Factors affecting the production of pantropic retroviral and lentiviral vectors

Successful gene delivery is important for gene therapy. In this study, several issues have been addressed to improve the production and transduction efficiency of retroviral and lentiviral vectors. First, the effects of pH on the transduction efficiency of NIH3T3 cells were investigated using Moloney murine leukemia viral (MoMuLV) vector and murine stem cell viral (MSCV) vector. A small increase in pH from 7.33 to 7.7 significantly increased the overall transduction efficiency, partly due to greater stability of protamine sulfate at higher pH. Second, the ratio of envelope protein (vesicular stomatitis viral glycoprotein or VSV-G) plasmid to vector plasmid in a transient pantropic retroviral vector production system was examined. VSV-G to vector plasmid ratios ranging from 0.053 to 0.2 resulted in optimal transduction. These ratios are far below the typically employed plasmid ratios. The transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed. Third, a third-generation inducible lentiviral vector producer cell line (clone 120C) was adapted to suspension culture and reduced serum levels, and was successfully scaled up to 5 L perfusion bioreactors. Finally, the effects of cholesterol supplementation on the production of retroviral and lentiviral vector were studied. Cholesterol is a major component in membrane microdomains called lipid rafts, which are involved in the assembly of many enveloped viruses. The infectivity of retroviral and lentiviral vectors produced in culture with cholesterol supplementation was greatly increased compared to those without cholesterol supplementation. The amount of vector produced was also greater with cholesterol for lentiviral, but not retroviral vector. Cholesterol must be added before and throughout the transfection or induction culture to obtain the maximum benefit. Both free and total cholesterol content in producer cells for retroviral vector production increased with cholesterol supplementation. The increase in the cholesterol content, and possibly other changes in membrane components, in producer cells might affect the vector assembly and budding process in lipid rafts, and thereby increase the infectivity of the viral vector. The results from this study will contribute to improving the production of viral vectors for both research and clinical use.