Development of a production method for scaling up lentiviral vector manufacture

描述(申请人提供)：来自慢病毒的基因治疗载体与其他基因转移载体相比具有许多优点，它们代表了一种治疗各种疾病的有前途的方法，如老年性黄斑变性、帕金森氏病和血细胞恶性肿瘤。不幸的是，由于缺乏稳定的包装细胞系，以及与标准瞬时转染法相关的低效，慢病毒载体很难大量生产。这项第一阶段的提案描述了一种可扩展的慢病毒载体生产工艺的开发，该工艺使用MaxCyte专有的流动电穿孔技术将编码牛免疫缺陷病毒(BIV)基因治疗载体组件的质粒DNA导入哺乳动物细胞。该工作计划包括优化电穿孔参数，以便在小规模和大规模将BIV组分质粒装载到贴壁的HEK 293T细胞中。这些实验旨在证明，流动电穿孔比其他方法(如磷酸钙沉淀法)更适合于慢病毒载体生产中常用的细胞转染方法。此外，还将开发将BIV组分质粒导入悬浮细胞的方案，这将为生产生物制品提供比贴壁细胞更多的优势。其目标是产生一种高效的、基于悬浮细胞的慢病毒生产方法，该方法可以扩大规模，以适应临床规模测试以及基于慢病毒的治疗产品的商业生产的需要。这些研究的结果将为第二阶段研究提供基础，以优化制造方案，这些方案可以用于GMP设施，用于商业规模生产慢病毒基因治疗载体。

DESCRIPTION (provided by applicant): Gene therapy vectors derived from lentiviruses offer a number of advantages over other gene transfer vectors, and they represent a promising approach for treating a variety of diseases, such as age-related macular degeneration, Parkinson's disease, and blood cell malignancies. Unfortunately, lentiviral vectors are difficult to produce in large numbers due to the lack of stable packaging cell lines and to inefficiencies associated with standard methods of transient transfection. This Phase 1 proposal describes the development of a scalable process for lentiviral vector production that uses MaxCyte's proprietary flow electroporation technology to transfect plasmid DNAs encoding components of bovine immunodeficiency virus (BIV) gene therapy vectors into mammalian cells. The workplan includes optimization of electroporation parameters for loading BIV component plasmids into adherent HEK 293T cells at both small and large scales. These experiments are intended to demonstrate that flow electroporation is superior to other methods (e.g., calcium phosphate precipitation) for transfecting cells that are commonly used in lentivector production. In addition, protocols will be developed for transfecting BIV component plasmids into suspension cells, which offer a number of advantages over adherent cells for manufacturing biological products. The goal is to generate an efficient, suspension cell-based method for lentivirus production that can be scaled up accommodate the needs of clinical-scale testing as well as commercial manufacturing of a lentivirus-based therapeutic product. The results of these studies will provide the foundation for Phase 2 studies to optimize manufacturing protocols that can by used in a GMP facility for producing lentiviral gene therapy vectors on a commercial scale.