Development of a scalable and consistent process for the production of lentivirus for gene-modified cell therapies

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Scalable and consistent production of stable cell lines for lentiviral vector (LV) production is necessary to ensure sufficient supply of high-quality vector for gene therapies and gene-modified cell therapies. This requires an integrated approach with both biological engineering of the cell lines and bioprocess engineering of the upstream and downstream processing being considered in parallel. The focus of the project is the development of scalable and consistent production of stable cell lines for LV production to ensure sufficient supply of high-quality vector for gene therapies and gene-modified cell therapies. Specifically, this will focus on the production of transient and stable cell lines for LV production to genetically modify clinically-relevant cells natural killer (NK) cells to express a chimeric antigen receptor (CAR). The project objectives include:Production of a LV coding for a marker gene, e.g. GFP, and a LV for anti-CD19 CAR via a stable, continuous LV small-scale production system Identification and investigation of the key parameters for improved LV upstream processing using the iCELLis Nano via design of experiment studies including a comparison against other adherent technology systems.Demonstration of end-to-end bioprocess for CAR-NK manufacture. This will be achieved via the transduction and expansion of the NK-cells with a chimeric antigen receptor (CAR) in the AllegroTM XRS system using LV vector produced in the iCELLis Nano. Due to the multiple parameters and complexity of the process, an initial screening experiment will be established using a DoE approach to monitor virus recovery using simple, robust assays (p24 ELISA, qPCR and FACS-based infectivity assays). By mapping the full experimental space, we enable the use of Quality by Design in the final scalable process.This aligns directly to the EPSRC Manufacturing the Future Theme, with a specific focus on manufacturing 21st century products. A major limitation at present for ATMP production is the lack of appropriate gene delivery vectors. This project seeks to address this critical bottleneck through scalable production of improved viral vectors.