent retrovirus (RCR). The required testing utilizes a combination of biologic assays and molecular tests using PCR. In the course of a human clinical gene therapy trial, we detected 4070A viral envelope sequences in CD34(+) peripheral blood stem cells 2 days after transduction using a PCR-based assay, suggesting the presence of RCR. The supernatant and producer cells used for vector generation had been negative in extensive screening using the extended S(+)/L(-) assay. The presence of a replication-competent virus was subsequently excluded by a combination of biologic and PCR analyses. The source of the 4070A viral envelope sequences was determined to be packaging cell line DNA in the vector supernatant. The analysis of a variety of vector supernatants by quantitative real-time PCR revealed 4070A envelope DNA sequences from the packaging cell line in concentrations equivalent to approximately 50-500 focus-forming units per milliliter of wild-type 4070A virus. When PCR was performed after reverse transcriptase treatment of supernatant (i.e., assessing both RNA and DNA content), 4070A envelope sequence concentrations ranged from 10(2) to 3.5 x 10(3) focus-forming units per milliliter of wild-type 4070A virus. Our data indicate that PCR should not be used to analyze transduced cells for RCR within the first 2 weeks of vector exposure. Furthermore, investigators using PCR to analyze transduction efficiency shortly after vector exposure may experience false-positive findings.

Rapid titer determination using quantitative real-time PCR.

Quantitative real-time PCR was utilized to evaluate retroviral vector titer. RNA was prepared from vector supernatant and run in a one-step RT-PCR reaction combining reverse transcription (RT) and amplification in one tube. Sample analysis was performed in the ABI Prism 7700 Sequence Detector. PCR was quantitative over a range of 101 to 6 x 105 vector particles per reaction (2 x 102 to 1 x 107 vector particles per millilites of supernatant) and closely corre- lated with biologic titers performed on the test material. The 96-well capacity of the machine and 2 h of running time permit titer determinations within 8 h, facilitating the processing of large sample numbers while greatly decreasing technician time. Real-time PCR improves titer quantification and the identification of high-titer producer cells. This methodology will help investigators meet the challenges of developing vectors which lack selectable markers.