Development of a production method for scaling up lentiviral vector manufacture

开发扩大慢病毒载体生产规模的生产方法

• 批准号: 7157407
• 负责人: Madhusudan Viswanath Peshwa
• 金额: $ 11.69万
• 依托单位: MAXCYTE, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7157407
• 关键词: Lentivirus; biotechnology; cell adhesion; cell line; cell population study; electroporation; gene therapy; plasmids; technology /technique development; tissue /cell culture; transfection /expression vector

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.

描述（由申请人提供）：源自慢病毒的基因治疗载体提供了优于其他基因转移载体的许多优点，并且它们代表了用于治疗多种疾病的有希望的方法，所述疾病例如年龄相关性黄斑变性、帕金森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和与瞬时转染的标准方法相关的低效率，慢病毒载体难以大量生产。该第1阶段提案描述了慢病毒载体生产的可扩展工艺的开发，该工艺使用MaxCyte专有的流式电穿孔技术将编码牛免疫缺陷病毒（BIV）基因治疗载体组分的质粒DNA转染到哺乳动物细胞中。该工作计划包括优化电穿孔参数，用于将BIV组分质粒以小规模和大规模加载到粘附的HEK 293T细胞中。这些实验旨在证明流式电穿孔优于其他方法（例如，磷酸钙沉淀）用于分离常用于慢病毒载体生产的细胞。此外，将开发将BIV组分质粒转染到悬浮细胞中的方案，其提供了用于生产生物制品的贴壁细胞的许多优点。目标是产生一种用于慢病毒生产的有效的基于悬浮细胞的方法，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制造的需求。这些研究的结果将为2期研究提供基础，以优化生产方案，该方案可用于GMP设施中以商业规模生产慢病毒基因治疗载体。