Project Motivation

CRC & Bacterial Therapy

As the iGEM team of the University of Lausanne (UNIL), we are at the hub of cancer research. As a result, our team naturally gravitated to a project regarding that problem. In Switzerland, one of the more prevalent and deadly forms of cancer is colorectal cancer (CRC), where polyps formed in the colon or rectum turn into cancer. The current treatments can be divided into three categories: local, systemic or a combination of these two. The local treatments are mostly surgeries to remove the cancerous tissue in early stages of the disease (stages 0 and 1). Systemic treatments are chemotherapies or targeted therapies that are taken orally or need to be directly injected into the bloodstream in high dosage. With advanced forms of this illness, a combination of the two treatments is necessary (e.g. a surgery followed by multiple chemotherapy sessions). With such an endemic issue and with treatments being so limited, we decided to contribute to the solution by offering an experimental treatment. We decided to combine local and systemic treatments in an innovative way by delivering a drug directly into the tumoral tissue. To do so, we decided to use a probiotic bacteria (E. coli Nissle 1917) that secretes azurin, a drug with anti-tumoral properties. We chose this nonpathogenic probiotic because it has been proven that it can penetrate tumors better than other strains because of its anaerobic characteristics. This strain of bacteria has already been used in a number of studies and few clinical trials (phase I) to deliver anticancer drugs [1] [2].

Chronotherapy & Oscillations

Our solution is also based on chronotherapy, which is a therapy that coordinates the drug delivery with the circadian cycle to maximize its efficiency, as well as to reduce its side effects [3]. We got inspired by a study in clinical phase II done with 5-fluorouracil and folinic acid (two drugs commonly used to treat CRC) given at a specific time point of the circadian cycle. They concluded that this form of treatment yielded an excellent therapeutic index [4]. So to add some innovation to this mechanism, we decide to couple this bacterial secretion system with a "repressilator" genetic circuit. The repressilator is a synthetic network that uses three transcriptional repressors to produce oscillations [5]. Recently, the original repressilator circuit has been revisited and optimised to produce oscillations that are highly robust and regular [6]. Indeed, cells containing this oscillator keep their phase for hundreds of generations, allowing cells in flasks and colonies to oscillate synchronously without any coupling between them. We decided to use this system in E. coli Nissle to create a secretion of azurin in an oscillatory manner (with a period of ~24h) and thus to deliver the drug with the chronotherapy approach.

Why this approach instead of the classic treatments?

We are creating a treatment where the bacteria would be taken orally and then delivered directly into the tumor, thus reducing the amount of drug necessary for it to be effective. With current treatments, there is a diffusion of the drug into the rest of the body (as it cannot be injected locally in the colon). Hence, there is a need to increase the dosage, which causes side effects that can further debilitate the health of the patient. For the independent chronotherapy aspect: oscillations can continue without external stimuli, so if the best time to deliver azurin is in the middle of the night, the patient will not need to wake up to take it.

So that was how our project was designed, bacterial therapy mixed with a chronotherapy approach: Bacterial Oscillation Therapy, B.O.T., was born!

Kill switch

While doing the project, we received some comments on the biosafety aspect of our treatment. Introducing a genetically modified living organism into the body could cause some unexpected consequences. That is why we decided to add a safety mechanism known as a kill switch. We got inspired by the paper [7] and by a discussion with the Ohio State iGEM team. Our kill switch uses two well-known toxin-antitoxin systems, coupled to two temperature thermosensors as well as a phosphate sensor. To decrease the risk of unwanted escape and spread of the GMO, the thermosensors detect a drop in temperature under 35°C (meaning outside of the body) and leads to a simultaneous increase in toxin production and a decrease in antitoxin production, which leads to the death of the bacteria. If the bacteria were to enter the bloodstream, which could lead to a septic shock, the phosphate sensor would detect the high phosphate concentration of the blood and stop the production of antitoxin, thereby killing the bacteria. This addition puts an extra layer of security into our system, making it more suitable for human use, and the environment.

References

[1] Yu X, Lin C, Yu J, Qi Q, Wang Q. Bioengineered Escherichia coli Nissle 1917 for tumour-targeting therapy. Microb Biotechnol. 2020 May;13(3):629-636. doi: 10.1111/1751-7915.13523. Epub 2019 Dec 21. PMID: 31863567; PMCID: PMC7111071.
[2] He, Lian et al. “Intestinal probiotics E. coli Nissle 1917 as a targeted vehicle for delivery of p53 and Tum-5 to solid tumors for cancer therapy.” Journal of biological engineering vol. 13 58. 28 Jun. 2019, doi:10.1186/s13036-019-0189-9
[3] https://www.merriam-webster.com/dictionary/chronotherapy
[4] Focan C, Kreutz F, Focan-Henrard D, Moeneclaey N. Chronotherapy with 5-fluorouracil, folinic acid and carboplatin for metastatic colorectal cancer; an interesting therapeutic index in a phase II trial. Eur J Cancer. 2000 Feb;36(3):341-7. doi: 10.1016/s0959-8049(99)00282-8. PMID: 10708935.
[5] Elowitz, M., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature403, 335–338 (2000). https://doi.org/10.1038/35002125
[6] Potvin-Trottier L, Lord ND, Vinnicombe G, Paulsson J. Synchronous long-term oscillations in a synthetic gene circuit. Nature. 2016;538(7626):514-517. doi:10.1038/nature19841
[7] "Rational Design of Evolutionarily Stable Microbial Kill Switches" by F. Stirling et al.