711. Development of a Clinically-Acceptable Lentiviral Vector for Cystic Fibrosis Airway Gene Therapy

Abstract

Background: Lentiviral (LV) vectors are a promising gene delivery vehicle for cystic fibrosis airway gene therapy. However, the development of a clinically-acceptable method suited to large-scale lentivirus production remains a barrier to the translation of gene therapy to the clinic. Current virus production methods have limited-scalability and often result in contamination of the vector preparation with potentially immunogenic components such as bacterially-derived plasmid DNA, and animal sera products. Accordingly, a scalable process for the production of clinical-grade vector is required.Methods: LV vector preparations were treated with Deoxyribonuclease I (DNase I) to remove residual plasmid DNA, with the effectiveness measured by PCR using primers to detect the presence of the cytomegalovirus promoter sequence. The potential for scalable LV vector production in serum-free conditions was investigated by performing transient transfection of HEK 293FT cells adapted to grow in serum-free, suspension culture. To determine the efficiency of the virus production system RNA titering was performed using a commercial qPCR kit, and reporter gene expression of GFP and Luciferase were quantified using FACS and IVIS bioluminescence imaging.Results: Plasmid DNA was detected in the vector preparations following DNase treatment, indicating that DNase I was ineffective at eliminating the residual plasmid DNA. Lentivirus was successfully produced using transient transfection of serum-free, suspension growing HEK 293FT cells, and was scaled-up to a one-litre culture capacity in shake flasks. Titres of up to 108 TU/mL were achieved in the suspension cell culture system, which is comparable to the yields obtained in the standard adherent culture system.Conclusions: The protocol developed in this study for serum-free suspension culture virus production resulted in high vector titres that were comparable to those routinely achieved using adherent cells. Furthermore, relative to traditional virus production methods, this protocol is easily scalable, less labour intensive, and has less potential for contamination of the final vector preparation. DNase I treatment was ineffective at removing residual plasmid DNA from the vector preparation, indicating that enzymatic removal of plasmid DNA may not be a feasible approach.Funding: The University of Adelaide; Cure4CF Foundation.