Quantitative real-time PCR was utilized to evaluate retrovilated with biologic titers performed on the test material. The ral vector titer. RNA was prepared from vector supernatant 96-well capacity of the machine and 2 h of running time and run in a one-step RT-PCR reaction combining reverse permit titer determinations within 8 h, facilitating the protranscription (RT) and amplification in one tube. Sample cessing of large sample numbers while greatly decreasing analysis was performed in the ABI Prism 7700 Sequence technician time. Real-time PCR improves titer quantifiDetector. PCR was quantitative over a range of 10 1 to cation and the identification of high-titer producer cells. 6 × 10 5 vector particles per reaction (2 × 10 2 to 1 × 10 7 vecThis methodology will help investigators meet the chaltor particles per millilites of supernatant) and closely correlenges of developing vectors which lack selectable markers.Keywords: retroviral vectors; vector production; vector titer; quantitative PCR Estimation of retroviral vector titer and identification of high-titer packaging cell lines have been aided by the incorporation of selectable markers within the vector genome. These markers have typically been drug selection genes 1-4 or molecules expressed on the cell surface. 5 Recent concern about the immunogenic potential of these gene products in animals models and human clinical trials have prompted investigators to remove drug selection genes from vector constructs. 6,7 To simplify titer determination, and facilitate rapid identification of hightiter producer clones, investigators have utilized PCR and RNA dot blot assays to estimate vector RNA in supernatant preparations. [8][9][10] These methods are helpful in separating vectors with large to modest differences in titer but remain semi-quantitative. The development of real-time PCR technology, which combines PCR and fluorescence detection of target sequences, may provide a method for quantitative PCR with increased accuracy over current methods. 11,12 In addition, this technology provides for processing of large number of samples (96 reactions per run), one-step RT-PCR reactions (reverse transcription and PCR amplification in one tube), and real-time read-out (without the need for electrophoresis or dot blot analysis). This article demonstrates how this methodology accurately predicts biologic titers, significantly decreases turn around time, and decreases technician time. It has greatly simplified the screening of vector producer cells for high titer clones. Real-time PCR was performed using the ABI Prism 7700 Sequence Detector (TaqMan; PE Applied Biosystems, Foster City, CA, USA) which combines PCR tech-

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.