The initial establishment of a new poxviral/lentiviral hybrid system for efficient lentiviral vector production

Efficient gene delivery and sustained gene expression are required for successful human gene therapy. Although viral vectors are considered the most efficient vehicles for gene transfer, currently available viral vectors have not fully achieved these two requirements. Lentiviral vectors (LVs) can integrate into host chromosomes, allowing long-term gene expression, in addition, these vectors are non-toxic and minimally immunogenic since no viral genes are encoded in the vector genome, but are still limited to in vitro or ex vivo gene delivery because of their relatively low titers using transient transfection experiments. In order to develop an efficient transient transfection method for large-scale production of high titer lentiviral vector stocks, a minimal lentiviral vector producing system based on vaccinia virus that synthesizes T7 RNA polymerase was developed. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelope plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000 (TM). After 4 days, the culture supernatant of lentiviral vectors was collected, the RNA from the supernatant was examined by the RT-PCR, the protein from the supernatant was examined by Western blot, and the supernatant was used to transfect normal 293T, HepG2 and Vero, which were observed by the immunofluorescence microscopy. The type of cell lines, plasmids dosage and the MOI (the proportion between cell numbers and virus copies) were considered so critical to the output of this system that 3x3x3 factorial design was used to explore the yield optimization of this system. As judged by the results of RT-PCR and the Western blot, lentiviral vectors were found in the culture supernatant; as judged by immunofluorescence with microscopy, 293T, HepG2 and Vero which were transfected by the supnantant expressed the report protein - green fluorescent protein (GFP), the results confirmed the valid infectivity of the lentiviral vector produced by the system. Eventually, the best titers of lentiviral vector stocks was up to 1.3x10(8) tu/ml, which is one order of magnitude higher than the output of classical manufacture system. The new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established. It provides the basis for the future development of industrial application.