The Validation of RT-PCR Assay for Detection of Replication-competent Lentivirus (RCL) in Vector Preparations Using HIV Vector Based Gene Delivery System

Yoo, Eun Sun

Abstract

PURPOSE: Unlike murine oncoretroviruses which can infect only dividing cells, lentiviral vector based on HIV have been shown to efficiently transfer genetic materials into a various cells which makes lentiviral-based vector systems promising candidates for clinical gene therapy, but have also raised safety concerns because of potential creation of replication-competent lentivirus (RCL) by uncontrolled recombination. In this study, I intended to validate RT-PCR assay for detection of replication-competent virus (RCV) in vector preparations using HIV vector based gene delivery system.
METHODS
We prepared a replication-competent lentiviral supernatant from a 293T cell line which had been transfected with plasmid pHIV-VSV G-IRES-eYFP as a positive control, by introducing VSV-G into a first-generation HIV-based vector. A variant of this virus, which has additional deletions of vif and vpr was also used as another positive control. As a negative control, VSV-G pseudotyped HIV-based vector encoding YFP was used. These viruses encode enhanced yellow fluorescent protein (eYFP). I used reverse transcriptase (RT)-PCR to detect VSV-G RNA sequences contained within viral supernatants produced from transiently transfected 293T cells using oligonucleotides specific for conserved for VSV-G element.
RESULTS
After production in 293T cells, titer of each virus were<107 IU/mL. eYFP expression in 293T cells increased according to passage with extremely fluorescent with replication-competent vector. Correctly-sized DNA products was always detected, even when using supernatants from cells separately transfected with VSV-G and replication-defective HIV.
CONCLUSION
These data suggested that RT-PCR based method is not appropriate for transient transfection of producers where cell lysis is routine. Instead we turned our attention to the development of other sensitive assays.