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

Jun Chen, Lilith Reeves, Nathan Sanburn, James Croop, David A. Williams, Kenneth Cornetta

Research output: Contribution to journalArticle

34 Scopus citations

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 102 to 3.5 × 103 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.

Original languageEnglish (US)
Pages (from-to)186-197
Number of pages12
JournalVirology
Volume282
Issue number1
DOIs
StatePublished - Mar 30 2001

    Fingerprint

ASJC Scopus subject areas

  • Virology

Cite this