A Robust Protocol for Bio-Safety Re-Classification of Cell Lines Transduced with Lentiviral Vectors

Lentiviruses are a subfamily of RNA retroviruses that stably integrate their genetic information into host genomes. The transduction of cultured cells with safety-engineered replication-incompetent lentiviral vectors is a common procedure for delivering stable transgene overexpression in a wide variety of target lines and primary cell types. Work with lentiviral vectors and lentiviral vector-transduced cell cultures is generally managed under safety classification BL2/S2 due to risks from insertional mutagenesis. The prolonged culture of cells transduced with replication-incompetent lentiviral vectors under BL2/S2 conditions poses significant limitations to experimental downstream procedures and is not necessary once infectious lentiviral particles are no longer present. A safe transition of transduced cell lines to gene-technology safety level BL1/S1 requires certainty about the absence of any infectious particles to guarantee absence of risk for laboratory personnel. We have established a robust RT-PCR protocol to detect presence or absence of lentiviral vector mRNA components VSV-G, RRE, gag and pol, and for the detection of potential Psi-gag rearrangements. Our procedure detects lentiviral vector particles in cell culture supernatants using different MOIs for adherent and suspension cell lines, and we have characterized outcomes after defined serial washing steps and passaging procedures. We demonstrate that cell culture supernatants are free of detectable lentiviral vector particles after less than two passages. Our protocol establishes a robust and safe downgrading procedure for lentiviral vector transduced cells from BL2/S2 to BL1/S1 that can be easily adapted to any custom experimental setting.