Applying synthetic biology to upscale and de-risk biologics production by CHO cell transient transfection.

哺乳动物细胞瞬时转染通常涉及使细胞经历导致转基因表达爆发的程序，所述转基因表达在48小时内强度增加，然后迅速消退。在哺乳动物细胞中瞬时表达异源蛋白质是快速产生蛋白质试剂的有力途径。然而，与将重组基因稳定整合到基因组中并分离高表达克隆的方法相比，它在历史上具有较差的产率和再现性。迄今为止，改善瞬时转染的这些度量的方法包括：i）使用实验设计（DoE）来快速测量多个参数对蛋白质表达水平的影响，ii）添加小分子如N，N-二甲基乙酰胺（DMA）来增强基因表达，以及iii）与编码增强蛋白质折叠的蛋白质的另外的转基因共转染。本项目旨在捕获瞬时转染试剂引起的生物休克导致的基因上调和下调的蛋白质组学数据。这些数据将提供一个有价值的知识基础的合成基因网络（SGN）的设计，打开，或间接触发，转染程序。这些SGN然后将使用精确的CRISPR技术插入到CHO细胞基因组中，以指导增强转染性能的蛋白质的表达，而不会对细胞的代谢负担产生负面影响。在这种高水平转基因表达的爆发期间，几乎没有工作来确定细胞的代谢状态和需求。该项目的另一个要素将涉及在瞬时转染期间捕获代谢数据和改进代谢模型。该项目的所有元素将在一系列大分子治疗蛋白质中得到验证，从高标准平台实体（如单克隆抗体（MAb））到难以表达的蛋白质（如用于替代疗法的高度糖基化酶）。将测试不同规模的培养，以建立准确的规模模拟，并证明该项目产生的新方法的鲁棒性和可扩展性。

Mammalian cell transient transfection typically involves subjecting cells to procedures that result in a burst of transgene expression that increases in intensity over 48 hours then rapidly fades. Transient expression of heterologous proteins in mammalian cells is a powerful way to rapidly generate protein reagents. However, it has historically suffered from poor yields and reproducibility compared to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. To date approaches to improve these metrics for transient transfection include: i) the use of design of experiments (DoE) to quickly measure the influence of multiple parameters on protein expression levels ii) addition of small molecules such as N, N-Dimethyl acetamide (DMA) to enhance gene expression and iii) co-transfection with additional transgenes encoding proteins that enhance protein folding. This project proposes to capture proteomic data on the up- and down- regulation of genes that results from the biological shock caused by transient transfection reagents. These data will provide a valuable knowledge base for the design of synthetic gene networks (SGNs) that are switched on, or indirectly triggered, by the transfection procedure. These SGNs would then be inserted into the CHO cell genome using precise CRISPR techniques to direct expression of proteins that enhance transfection performance, without contributing negatively to metabolic burdens placed on the cell.Little work has been done to characterise the metabolic status and requirements of cells during this burst of high-level transgene expression. A further element of the project will involve capturing metabolic data during transient transfection and refining metabolic models. Outputs will inform media formulation and supplementation with the goal of shortening the expression burst period to the first 24 hours whilst preserving or increasing total yield.All elements of the project will be validated across a suite of macromolecular therapeutic proteins ranging from highly standard platform entities such as monoclonal antibodies (MAbs), to difficult-to-express proteins such as highly glycosylated enzymes for replacement therapies. Different scales of cultivation will be tested in order to establish accurate scale mimics and to demonstrate the robustness and scalability of novel methods arising from the project.