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IBD: Crohn's disease and Ulcerative colitis

What is IBD?

Worldwide, over 6 million people are suffering from Inflammatory Bowel Disease (IBD) 1 characterised by chronic intestinal inflammation and tissue destruction triggered by an aberrant immune response. IBD comprises mainly two conditions: Crohn’s disease (CD) and Ulcerative colitis (UC). Patients diagnosed with CD might show inflammation throughout the entire gastrointestinal tract (GIT), while in patients who suffer from UC, the inflammation is localized mainly in the colon and is continuous. As of today, the exact cause for IBD is still unknown. Research on the exact pathogenesis of IBD suggests a multifactorial mechanism involving genetic, gut microbial and environmental factors. 2 The highest prevalence of IBD has been reported in Europe and Northern America with increasing incidence in newly industrialized countries. 3 Patients suffering from IBD experience symptoms like diarrhea, bloody stools, and abdominal pain. However, symptoms are not confined to the inflamed areas and patients often suffer from other extraintestinal manifestations including mental health problems. Therapeutics such as aminosalicylates, corticosteroids, immunomodulators and inhibitory monoclonal antibodies are being used to alleviate the symptoms. However, these treatment options are known to cause systemic side effects and several cases of therapy resistance have been reported. 4 Therefore, the need to develop a novel specific and targeted treatment option causing less side effects is of utmost importance.

Molecular pathology

In a healthy individual, the epithelial cells of the GIT are perfectly aligned to form an impermeable barrier that bacteria from the lumen cannot cross. Histologically, cells are found tightly bound to each other. 2 However, this epithelial barrier and the protective mucus layer are disrupted in patients suffering from IBD. Spaces between the cells arise and bacteria from inside the gut can invade the mucosa, where they encounter immune cells, initiating inflammation. Inflammation then further damages the epithelial layer, allowing for more bacteria to penetrate the gut wall, increasing the inflammation levels even more. 2 A schematic of the intestinal pathology is shown in figure 2. Very often, IBD is a life-long ongoing vicious cycle of alternating active inflammation and remission phases. 2

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Why IBD?

Choosing our iGEM project was not straightforward because we identified many possible issues we could tackle with synthetic biology. The inspiration for IBD NanoBiotics came from the death of a team member's friend who passed away at the age of 21 because of colorectal cancer that developed out of Crohn's disease. The emotions related to this tragic event pushed the team to design a treatment option and help improve the quality of life of many patients in Switzerland and worldwide suffering from IBD. We are convinced that the microbiome harbours an infinite number of resources we can harness to our benefit. Additionally, by implementing the mindset and tools synthetic biology has given us, we can contribute to pioneering a new field of research that holds unrivalled potential, as our results show.

Our Solution: an engineered probiotic bacterium

We, the iGEM team 2022 from the University of Zurich, want to tackle the lack of specific treatment for IBD and to find a novel solution to improve the daily life of patients by means of synthetic biology. Therefore, we have engineered the probiotic strain Escherichia coli Nissle 1917 to recognize inflammation areas in the intestine via a nitric oxide sensor. This will trigger the local production and secretion of single domain antibodies (nanobodies) against the pro-inflammatory cytokine tumor necrosis factor (TNFα).

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Our engineering chassis

E. coli Nissle 1917 is a probiotic bacterium that can be found as a member of the human microbiota. It was first discovered in 1917 during the First World War by the German medical doctor Alfred Nissle in a soldier who did not show any symptoms after exposure to the Shigella bacterium, known to cause diarrhea and fever. Dr. Nissle discovered that the E. coli strain, which he had previously isolated from the soldier’s stool, could prevent the spread of Shighella bacteria on Petri dishes. 6

Tumor necrosis factor (TNF): an important mediator of inflammation

The TNFα molecule is a crucial regulator for the immune system's inflammatory response. It is mainly secreted by monocytes, macrophages, and T-cells and impacts the proliferation and differentiation of various cell types. Additionally, it stimulates the production of further inflammatory cytokines and chemokines, enhancing the already existing inflammation. In the inflamed areas of the intestine, TNFα recruits immune cells, causing intestinal fibrosis. 2 Blocking the action of TNFα is already the goal of approved anti-TNFα therapies for IBD. However, it is known that patients receiving anti-TNFα biologics, such as the monoclonal antibodies infliximab or adalimumab, develop after a certain time of consistant exposure an antibody resistance which drastically reduces the efficacy of the therapy. Suppression of the immune system or switching to an alternative treatment option are unavoidable and further increase the discomfort of IBD patients. 8

Nitric oxide as an inflammation marker

Our modified E. coli Nissle 1917 is able to sense the inflammation locally through a genetic circuit that responds to nitric oxide (NO), whose level is significantly increased at and near inflammation sites. 2 Moreover, this molecule is able to diffuse through the bacterial cell wall making the use of a particular surface receptor irrelevant. For these reasons, we focused on the design of a NO-sensor using the recently described pNorVβ promoter 5 coupled to a positive feed-back loop with NorR (its corresponding transcription factor).

Nanobodies: potent single domain antibodies

Once sensed, NO will induce the production of nanobodies. Nanobodies, also called single domain antibodies or VHH, only consist of a single heavy chain variable domain, which is able to bind to a specific antigen. The small size of nanobodies (15 - 20 kDa) gives them special abilities, which clearly distinguish them from conventional antibodies. They can locally penetrate barriers (such as tissues) more easily and can withstand extreme environmental conditions, such as high temperatures and low pH. 9 They show high affinity and stability, and their recombinant expression has revolutionized the biotechnology field. Nanobodies have already been discovered in camelid animals back in the '90s. Usage of these nanobodies in the clinic often requires an additional step called "humanization" in order to reduce unwanted immunological reactions upon administration. This step describes the exchange of one or a few specific amino acids that are recognized as "foreign" by the human immune system. 10 Still today, camelid animals are infected with the antigen of choice and effective nanobodies are obtained from their blood. However, new manufacturing technologies have been developed, allowing the screening of new candidates by using naive or synthetic libraries in combination with phage and ribosome display. The usage of synthetic libraries results in the generation of so-called "sybodies". 11

Because of their small size, nanobodies can be produced and secreted to the extracellular environment from bacteria. 12 For our project, we have worked with humanized anti-TNFα antibodies 10 to avoid a possible immune reaction in vivo. We have coupled these nanobodies to a tag that allows their selective secretion via the hemolysin A secretion system, a type I secretion system found in uropathogenic E. coli strains. This one step secretion system consists of three proteins: TolC, hemolysin B (HlyB) and hemolysin D (HlyD). These three proteins build a continuous channel through which the HlyA toxin would originally be secreted in a one-step manner. Interestingly, the secretion signal is not found on the N-terminal site as it is for most secretion tags, instead it is found at the C-terminal end and the signal sequence is not removed during secretion. Scientists have identified the secretion signal and were able to secrete various proteins of different sizes with the hemolysin A secretion machinery. 12

IBD Nanobiotics: an engineered E.coli Nissle 1917 to fight inflammation in IBD patients

Summarized, the engineering of our chassis E. coli Nissle 1917 consists of:

However, IBD NanoBiotics could not have taken its shape without the vital contributions of the human practices and dry lab team. The human practices team talked to experts, patients, and doctors to implement their experiences on our therapy design and connect the world to our project. The dry lab team simulated the interactions in the GIT between IBD NanoBiotics and inflammation sites to conduct an in-silico proof of concept.

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Integrating a mathematical IBD model

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Connecting IBD NanoBiotics to the world

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Further reading:

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References:

1. GBD 2017 Inflammatory Bowel Disease Collaborators (2020). The global, regional, and national burden of inflammatory bowel disease in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. The lancet. Gastroenterology & hepatology, 5(1), 17–30
2. Zhang YZ, Li YY, Inflammatory bowel disease: pathogenesis, World J Gastroenterol. 2014;20(1), 91-99
3. Qingdong Guan, 2019, “A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Disease
4. Zhaobei Cai, et al., 2021, “Treatment of Inflammatory Bowel Disease: A Comprehensive Review”, Frontiers in Medicine
5. Xiaoyu J. Chen et al., 2021, Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells
6. Ulrich Sonnenborn, 2017, "100 years of E. coli strain Nissle 1917", Oxford University Press's blog
7. St-Pierre F. et al., 2013, One-Step Cloning and Chromosomal Integration of DNA
8. Pithadia, A. B., & Jain, S. (2011). Treatment of inflammatory bowel disease (IBD). Pharmacological reports : PR, 63(3), 629–642.
9. Harmsen, M.M., De Haard, H.J. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77, 13–22 (2007).
10. Silence, Karen, Lauwereys, Marc, De Haard, Hans, et al. "Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor", Int. Publication Number: WO 2004/041862 A2, 21 May 2004
11. Zimmermann, I., Egloff, P., Hutter, C.A.J. et al. Generation of synthetic nanobodies against delicate proteins. Nat Protoc 15, 1707–1741 (2020).
12. Ruano-Gallego, D., Fraile, S., Gutierrez, C. et al. Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact 18, 47 (2019).