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.
Chen
J
J
Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis 46202, USA.
Reeves
L
L
Sanburn
N
N
Croop
J
J
Williams
D A
DA
Cornetta
K
K
D002460:Cell Line; D002986:Clinical Trials as Topic; D004340:Drug Contamination; D005189:False Positive Reactions; D015316:Genetic Therapy / Q000592:standards*; D005822:Genetic Vectors / Q000592:standards*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000592:standards*; D012367:RNA, Viral / Q000032:analysis; D012190:Retroviridae / Q000235:genetics / Q000302:isolation & purification; D012192:Retroviridae Infections / Q000517:prevention & control; D012449:Safety; D014759:Viral Envelope Proteins / Q000235:genetics
D012367:RNA, Viral; D014759:Viral Envelope Proteins