Team:Peking/Human Practices
Integrated Human Practices
Microbial therapies are derived from naturally occurring microorganisms that have often been genetically modified to reduce systemic pathogenicity and increase anti-cancer efficacy. Compared with most other therapeutics, the effectiveness of tumour-targeting bacteria is not directly affected by the ‘genetic makeup’ of a tumour. Bacteria initiate their direct antitumour effects from deep within the tumour, followed by innate and adaptive antitumour immune responses. As microscopic ‘robotic factories’, bacterial vectors can be reprogrammed following simple genetic rules or sophisticated synthetic bioengineering principles to produce and deliver anticancer agents on the basis of clinical needs (Figrue 1) [1].
Figure .1 Current design of bacteria cancer therapy.
Safety is an important issue on bacteria therapy, which has been mentioned in many literatures [2][3], and has aroused wide concern (Figure 2). One of the major problems of bacteria therapy owe to their infectious nature, especially when the therapeutic effect is strongly associated with toxicity. In many cases, bacteria showing satisfying safety profile didn’t have any significant therapeutic effect [4]. To find out precisely how urgent safety concern is, and whether our system can help clinical researches, we interviewed some experts in corresponding fields including synthetic biology and microbiology. Their suggestions helped us better comprehend the key points in bacteria therapy and motivated us to optimize our project.
Figure. 2 Studies of the safety issues on bacteria therapy are increasing
Prof. Zhang Lixin highly appreciated our project which could easily get a bacterial strain of stable slow growth rate. He attached great importance to the precise control of cell density. Current methods of obtaining therapeutic bacteria of stable slow growth rate include changing the nutrient content of culture medium, or adding antibiotics that take effect by blocking specific biological process (for example, sublethal dose of chloramphenicol inhibits translation) [5]. While these treatments helped to reveal the coupling between general growth parameters, they have their disadvantages. Nutrition-limiting culture medium can significantly slow down biomass accumulation and expression of protein of interest; preparing medium containing different levels of nutrients can also be too complex to operate. The use of antibiotics poses the risk of drug abuse and antibiotic resistance. Their limitations make them even more unpractical if we hope the engineered bacteria to function in human body. Another method of obtaining slow growing therapeutic bacteria is site-directed random mutagenesis, which can be extremely burdensome or time-consuming. Thus, he thought our system may have a broad application prospect. He provided some literatures to help us confirm the method of demonstrating CRISPRri [6][7]. He also took special interest in whether the growth inhibition effect could be reversed. With his suggestions, we designed an experiment to reverse the slow-growing state of our bacteria. See our Demonstration page for more detailed data and analysis.
Prof. Li Tong reminded us that if we wanted to apply our system to clinical scenarios, the stability of CRISPR in chassis would be very important. As horizontal gene transfer (HGT) is a quite a normal phenomenon, we plan to knock our system into bacterial genome to get a more stable strain. See our Future Plan page for planned characterizations.
Another interviewee, Dr. Dong Yuxuan agreed that bacteria density control could improve the safety of engineered bacteria. By controlling cell density to a predefined level, we can keep the balance between bacterial toxicity and therapeutic effect, which would be important in the clinical studies of microbial anti-cancer therapy. After sharing preliminary data with him, he was curious about the physical conditions of our bacteria. He underlined the physical condition of bacteria in maintaining its normal function, which prompted us to analyses the transcriptome of bacteria in the future. See our Future Plan page for planned characterizations.
Knowing how to measure the effect of our system is as important as knowing what we should do. Throughout June and July, we made effort in characterizing the effect of our system with OD600, measured by microplate readers. But the results were not so satisfying. Finally, we found that our bacteria were significantly elongated, which made OD600 not a suitable parameter for cell number. So we used traditional microscope and hemocytometer to measure cell number and cell morphology, which was troublesome. Conversation with Prof. Luo Chunxiong enlightened us to consider more advanced and automated devices. Prof. Luo suggested us using microfluidic chips to measure cell-growth and track single cell’s behavior. With his help, we used microfluidic chips as our major method to characterize our system and generated large mass of quantitative results.
In conclusion, by accessing literatures and consulting with experts, we confirmed that safety is very important in microbial therapy, and that cell density control could be helpful to balance safety and therapeutic effect in bacteria. Our project provides a universal and convenient tool to control the growth of bacteria, which is meaningful in bacteria therapy and many other scenarios. Through controlling DNA replication initiation, we can control growth rate, unrelated gene expression level, length and even adhesion of engineered bacteria, which will be of great help to the preclinical and clinical studies about bacterial therapy.
Integrated human practices helped us find the application prospects for our project and proposed the optimization of the project design. From the start to the end, we learned a lot from human practices.
Reference:
[1]Liu, Sai et al. “Tumor-targeting bacterial therapy: A potential treatment for oral cancer (Review).” Oncology letters vol. 8,6 (2014): 2359-2366. doi:10.3892/ol.2014.2525.
[2] Forbes et al. White paper on microbial anti-cancer therapy and prevention. Journal for Immuno Therapy of Cancer (2018) 6:78
[3] Shibin Zhou et al. Tumour-targeting bacteria engineered to fight cancer. Nature review. (2018)18:727
[4] Toso et al. Phase I Study of the Intravenous Administration of Attenuated Salmonella typhimurium to Patients With Metastatic Melanoma. Clin Oncol . 2002 January 1; 20(1): 142–152.
[5] Scott, M., Gunderson, C. W., Mateescu, E. M., Zhang, Z. & Hwa, T. Interdependence of Cell Growth and Gene Expression: Origins and Consequences. Science (80-. ). 330, 1099–1102 (2010).
[6] Hansen FG and Atlung T (2018) The DnaA Tale. Front. Microbiol. 9:319
[7] Flemming G. Hansen, et al. Insights
into the Quality of DnaA Boxes and Their Cooperativity. J. Mol. Biol. (2006) 355, 85–95