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

LV的可规模化和一致性生产对于确保基因治疗和基因修饰细胞治疗的高质量载体的充足供应至关重要。该项目的重点是通过研究灌注和建立高通量，可扩展的悬浮平台来优化慢病毒载体（LV）生产，用于悬浮适应的稳定慢病毒生产细胞（HEK 293 T衍生）。产生的LV将用于扩增目标靶细胞（造血干细胞）。生成的数据将支持数据分析和流程模型的建立。慢病毒将用于扩增造血干细胞，并对这些细胞进行扩增和表征，在小规模搅拌罐生物反应器系统（100-250 mL）中建立的工艺条件将在大规模搅拌罐生物反应器系统（高达5 L）中进行验证。产生的LV将用于扩增目标靶细胞（造血干细胞-HSC）。具体而言，该项目将建立在CSL开发悬浮适应性HEK细胞系的先前工作的基础上，并在调查关键工艺参数的大规模DoE研究后建立稳健的灌注工艺。工作将开始调查一系列的平台，以建立一个合适的高通量，小规模的悬浮平台的LV生产研究。一旦建立，将开发灌注过程，并将测试所生产的LV对目标造血干细胞的功能性。这符合EPSRC的医疗保健技术和制造未来主题，特别是“开发未来疗法”和“21世纪世纪产品”的重大挑战

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