Development of Replicon Cell Pools Bearing a Flavivirus RNA Replicon as a Source of HIV-1 Gag-Pol for Lentiviral...

Lentiviral vectors (LVs) are indispensable tools in cell and gene therapy. Rising demand has created a global shortage of LVs, driving the development of novel packaging approaches. We report a novel vector packaging approach using autonomously replicating cytoplasmic RNAs (replicons) to express packaging proteins. Yellow fever virus (YFV) was used as a source of replicons encoding the HIV-1 Gag-Pol polyprotein together with reporter or selectable markers. YFV replicons were able to establish chronic infection in HEK293FT cells. Replicons expressing HIV-1 Gag-Pol containing the wild-type HIV-1 protease caused strong cytotoxicity, which prevented the selection of polyclonal cell pools harboring the replicon. In contrast, a replicon carrying the T26S mutation in the HIV-1 protease gene showed no measurable cytotoxic effects, enabling the generation of stable replicon-containing cell pools. The replicon cell pools were established using antibiotic selection and maintained Gag-Pol expression for at least ten passages under selection pressure. Using these first-generation replicon cell pools as packaging cells, LV production required only transient transfection of a transfer vector, a Tat/Rev plasmid, and an envelope plasmid. Yields reached ~10⁶ TU/mL prior to concentration and ~10⁹ TU from multilayer cell stacks, which fall within the range typically reported for conventional transient transfection systems under similar culture conditions. The resulting vectors efficiently transduced target cells, and no replication-competent lentivirus (RCL) was detected using a two-phase RCL assay with p24 ELISA detection. This demonstrator platform utilizing replicon cell pools represents a novel approach for LV packaging.