Lentivirus Production Is Influenced by SV40 Large T-Antigen and Chromosomal Integration of the Vector in HEK293 Cells

Currently, lentiviral vectors for research and gene therapy are produced from 293-T cells that are transiently transfected with plasmids encoding the vector and helper functions. However, transiently transfected vectors as well as the presence of SV40 virus large T-antigen (T-Ag) cause serious technical and safety considerations. We aimed to exploit single copy integration sites in the HEK293 genome supporting lentiviral vector production. We found that lentiviral vectors result in minimal infectious particle production from single copy integrants in HEK293. Moreover, once this cell line harbors single copy integrations of lentiviral vectors, its ability to transiently produce lentiviral vectors becomes strongly impaired. T-Ag has a dramatic effect on virus production. Low levels of constitutive T-Ag expression can overcome the production restriction imposed by integrated lentiviral vectors copies. Interestingly, T-Ag does not exert its role at the level of transcriptional activity of the vector; rather, it seems to impose an indirect effect on the cell thereby enabling lentiviral vector production. Altogether, our study highlights the restrictions for integrated lentiviral vectors that are relevant for the establishment of stable and safe producer cell lines. Gama-Norton and colleagues evaluate the feasibility of lentiviral vectors that are commonly used in transient production protocols to generate infectious viral particles on low copy integration into the HEK293 genome. They show that HEK293 cells do not support efficient lentiviral vector production when the vector is integrated at a low copy number in the cellular genome and that this impairment is partially dependent on the SV40 large T antigen.