Packaging Cell Line DNA Contamination of Vector Supernatants: Implication for Laboratory and Clinical Research

Abstract

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.

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

“…4070A envelope sequences have never been detected in any sample after transplantation. As previously reported, this DNA is not persistent, 26 but our results indicate that PCR can be problematic when screening transduced products for replication competent retrovirus (RCR). 30 In terms of toxicity related to the chemotherapy administration, three of five subjects developed pulmonary function test abnormalities with modest decreases in their diffusion capacity prior to the fourth cycle of intensified PCV.…”

“…Prior to infusion of the transduced stem cell product, cells were evaluated for replication competent retrovirus using the extended S þ /LÀ assay 26,27 and by PCR using primers for the retroviral 4070A envelope sequence. After infusion, peripheral blood and bone marrow from study participants were evaluated for retroviral 4070A envelope sequence by PCR.…”

“…After infusion, peripheral blood and bone marrow from study participants were evaluated for retroviral 4070A envelope sequence by PCR. 26 Serum samples from study participants were also evaluated periodically for the development of antibodies against fibronectin fragment CH-296 (Tanox, Houston, Texas). 16 Median survival was determined using the JMP version 3.1.5 statistical program (SAS Statistical Institute, Cary, NC).…”

“…After extensive evaluation, it was determined that the 4070A envelope DNA detected by PCR represented producer cell line DNA contained in the vector supernatant and was not related to replication competent retrovirus. 26 During this evaluation, no stem cell products were infused and enrolled patients received untransduced products until this issue was resolved. This led to one participant receiving MGMT-modified cells during cycles 2 and 3 and a second patient receiving MGMT-modified cells during cycles 3 and 4.…”

Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods

“…[4][5][6]9 However, the RNA titers thus obtained may overestimate the number of functional vector particles due to the presence of defective interfering particles and packaging cell line DNA. 10,17,20 Also, as shown by our studies above with the amp r PCR assay, carry-over of plasmid DNA is of concern in vectors produced by transient transfection methods.…”