Development of a production method for scaling up lentiviral vector manufacture

描述（由申请人提供）：来自慢病毒的基因治疗载体提供了许多优于其他基因转移载体的优点，并且它们代表了治疗各种疾病的有希望的方法，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标准瞬时转染方法相关的低效率，慢病毒载体很难大量生产。该i期提案描述了一种可扩展的慢病毒载体生产工艺的发展，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）基因治疗载体成分的质粒dna转染到哺乳动物细胞中。工作计划包括优化将BIV成分质粒加载到贴壁HEK 293T细胞上的电穿孔参数。这些实验旨在证明流动电穿孔优于其他方法（例如，磷酸钙沉淀），用于转染细胞，通常用于慢载体生产。此外，将开发将BIV成分质粒转染到悬浮细胞中的方案，悬浮细胞在制造生物制品方面比贴壁细胞具有许多优势。目标是产生一种高效的、基于悬浮细胞的慢病毒生产方法，这种方法可以扩大规模，以适应临床规模测试的需要，以及慢病毒治疗产品的商业生产。这些研究的结果将为第二阶段研究提供基础，以优化生产方案，这些方案可以在GMP设施中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.