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

对生物制剂的需求正以前所未有的速度增长。因此，预计到2020年，疫苗市场规模将从240亿美元增加到610亿美元（全球研究-研究全球化中心）。全球范围内的病毒威胁仍然无处不在，并且不断变化。仅在过去二十年中，就有四种病毒引发了重大疫情：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、规模化和可控生产病毒疫苗的最具吸引力的生产平台。本研究计划旨在通过开发可用于Vero细胞和昆虫细胞（Sf9）平台的具有良好特征的荧光蛋白（FPs）工具箱，分别对病毒和病毒样颗粒（vlp）的生产进行实质性改进。为了改进Vero平台，我们的目标是利用细胞培养条件和基因敲低策略的组合开发单细胞悬液适应的Vero细胞系。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的控制表达。这种控制将通过研究替代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提高这两个平台的细胞特异性生产力，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。该研究项目旨在培训生物产品和系统控制方面的科学家和工程师。因此，学生们接触到：操纵DNA；处理细菌(

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 (