The project of the iGEM Team Tübingen 2019 is the development of a microbial chassis, which can be used as a probiotic E.Coli Nissle 1917, that secretes a drug against Type 2 Diabetes Mellitus. The microbial chassis will consist of a CRISPR/Cas3 based kill switch system, which senses the environmental conditions it is exposed to, consequently determining whether the survival, and integrity of the nucleic acids, of the bacterium can be provided. This kill switch system should enable the use of a genetically engineered organism (GMO) in therapy, as the threat of release into the environment is reduced, since it cannot survive outside the human gut. Thus, it is a concept that can be used for a diverse spectrum of therapies by exchanging the drug and the conditions.

The gene of interest we are focusing on, Exendin-4, serves as a drug for Type 2 Diabetes Mellitus and is an incretin mimetic originating from a lizard called Gila monster (Heloderma suspectum). This incretin mimetic is already used in the pharmaceutical industry for the therapy of Type 2 Diabetes Mellitus and is also known as Exenatid. The human analogue to Exendin-4 is glucagon like peptide 1 (GLP-1), which is secreted from the L-cells in the small intestine, if nutrients are available in the gut. GLP-1 and its analogues cause the so-called incretin effect, which means that the release of insulin as response to sugar is higher, when sugars are consumed orally in contrast to when they are given intravenous.

Project Inspiration

When committing to iGEM, we decided we wanted to a) design a project which uses a new system of the field of synthetic biology and b) make an impact with a product, which can actually be of use to the society. So when Dr. Pengfei Xia, one of our instructors proposed to design a new kill-switch system for bacteria, based on the Type I CRISPR system of CRISPR/Cas3, we were determined to not only implement the system, but also find a way of making it a useful, universally applicable, tool.

As there are four students from the field of molecular medicine within our team, we rapidly got stuck on brainstorming in the field of medical applications for our Kill-Switch systems, whether there was a disease which could easily profit from a new therapeutic strategy. This led us to Diabetes Mellitus, one of the main diseases responsible for premature death worldwide [16]. In 2014, there were 422 Million adults with a diagnosed Diabetes Mellitus worldwide [1], an alarmingly high number, which inspired us to look into alternative treatment strategies of Type 2 Diabetes Mellitus patients, as they make up the largest group of Diabetes patients. There are currently many options for treatment available, however all of these treatments require the patient to actively participate and comply to his or her therapy scheme. As compliance and persistence are key factors for therapeutic success, we, the iGEM Team Tübingen, want to revolutionize the treatment and its administration by using Synthetic Biology and our CRISPR/Cas3 system.

In order to do so, we thought about changes in therapy, which would make it easier to comply to the therapy scheme and easily came up with issues about daily injections and multiple drugs, that need to be taken at certain times a day. Overall, we came to the conclusion that a probiotic bacterium, that synthesises a drug when it is needed and therefore lifts the burden of application from the patient, was our way to go. On top of that, we realized that in 2017 Team AQA_Unesp had already tried to target Type 1 Diabetes Mellitus with a probiotic, hence we felt that the viability of the idea was supported by this [7]. Additionally, this approach would allow us to not only use our bacterium with the CRISPR/Cas3 system as biofactory, but also make it the therapeutic agent which is applied, making the biosafety function of CRISPR/Cas3, not only a tool, but also a requirement for safe application.

The idea of using GMOs as probiotics, is, in general, very interesting for chronic diseases. Therefore, we want to use the application in Type 2 Diabetes Mellitus as an example of the strengths and limitations of such a system, and use our public involvement to gather the society’s perception and opinion of such a therapeutic strategy. Consequently, we set ourselves some goals for the project, which are i) the proof of principle of the CRISPR/Cas3 system ii) the production of Exendin-4 and iii) the incorporation of the public and several sub-projects for communication and education into our project.

Overall, we consider our project to be an important trial for the use of synthetic biology in long-term therapy, where a patient is not restricted to stay within a facility, but can live normally and without a financial or other burden of his or her disease, as the probiotic bacteria in the microbiome will do their job for the medication. We hope, to have designed a safe system which will prohibit the survival of GMOs within the environment and enables the use of probiotics.

Project Description

Type 2 Diabetes Mellitus and Incretin Mimetics

GLP-1 and Exendin-4 will bind to their receptor, a G-protein coupled receptor on the pancreas surface, which will cause an intracellular cascade activating an Adenylate Cyclase. If ATP is available within the cell, which means if the cell has already taken up sugar and run through glycolysis, cAMP will be produced. Overall, this will result in prolonged depolarization and calcium ion influx and consequently more secretion of insulin from the beta cell [2]. This increase in insulin secretion can overcome the relative lack of insulin in the periphery, which occurs in Type 2 Diabetes Mellitus.

GLP-1 and its analogues are especially interesting for the treatment of Type 2 DM, since they do not only have a direct effect on the pancreas, but also decrease gastrointestinal motility and secretion, hunger and the emptying of the stomach [2], thus helping in weight reduction. Additionally, it is considered to be cardioprotective [2]. This is very important for the therapy, since many cases of Type 2 DM are caused by an unhealthy lifestyle and obesity. On top of that, this effect proposes the outlook of using the drug as medication against obesity and adipositas, without the indication of diagnosed type 2 DM or in stages of prediabetes, in addition to a lifestyle intervention.

Generally, Type 2 Diabetes Mellitus is a metabolic disease, which affects about 10% of the population worldwide, with increasing numbers. One of its major symptoms is the relative lack of insulin, which causes decreased uptake of glucose into cells, resulting in an energy crisis within the cells. In Type 2 DM, the body compensates this relative lack of Insulin by secreting more Insulin, which causes stress for insulin-producing beta cells of the pancreas. This increased stress will lead to the destruction of the beta cells, leaving the body with an absolute lack of Insulin, making insulin substitution necessary.

In Type 2 DM the relative lack of Insulin can have multiple reasons. It can be induced by overconsumption of sugar-containing food, decreasing the sensitivity of the Insulin receptors, as they are downregulated. But it can also have genetic reasons, like a mutation in an essential gene.

We decided to formulate our drug as a probiotic, since it bears multiple advantages. First of all, a probiotic can implement itself in the human guts microbiome, making it possible that the drug does not have to be taken daily according to a strict therapy scheme. This will also address the group of patients, who are not able to stick to a therapy scheme, for instance the elderly or people with fear of other forms of formulation. Also, it was shown by multiple studies, that probiotics can, in general, positively influence the course of Diabetes mellitus [5,6].

As a consequence, our probiotic can be released into the environment due to excretion. For biosafety reasons and for strict control of our therapy, we wanted to implement a new biocontainment chassis, based on a CRISPR/ Cas 3 system.

CRISPR/Cas3-system

Exendin-4, a GLP-1 analogue

Wetlab Project Plan

Modelling and Simulation

Drylab

Human Practice

While checking the scientific possibilities to produce a therapeutic probiotic, we soon realized that we would have to engage the public with the goal of designing a product and Human Practice had to be an important part of our project.

Therefore, we divided our Human Practices project into four phases:
Phase I: General Considerations
Phase II: Human Practice Events of the iGEM Team Tübingen
Phase III: Implementing new strategies based on our public integration and outreach
Phase IV: Conclusion and Reflection

Our Human Practice projects therefore are:

Biosafety measures

Obviously, biosafety is very important if a GMO is used as a probiotic. That’s why, on the one hand we have invested a lot of energy into the design of our CRISPR/Cas3 kill switch system, and on the other hand have intensively confronted ourselves with the iGEM regularies and the german law. As a support, Jörg Schibel and Brigitte Walderich helped us evaluate the safety for parts, we weren’t quite sure whether they were Biosafety Level 1 conform, like the CPP.

Political involvement

Since a GMO as a probiotic is currently not allowed to be used in Germany or the European Union, we are interested in whether politics is moving in this field and what has to be done to achieve change. Hence, we are currently planning a so-called “Fishbowl”, which is a discussion round with one seat that can be taken by anyone from the audience. We want to invite people from the group of Young Europeans, who belong to different parties, and discuss Synthetic Biology, the dynamic of the European Law etc. with them, in order to grasp where we need to take action to promote change, which allows a more diverse usage of Synthetic Biology outside the lab.

Social impact and public reach out

From the beginning, we wanted to choose a project that matters not only to us but can be helpful for the public. Since, Diabetes is now considered a pandemic with a substantial threat to human health around the world and currently, at least 400 Million people worldwide suffering from Diabetes [16], we are sure to have chosen a topic, which requires more attention. Especially, with Germany as a comparatively small country, being currently on rank 9 of total diabetes cases, with 7.5 Million patients [16].

This pandemic is a result of an unhealthy and sedentary lifestyle, which promotes obesity and lack of overall fitness, resulting in not only a health, but a socioeconomic crisis as well. That’s why we want to start a preventive campaign for Diabetes and Adipositas in cooperation with Dr. Eike Latz, Institute Director of the Institute of Innate Immunity Bonn and his partner Dr. Anette Christ. Additionally, we have met with Prof. Andreas Fritsche, the deputy director of the Institute for Diabetes Research and Metabolic Disease of the Helmholtz Institute Munich at the Eberhard-Karls University Tuebingen, who has advised us to set another focus on solely adipose people, since our drug has the potential to help them lose weight. Through Prof. Fritsche, we also got insight into the clinical aspects and patient’s acceptance of our product. Overall, Prof. Fritsche made an estimation, that about two thirds of his Type II DM patients would probably be willing to try a new therapy, such as ours. However, he is concerned that the general belief that genetically modified organisms are dangerous and bad, may pose a threat to our idea.

Consequently, we decided to launch a survey [19]. To design our survey, we wanted to fulfill the criteria proposed by iGEM. Hence, informed consent and privacy/ data protection are highly important in survey conduction. Also, our survey design had to be specific and unambiguous, with the goal of not compromising our survey by insinuating a “correct” response to any question. For this purpose, we used the tools provided for free for students by www.umfrageonline.com. As in our case, the participants volunteered to conduct our survey, our survey is a “nonprobability” survey and the results do not represent a randomized group. However, our study had the purpose to help us with refining our project and give us insight into the perception of new therapies and Synthetic Biology. We launched the survey when one of our teammates explained the project at a science slam, which gave us a great audience and allowed for data collection. However, the students present were not really a group of high Diabetes Mellitus prevalence, therefore, we also asked several Diabetologists in Tübingen to spread the survey amongst their patients. The data is currently being evaluated, so we can draw our conclusions to the project.

Lastly, Prof. Fritsche proposed us to hold a talk about our project in September 2019 for the Diabetes patients, who have taken part in his studies. We are looking forward to presenting the results of our research and getting their opinion on our project.

Sources

[1] World Health Organization. (2016). Global Report on Diabetes. [online]Available https://apps.who.int/iris/bitstream/handle/10665/204871/9789241565257_eng.pdf;jsessionid=8D5E18D2A5627102F238B7D2DA7AD2C4?sequence=1 [Accessed 26.03.2019].
[2] Jens Juul Holst. The Physiology of Glucagon-like Peptide 1. (2007). Physiological Reviews. p.1409-1439. https://www.physiology.org/doi/abs/10.1152/physrev.00034.2006
[3] Lim, Gareth E., Brubaker, Patricia L. Glucagon-Like Peptide 1 Secretion by the L-Cell. (2006). 10.2337/db06-S020. Diabetes. p. S70-S77
[4] Copley, Kathrin & McCowen, Kevin & Hiles, Richard & L Nielsen, Loretta & Young, Andrew & Parkes, David. (2006). Investigation of Exenatide Elimination and Its In Vivo and In Vitro Degradation. Current drug metabolism. 7. 367-74. 10.2174/138920006776873490.
[5] Duan F, Curtis KL, March JC. Secretion of insulinotropic proteins by commensal bacteria: rewiring the gut to treat diabetes. Appl Environ Microbiol. (2008);74(23):7437–7438. doi:10.1128/AEM.01019-08
[6] Duan FF, Liu JH, March JC. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes. (2015);64(5):1794–1803. doi:10.2337/db14-0635
[7] http://2017.igem.org/Team:AQA_Unesp
[8] http://2012.igem.org/Team:NTU-Taida/Project/Circuit
[9] Sicard JF, Le Bihan G, Vogeleer P, Jacques M, Harel J. Interactions of Intestinal Bacteria with Components of the Intestinal Mucus. Front Cell Infect Microbiol. (2017);7:387. Published 2017 Sep 5. doi:10.3389/fcimb.2017.00387
[10] Barnhart MM, Lynem J, Chapman MR. GlcNAc-6P levels modulate the expression of Curli fibers by Escherichia coli. J Bacteriol. (2006);188(14):5212–5219. doi:10.1128/JB.00234-06
[11] Konopka JB. N-acetylglucosamine (GlcNAc) functions in cell signaling. Scientifica (Cairo). (2012);2012:489208. doi:10.6064/2012/489208
[12] https://www.uniprot.org/uniprot/P0A8V6 accessed: 19 Jun 2019
[13] Feng Y, Cronan JE. Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PLoS One. (2012);7(9):e46275. doi:10.1371/journal.pone.0046275
[14] Federle MJ. Autoinducer-2-based chemical communication in bacteria: complexities of interspecies signaling. Contrib Microbiol. (2009);16:18–32. doi:10.1159/000219371
[15] Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; (2000). Catabolite repression of the lac operon: positive control. Available from: https://www.ncbi.nlm.nih.gov/books/NBK22065/
[16] IDF (International Diabetes Federation. IDF Diabetes Atlas. (2017). Eighth Edition. Available from: https://diabetesatlas.org/resources/2017-atlas.html
[17] https://www.experimenta.science/
[18] https://www.un.org/sustainabledevelopment/ accessed: 01 May 2019 logos retrieved from here according to their guidelines
[19] https://igem-tuebingen.com/human_practices survey available under the link
[20] https://2019.igem.org/Team:Costa_Rica/SDG-Challenge iGEMxSDGs page
[21] https://forms.gle/xtoeMHuAMpDHeaCc9 Team Taipei video conference

Team:Tuebingen/Description

Project Inspiration and Description

Abstract Project Description