A fluorescence protein toolbox for improving virus and virus-like particle-based biologics production in animal cells

用于改善动物细胞中病毒和病毒样颗粒生物制剂生产的荧光蛋白工具箱

• 批准号: RGPIN-2017-04211
• 负责人: Aucoin, Marc
• 金额: $ 2.04万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=637941
• 关键词: fluorescence; protein; toolbox; improving; virus

The demand for biologics is increasing at an unprecedented rate. As a result, the vaccine market is expected to increase from 24 billion dollars to 61 billion dollars by 2020 (Global Research – Center for Research Globalization). Viral threats on a global scale remain omnipresent and ever-changing. In the last two decades alone, four types of viruses have been the cause of major outbreaks: SARS coronavirus; avian and swine influenza viruses; Ebola virus; and most recently, Zika virus. Cell culture is the most attractive manufacturing platforms for rapid, scalable and controlled production of viral vaccines. This research program aims to bring substantial improvement to the production of viruses and virus-like particles (VLPs) in the Vero cell and insect cell (Sf9) platforms respectively, by developing a toolbox of well-characterized fluorescent protein (FPs) that can be used in both platforms. To improve the Vero platform, we aim to develop a single-cell suspension-adapted Vero line using a combination of cell culture conditions and gene knockdown strategies. We will also use our FP-toolbox to engineer combinations of FP that are responsive to the state of the cell to understand the effects of culture conditions and infection on the health of the cell. To improve the baculovirus expression vector – insect cell system, we aim to use the FP toolbox to gain insight on controlled expression of multiple proteins inside the cell. This control will be achieved by investigating alternative and novel promoter sequences to drive protein expression, as well as the use of CRISPR-Cas9 to modulate and downregulate protein expression. It is a goal of this program to increase the cell specific productivity of both of the platforms at increasing cell densities, and it is believed that the FP toolbox that will be developed will play an integral role in understanding changes in gene expression and state of the cell. This research program is geared towards training scientists and engineers in the control of biological products and systems. As a result, students get exposed to: manipulating DNA; working with bacteria (

对生物制剂的需求正以前所未有的速度增长。因此，到2020年，疫苗市场预计将从240亿美元增加到610亿美元（全球研究中心）。全球范围内的病毒威胁仍然无处不在，而且不断变化。仅在过去的二十年里，四种类型的病毒已经成为主要疫情的原因：SARS冠状病毒;禽流感和猪流感病毒;埃博拉病毒;以及最近的寨卡病毒。细胞培养是快速、可规模化和可控生产病毒疫苗的最具吸引力的生产平台。该研究计划旨在通过开发可用于两个平台的良好表征的荧光蛋白（FP）工具箱，分别在Vero细胞和昆虫细胞（Sf 9）平台中大幅改善病毒和病毒样颗粒（VLP）的生产。为了改进Vero平台，我们的目标是使用细胞培养条件和基因敲低策略的组合来开发单细胞悬浮适应的Vero系。我们还将使用我们的FP工具箱来设计对细胞状态有反应的FP组合，以了解培养条件和感染对细胞健康的影响。为了改进杆状病毒表达载体-昆虫细胞系统，我们的目标是使用FP工具箱来了解细胞内多种蛋白质的受控表达。这种控制将通过研究替代的和新的启动子序列来驱动蛋白质表达，以及使用CRISPR-Cas9来调节和下调蛋白质表达来实现。该计划的目标是在增加细胞密度的情况下增加两个平台的细胞比生产率，并且相信将开发的FP工具箱将在理解基因表达和细胞状态的变化方面发挥不可或缺的作用。该研究计划旨在培养科学家和工程师控制生物产品和系统。结果，学生们接触到：操纵DNA;与细菌（