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

哺乳动物细胞瞬时转基因通常包括使细胞经历导致转基因表达突然爆发的过程，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是一种快速产生蛋白质试剂的有效方法。然而，与将重组基因稳定整合到基因组中并分离出高表达克隆的方法相比，它历史上一直存在产量和重复性较差的问题。到目前为止，改进这些瞬时转染指标的方法包括：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；iii)与额外的编码蛋白质的转基因共转染，以增强蛋白质折叠。该项目建议捕获瞬时转染剂引起的生物休克引起的基因上调和下调的蛋白质组学数据。这些数据将为合成基因网络(SGN)的设计提供有价值的知识基础，这些SGN由转基因过程启动或间接触发。然后使用精确的CRISPR技术将这些SGN插入到CHO细胞基因组中，以指导蛋白质的表达，从而提高转染性能，而不会对细胞施加的代谢负担产生负面影响。在这种高水平的转基因表达爆发期间，几乎没有人做过工作来表征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基因过程中捕获代谢数据，并改进代谢模型。结果将为媒体配方和补充提供信息，目标是将表达突发期缩短到最初24小时，同时保持或增加总产量。该项目的所有要素将在一系列大分子治疗性蛋白质中得到验证，范围从单抗(MAb)等高度标准的平台实体到难以表达的蛋白质(如用于替代疗法的高度糖基化的酶)。将对不同规模的种植进行测试，以建立准确的规模模拟，并展示该项目产生的新方法的稳健性和可扩展性。

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.