- Authors: Leinonen HM. et al.
- Year: 2019
- Journal: Meth Clin Dev 15 63-71
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Dish/vessel: iCELLis 500/100m²or/330m² High/Low Compaction. Growth medium: DMEM; L-glut; P/S. Seeding: 7.000 cells/cm² 4 days before transfection. DNA : 300ng/m² or 200ng/m². Reagent Amount: 300ng/m² or 200 ng/m². Ratio: 1:1. Complexation. Volume cell culture: 65 ml. Transfection Day 4. Perfusions aiming 0.5 g/L glucose & endonuclease treatment after harvest. medium change 24h afer transfection before virus harvest.
The therapeutic efficacy of a lentiviral vector (LV) expressing the herpes simplex virus thymidine kinase (HSV-TK) was studied in an immunocompetent rat glioblastoma model. Intraperitoneal ganciclovir injections (50 mg/kg/day) were administered for 14 consecutive days, resulting in reduced tumor volumes as monitored by MRI. Survival analyses revealed a significant improvement among the LV-expressing HSV-TK (LV-TK)/ganciclovir-treated animals when compared to non-treated control rats. However, a limiting factor in the use of LV has been the suboptimal small-scale production in flasks. Our aim during the translation phase, prior to entering the final pre-clinical and early clinical phases, was to develop a scalable, robust, and disposable manufacturing process for LV-TKs. We also aimed to minimize future process changes and enable production upscaling to make the process suitable for larger patient populations. The upstream process relies on fixed-bed iCELLis technology and transient plasmid transfection. This is the first time iCELLis 500 commercial-scale bioreactor was used for LV production. A testing strategy to determine the pharmacological activity of LV-TK drug product by measuring cell viability was developed, and the specificity of the potency assay was also proven. In this paper we focus on upstream process development while showing analytical development and the proof-of-concept of LV-TK functionality.