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

为确保基因治疗和基因修饰细胞治疗提供足够的高质量载体，需要可扩展和一致地生产稳定的慢病毒载体细胞系。这需要一种综合的方法，同时考虑细胞系的生物工程和上游和下游加工的生物工艺工程。该项目的重点是为LV生产开发可扩展和稳定的细胞系，以确保为基因治疗和基因修饰细胞治疗提供足够的高质量载体。具体来说，这将集中在生产瞬时和稳定的LV细胞系，以基因修饰临床相关细胞自然杀伤细胞（NK细胞）以表达嵌合抗原受体（CAR）。项目目标包括：通过稳定、连续的LV小规模生产系统生产编码标记基因（如GFP）的LV和抗cd19 CAR的LV，通过实验研究设计，包括与其他贴壁技术系统的比较，确定和研究使用iCELLis Nano改进LV上游处理的关键参数。CAR-NK制造的端到端生物工艺示范。这将通过在AllegroTM XRS系统中使用iCELLis Nano生产的LV载体与嵌合抗原受体（CAR）进行nk细胞的转导和扩增来实现。由于该过程的多个参数和复杂性，将采用DoE方法建立初始筛选实验，使用简单、可靠的检测方法（p24 ELISA、qPCR和基于facs的传染性检测）监测病毒恢复。通过绘制完整的实验空间，我们可以在最终的可扩展过程中使用质量设计。这与EPSRC制造未来的主题直接一致，特别关注制造21世纪的产品。目前ATMP生产的一个主要限制是缺乏适当的基因传递载体。该项目旨在通过可扩展生产改进的病毒载体来解决这一关键瓶颈。

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.