Understanding and manipulating lactate metabolism in single cells

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.

Chinese Hamster Ovary (CHO) cells are used in the production of more than half of therapeutic glycoproteins. Lactate is a key waste metabolite in CHO cultures and shows complex dynamics over time. Early in culture lactate accumulates, but towards stationary phase, cells undergo a metabolic shift to lactate consumption. This 'lactate switch' is associated with high productivity, but different cell lines do not show uniform behaviour with respect to lactate metabolism and the biological basis for the lactate switch is not currently known. Further, high concentrations of lactate damage cell growth and productivity. The proposed work would develop tools to understand and manipulate lactate metabolism in CHO cells, which will underpin the development of higher performance cultures with optimised metabolism.

To facilitate understanding, we will develop a genetically-encoded biosensor for intracellular lactate concentration. The biosensor will enable both the high throughput measurement of lactate concentration changes over time as well as single-cell measurements to gain insight into the population variability of metabolism under different bioprocessing conditions. This information is unobtainable by conventional assays and can lead to the design of better bioprocesses. To manipulate lactate concentration, we will use a Cas9-VPR fusion, where the length of the guide RNA and the target location allow for the simultaneous up- and downregulation of gene expression in the same cells. We will use this to change the expression level of selected targets to artificially flip the lactate switch. Finally, both sets of tools will be connected to develop self-regulating cells that can sense their own lactate concentration and modulate gene expression in response. Such a cell line could cause a paradigm shift in bioprocessing where currently offline analyses are used to monitor cell metabolism and an operator is required to make any changes.

The proposed work will develop tools for the measurement of lactate concentration in single cells, modulate the expression level of metabolic targets associated with lactate metabolism, and combine these two strands to develop self-regulating cells. The potential beneficiaries from the work are described below.

Staff Training: The PDRA would benefit from the multidisciplinary environment of the Imperial College Centre for Synthetic Biology and interactions with the Network of Excellence in Industrial Biotechnology. S(he) would also gain cutting-edge skills in synthetic biology and the principles of guide RNA design, which are translatable to genome editing projects in the future. The PDRA would also have access to the unique training programs of SynBiCITE: the 4-Day MBA and LeanLaunchPad, enhancing their transferable skillset.

Researchers in synthetic biology, biochemistry/metabolism and bioprocess engineering: The knowledge generated by the proposed work would benefit a number of different research communities. It would lead to a greater understanding of mammalian cell metabolism and the extent of population variability of intracellular metabolite concentrations. It is likely that different cell line engineering or bioprocessing strategies will result in more or less population variability, which could also serve as a driver for evolution. Understanding this interplay could allow for the design of better, more resilient bioprocesses. The proposed work would also provide a basis for testing various hypothesis about the origin of the lactate switch in CHO cells, which is currently not understood. Finally, the development of self-regulating cells with a clear industrial application would be of interest to researchers in synthetic biology.

Industrial R&D: The tools developed as part of the proposed work could benefit scientists and bioprocess engineers working in industrial research and development. The lactate biosensor could be useful for process development as it would allow rapid, non-destructive, high sensitivity measurements of lactate concentration in small volumes or single cells. The fluorescent readout enables high throughput measurements and so could be useful for empirical screening of process conditions and media/feed formulations. Furthermore, the methodology for modulating the expression of metabolic targets could be useful for cell line development- both for inducing changes in lactate metabolism using our designed guide RNAs and also for extending the methodology to other parts of metabolism relevant to productivity and/or product quality. Finally, in the long-term, the results from the third objective (develop self-regulating cells) could revolutionise biological manufacturing in industry, where offline analyses and manual adjustment of culture conditions are the current norm. Once the concept has been successfully demonstrated for lactate, it could also be adopted for other metabolic waste products and intermediates. The concept could also be extended beyond the production of recombinant proteins in CHO cells to other hosts producing different product classes including small molecules, biofuels, and materials where the control over the accumulation of intermediates can be vital to increasing productivity.

Cancer biologists: Metabolism is highly conserved among mammalian cells and CHO cells share many metabolic phenotypes with cancer cells. Thus, cancer biologists will benefit from the tools developed in Objectives 1 and 2 of this proposal, which could be redeployed to study metabolism of cancer cells in vitro and in vivo.

Healthcare funders, and patients: In the long-term, the work proposed could lead to improved CHO systems with higher production yields of therapeutic proteins, which could decrease the overall cost of these lifesaving treatments many of which are currently extremely expensive. This would in turn reduce the costs to the healthcare system and would increase patient access.