A New Poxviral/Lentiviral Hybrid System Capable Of Efficient Lentiviral Vector Production

Objective: Lentiviral vectors are attractive tools for human gene therapy. The goal of the present study was to develop an efficient transient-hybrid system for large-scale production of high titer lentiviral vector stocks.Methods: In this system, BHK₂₁ was co-transfected by three main plasmids consisting of transuding plasmid pVECRNA, packaging plasmid pgagpol, envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000?. After 4 days, the culture supernatant of lentiviral vectors was collected and then followed by filtration through 0.22-μm microporous membrane.Results: The culture supernatant was confirmed by the fragment of 96bp generated by RT-PCR and sequencing. After 48h, BHK₂₁ was co-transfected by the three main plasmids and the helper virus. The expression of p24 was observed in BHK₂₁ through indirect immunofluorescence by confocal microscopy, the p24 was mainly distributed in cytoplasm, rarely in cell nucleus. GFP was also expressed in BHK₂₁ and 293T 48h later after they were infected with the culture supernatant.3*3*3 factorial design was used to explore the yield optimization of this system. The type of cell lines, plasmids dosage and the MOI (the proportion between cell numbers and virus copies) were considered critical to the output of this system. The SPSS analysis results demonstrated that the three factors have significant effects on the output of the system respectively, and these three factors have significant interaction.3*3 factorial design was used to explore the yield optimization of this system when BHK21 was utilized as the production cell line for that it was the best cell line for the LV production among the three mentioned cell lines containing BHK₂₁, Vero, and HepG₂. the SPSS analysis results illustrated that the plasmids dosage and the MOI have significant effects on the system output respectively, and these two factors have significant interaction.