Optimization of production and purification methods of 3rd generation hiv based lentiviral vectors for in vivo applications

Aim: Lentiviral vectors (LVV) are gene transfer tools, which have been preferred in clinical gene therapy trials in terms of their safety, efficacy and long-term gene expression. Despite the therapeutic effectiveness of these vectors is mostly dependent on the genes they carry, they must be produced in high quantity and purity for their successful in vivo applications. Thus, there is a need for a practical, scalable, a robust vector production method applicable to any basic laboratory settings. Thus, the aim of this study is to design a lentiviral production and purification technique to obtain high titered, bio-safe, 3rd generation LV vectors for in vivo gene transfer applications. Methods: The 3rd generation LVV was produced in roller bottles, by temporarily transfecting 293T cells with packaging and transfer plasmids in the presence of Chloroquine and CaPO4. Following clearance of cellular debris by low-speed centrifuge and filtration, virus was concentrated by high-speed ultracentrifugation in the presence of sucrose-cushion. Concentrated viral samples were then purified by anion-exchange chromatography after benzonase treatment to remove cellular DNA. Viral titers were quantified by RT-PCR, purity was determined by SDS-PAGE, and effect of virus on cellular viability was assayed by MTT test. Results: Transfections performed in IMDM containing 10% FBS and 25µM Chloroquine resulted in successful production of LVVs in OPTIMEM with 10% FBS. Ultracentrifugation resulted in 200-fold concentration of viral vectors allowing samples to be loaded onto anion exchange columns. Following anion exchange chromatography, 90% purification was achieved resulting in a titer of 6.2x10^10 TU/ml. p24 ELISA assays suggested a vector recovery rate of 53% after chromatography. Conclusion: Based on our results, here we report a practical, optimized, scalable production of LVV suitable for laboratory scale lentiviral production.