Understanding and manipulating lactate metabolism in single cells

了解和操纵单细胞中的乳酸代谢

• 批准号: BB/S006206/1
• 负责人: Karen Polizzi
• 金额: $ 71.45万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS006206%2F1
• 关键词: Understanding; manipulating; lactate; metabolism; single

Many of the medicines prescribed today are proteins that are manufactured in animal cell cultures. Decades of research has examined ways to increase the productivity of these cultures by changing the nutrients the cells are fed, genetic engineering of the cells themselves and other strategies. However, there are still challenges that limit the overall amount of protein produced. One of these challenges is the accumulation of the metabolic waste product, lactic acid, the same waste product that causes your muscles to be sore after strenuous exercise. The concentration of lactate in animal cell cultures changes over time. Early on, it accumulates due to rapid metabolism, but later the cells use it to make energy and amino acids. This is called the lactate switch. Different batches of cells undergo the lactate switch at different times and the trigger for flipping the switch is not well understood. However, with respect to the production of protein medicines, the lactate switch is a positive trait associated with higher protein production.The proposed work seeks to create a set of tools for understanding and manipulating the lactate switch. The first goal is to create a non-invasive indicator that allows measurement of lactate concentration in individual cells. This tool would allow us to measure the extent of variation between cells, something that is currently not possible with standard techniques. We will use it to understand how different culture conditions affect lactate accumulation in the cells to try and identify how much variability there is in each scenario. Overall, this information can be used to choose manufacturing conditions with less difference between cells.The second goal is to develop tools that allow us to change the expression level of individual enzymes associated with lactate production and consumption. With these tools, we will be able to test hypotheses about which enzymes are associated with the lactate switch by controlling them with an external signal. Once the enzymes are identified, we could use the system to control when the lactate switch is flipped, making sure that all cells in the culture do switch from lactate production to consumption and making sure the switch flips at the best time to ensure high protein production. Finally, we will merge the two sets of tools to create cells that sense their own lactate concentration and when it gets too high, regulate their own lactate metabolism genes. This will create cells that do not make too much lactate, which should increase the amount of protein that they produce. The 'self-regulating cells' will also serve as a model for how to do this with other traits of interest in the future. This project could, therefore, change the way cells are developed for manufacturing purposes.

今天的许多药物都是由动物细胞培养的蛋白质制成的。几十年的研究已经探索了通过改变细胞的营养物质、细胞本身的基因工程和其他策略来提高这些培养物的生产力的方法。然而，仍然存在限制蛋白质生产总量的挑战。其中一个挑战是代谢废物乳酸的积累，同样的废物会导致你的肌肉在剧烈运动后酸痛。动物细胞培养物中的乳酸浓度随时间变化。早期，由于快速的新陈代谢，它积累起来，但后来细胞用它来制造能量和氨基酸。这被称为乳酸开关。不同批次的细胞在不同的时间经历乳酸开关，翻转开关的触发机制尚不清楚。然而，就蛋白质药物的生产而言，乳酸开关是与更高蛋白质产量相关的积极特征。提出的工作旨在创建一套工具来理解和操纵乳酸开关。第一个目标是创造一种非侵入性指标，可以测量单个细胞中的乳酸浓度。这个工具将使我们能够测量细胞之间的变异程度，这是目前标准技术无法做到的。我们将用它来了解不同的培养条件如何影响细胞中的乳酸积累，以尝试确定每种情况下的可变性。总的来说，这些信息可以用来选择制造条件，在单元之间的差异较小。第二个目标是开发工具，使我们能够改变与乳酸生产和消耗相关的单个酶的表达水平。有了这些工具，我们将能够通过外部信号控制哪些酶与乳酸开关相关来测试假设。一旦确定了酶，我们就可以使用该系统来控制乳酸开关的翻转时间，确保培养中的所有细胞都从乳酸生产切换到消耗，并确保开关在最佳时间翻转，以确保高蛋白生产。最后，我们将合并这两套工具，创造出能够感知自身乳酸浓度的细胞，当乳酸浓度过高时，调节自身乳酸代谢基因。这将使细胞不会产生过多的乳酸，从而增加它们产生的蛋白质的数量。“自我调节细胞”也将成为未来如何对其他感兴趣的特征做到这一点的一个模型。因此，这个项目可能会改变用于制造目的的电池的开发方式。

项目成果

Rapid Antibody Glycoengineering in CHO Cells Via RNA Interference and CGE-LIF N-Glycomics.

• DOI: 10.1007/978-1-0716-1685-7_7
• 发表时间: 2021-10
• 期刊: Methods in molecular biology
• 作者: Pavlos Kotidis;Masue M Marbiah;Roberto Donini;Itzcóatl A. Gómez;Ioscani Jimenez Del Val;S. Haslam;K. Polizzi;C. Kontoravdi

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

中国仓鼠卵巢细胞中的快速抗体糖工程

• DOI: 10.3791/63872-v
• 发表时间: 2022
• 期刊: Journal of Visualized Experiments
• 作者: Kontoravdi C

A call for caution in analysing mammalian co-transfection experiments and implications of resource competition in data misinterpretation.

• DOI: 10.1038/s41467-021-22795-9
• 发表时间: 2021-05-05
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Di Blasi R;Marbiah MM;Siciliano V;Polizzi K;Ceroni F
• 通讯作者: Ceroni F