468. Evaluation of Factors Influencing the Production Titers of SIV-Derived Lentiviral Vectors
Authors: Nora Naumann, Suk See De Ravin, Morvarid Moayeri, Uimook Choi, Yasuhiro Ikeda, Harry L. Malech
Journal: Molecular Therapy (2006)

Lentiviral vectors are capable of transducing non-dividing/slow proliferating cells. Thus, they are suggested to more efficiently transfer genes into long-term repopulating hematopoietic stem cells (HSCs). Human immunodeficiency virus (HIV)-1-based vectors have so far dominated the field of lentiviral vector development for transduction of HSCs. Because HIV is a known human pathogen, alternative lentiviral vectors e.g. derived from simian immunodeficiency virus (SIV) might be preferred for application in humans if similar efficiency could be achieved. We investigated different variables influencing the titer of transiently produced green fluorescent protein (GFP) encoding SIV-derived lentiviral vectors pseudotyped with different envelopes. 293T HEK cells were co-transfected with transfer vector (v), gag-pol encoding (gp), rev-tat encoding (rt), and envelope encoding (e) plasmid DNA at different concentrations. The virus titer produced was dependent on the ratio of the components with the best results at 5 (v) : 3 (gp) : 1.2 (rt) : 1 (e) for VSV-G and a MLV-amphotropic envelope and 4 (v) : 2.4 (gp) : 1 (rt) : 2 (e) for a modified RD114 envelope (RD114+). DNA quality is very critical and if transfection efficiency is high but virus production poor endotoxin contamination or other problems with the purity of the plasmid DNA should be considered. The application of chloroquine or sodium butyrate did not result in a benefit. In our studies the use of IMDM supplemented with 10% FBS was the best medium to use compared to DMEM + 10% FBS and IMDM + 5% or + 0% FBS. FuGENE6-based transfection did not outperform our calcium-phosphate-precipitation protocol. We also found that different individuals following the same protocol had significantly different production efficiencies, and that individuals over time showed greatly increased ability to produce much higher titers presumably through a "learning" process, but could not always pinpoint why their results improved. Concentration of large volumes of virus production medium containing virus particles could be achieved by high-speed (non-ultra-) centrifugation (18,600 g for 4 h) for both VSV-G and modified RD114 pseudotyped vectors and by lower speed centrifugation (8,000 g for 3 h) for MLV-amphotropic vectors with excellent recovery. Thus, under best conditions and after concentration (150 to 200-fold) vector titers of approximately 2x10E7 IU/ml could be achieved as determined using the human erythroleukemia cells K562 as a target. We demonstrated excellent performance of the SIV vectors in human, rhesus, and canine CD34+. It was possible to achieve ex vivo transduction rates in human CD34+ cells of greater than 90% after three overnight transductions. It is of note that mouse bone marrow cells could not be transduced with VSV-G pseudotyped SIV-vectors in contrast to a similar HIV-1- based vector demonstrating profound host cell difference in these vectors. In conclusion, these studies might be helpful to optimize SIV-derived lentiviral vector production and performance with a special focus on their use for human and large animal (rhesus, dog) HSCs.