Improving the production and quality of lentivirus for the transduction of haematopoeitic stem cells via a perfusion culture process

可扩展和一致的LV生产对于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至关重要。该项目的重点是通过研究灌流和为悬浮适应的稳定慢病毒产生细胞(HEK293T衍生)建立高通量、可扩展的悬浮平台来优化慢病毒载体(LV)的生产。所产生的LV将用于转导感兴趣的靶细胞(造血干细胞)。产生的数据将为数据分析和建立流程模型提供支持。慢病毒将用于转导造血干细胞，这些细胞具有扩增和鉴定的范围。在小型搅拌槽生物反应器系统(100-250毫升)中建立的工艺条件将在较大规模的搅拌槽生物反应器系统(高达5L)中得到验证。产生的LV将用于转导感兴趣的靶细胞(造血干细胞-HSCs)。具体地说，该项目将建立在CSL开发悬浮适应的HEK细胞系的先前工作的基础上，并在对关键工艺参数进行大规模的能源部研究之后，建立一个稳健的灌流过程。工作将从调查一系列平台开始，以建立适合LV生产研究的高通量、小规模悬浮平台。一旦建立，将开发一种灌流过程，并将对所生产的LV在目标造血干细胞上进行功能测试。这与EPSRC的医疗技术和制造未来的主题，特别是“开发未来疗法”和“21世纪产品”的重大挑战相一致

Scalable and consistent production of LV is critical to ensure sufficient supply of high-quality vector for gene therapies and gene-modified cell therapies. The focus of the project is to optimise lentiviral vector (LV) production through the investigation of perfusion and the establishment of a high-throughput, scalable suspension platform for suspension-adapted stable lentiviral producer cells (HEK293T-derived). The LV produced will be used to transduce the target cells of interest (hematopoietic stem cells). The data generated will support data analytics and establishment of process models. The lentivirus will be used to transduce hematopoietic stem cells, with scope for expansion and characterisation of these cells.The process conditions established in the small scale stirred-tank bioreactor systems (100-250 mL) will be validated in larger-scale stirred-tank bioreactor system (up to 5L). The LV produced will be used to transduce the target cells of interest (hematopoietic stem cells - HSCs). Specifically, this project will build on previous work undertaken at CSL developing a suspension-adapted HEK cell line and establish a robust perfusion process following large-scale DoE studies investigating the key process parameters. Work will begin by investigating a range of platforms to establish a suitable high-throughput, small-scale suspension platform for the LV production studies. Once established, a perfusion process will be developed and the LV produced will be tested for functionality on the target haematopoietic stem cells.This aligns to the EPSRC's Healthcare Technologies and Manufacturing the Future Themes, specifically the 'Developing Future Therapies' and '21st century products' Grand Challenges