816. Rapid, Consistent and Scalable Production of Viral Vectors in Suspension Cells by Cotransfection of Viral Component Plasmid DNA or RNA
Authors: James Brady, Rama Shivakumar, Matthew Malehorn, Stephanie Feller, Cornell Allen, Linhong Li, Nicholas Chopas, Sergey Dzekunov, Madhusudan V. Peshwa, Linda N. Liu
Journal: Molecular Therapy (2006)

Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte's proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte's high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy. Viral vectors show promise as gene therapy treatments for a number of diseases. However, it has been difficult to develop a robust system for generating large volumes of high-titer viral vectors based on lentivirus, alphavirus and adeno-associated virus (AAV) that are needed for clinical applications. Problems stem from the lack of stable producer cell lines and inherent inefficiencies associated with introducing multiple plasmids into adherent monolayer cells by conventional transient transfection techniques (i.e., calcium phosphate precipitation or cationic lipids). Other researchers have demonstrated that suspension cells of lymphoid origin, which can be grown at much higher density than adherent cells, can be converted into efficient retroviral vector producer cell lines (Chan et al., 2001. Gene Ther. 8: 697). However, commercially available transient transfection systems are not optimal for large volume co-transfection of suspension cells, especially hematopoietic cells. MaxCyte's proprietary high-throughput electroporation technology provides a closed, robust, scalable system for high efficiency transfection of both adherent and suspension cells. The MaxCyte system previously was used to produce high titer alphaviral vectors from >10 x109 Vero cells and lentiviral vectors from >1 x109 adherent HEK 293T cells. To improve the efficiency of lentiviral vector production at large scale and to demonstrate the feasibility of lentivector production in suspension cells, we used the MaxCyte system to transfect large volumes (38 x109 cells in 380 mL) of K562 human hematopoietic cells rapidly (within 20 minutes) with a plasmid encoding the green fluorescent protein (GFP) marker gene. The transfected cells were collected in fractions of 35-40 mL. Cells were cultured from each fraction or pooled samples to monitor consistency of the high- throughput electroporation. Greater than 90% of the cells expressed GFP when analyzed by flow cytometry at 48 hrs post-electroporation for all fractionated and pooled cell samples. Propidium iodide (PI) exclusion revealed the cell viability of all the fractionated and pooled cell samples to be greater than 90%. In addition, we were able to produce lentivirus in K562 cells by small- scale electroporation of 10 x106 cells with four plasmids encoding components of a lentiviral vector derived from HIV carrying the eGFP marker gene. In conclusion, these studies will demonstrate that use of suspension cells processed by MaxCyte's high- throughput electroporation-based transfection system is a feasible approach for generating large volumes of high-titer viral vectors suitable for clinical / commercial applications in human gene therapy.