While inflammatory bowel disease (IBD) manifests in the gut, the drugs available to treat it are less focused. “The biggest problem with the drugs that are actually used is that they have global effects,” said Cauã Westmann, a synthetic biologist at the University of Zurich and a mentor to the 2022 UZurich IBD NanoBiotics International Genetically Engineered Machine (iGEM) Team. An iGEM team consists of a group of students that participates in a yearly synthetic biology competition where they build biological systems to solve real-world problems. The 2022 UZurich IBD NanoBiotics iGEM team decided to try to find a more localized solution to treating IBD by engineering bacteria to sense and respond to gut inflammation, a common feature of IBD (1). To do this, they focused on one biomarker of inflammation: nitric oxide (NO).

Westmann and his team chose E. coli Nissle 1917 (EcN) as the vehicle for their targeted IBD drug because it’s a probiotic that has been studied as a member of the gut microbiome for over one hundred years. The team engineered EcN to house three biological parts that work together: a nitric oxide sensor to detect inflammation, a nanobody that binds tumor necrosis factor alpha (TNFα), and a secretion system to expel the nanobody from the bacterium. As TNFα is a pro-inflammatory cytokine with a role in IBD, the team hoped that sequestering it with a nanobody would reduce inflammation.  

The team began by characterizing each component individually before combining the three together. First, they assessed the NO sensing system using a fluorescent reporter. Then, they measured nanobody activity using in vitro binding assays and cell-based assays and compared the nanobody’s activity to adalimumab, a monoclonal antibody currently on the market to treat IBD. They found that the nanobodies bound to TNFα as well as adalimumab did. To verify their effect on cellular functions, the researchers found that the nanobodies decreased the gene expression of the pro-inflammatory cytokine interleukin-1 β in cells exposed to TNFα. Lastly, they tested the components together, finding that the engineered bacterium produced and secreted nanobodies based on the NO concentration.

“[We] put together different modules that already exist … [and] characterized them in a new context,” said Westmann. 

In addition to designing their circuit, the team developed a mathematical model to analyze the interactions between their engineered bacteria and inflamed tissue. This model uses data from previous literature to analyze parameters such as NO concentration, the number of bacteria present, TNFα production, and how well the nanobody binds to TNFα to predict how their engineered system would behave in the gut.

“The model is very simple. There are complicated mathematical models in synthetic biology, but there was nothing on that specific topic of gut colonization and the diffusion of nanobodies and nitric oxide,” said Westmann.

The model is very simple. There are complicated mathematical models in synthetic biology, but there was nothing on that specific topic of gut colonization and the diffusion of nanobodies and nitric oxide. 
- Cauã Westmann, University of Zurich

Wei Huang, a synthetic biologist at the University of Oxford who previously developed a nitric oxide biosensor in EcN, said, “This is a very nice paper and is a good demonstration [of applying] the gene circuit biosensor to respond to the disease biomarker and release the nanobody to treat disease” (2). He added, “[It] is a very good showcase [of] synthetic biology.” 

Recent work from another group at Massachusetts General Hospital used an endogenous gut bacterium to produce nanobodies in mice (3). Although the bacterium expressed the nanobody all the time, the team saw inflammation decrease in the gut.

Westmann’s team has not tested their system in vivo yet. While he thinks that engineered probiotics won’t be on the market for some time, he is hopeful about the future for such a drug delivery system.

References

1. Weibel, N. et al. Engineering a Novel Probiotic Toolkit in Escherichia coli Nissle 1917 for Sensing and Mitigating Gut Inflammatory Diseases. ACS Synth Biol  13, 2376–2390 (2024).
2. Chen, X. et al. Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells. ACS Synth Biol  10, 2566–2578 (2021).
3. Lynch, J. et al. Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut. Cell Host Microbe  31, 634-649 (2023).