Background

Inflammatory bowel disease (IBD) is a disease that causes chronic inflammation of the gastrointestinal mucosa and is difficult to cure completely. IBD is classified into ulcerative colitis (UC) and Crohn’s disease (CD), which cause inflammation in the colon and the entire digestive tract, respectively, and lead to long-term symptoms such as bloody stool, diarrhea, and abdominal pain. IBD develops due to abnormalities in the immune system caused by genetic and environmental factors, but the detailed causes are still unknown [1][2]. Severe IBD can lead to colorectal cancer.

Patients are increasing worldwide. Currently, more than 7 million people suffer from IBD globally [3]. In industrialized countries such as North America, Europe, and Oceania, it is predicted that 1% of the population will be affected by IBD within the next 10 years [4]. In Asia, including Japan, 0.03–0.1% of the population currently has IBD, but within the next 10 years, it is estimated that 0.36–0.66% of the population will be affected [4][5]..

Current Treatments

In current IBD treatment, both UC and CD are mainly treated by combining multiple therapies to control symptoms, induce remission, and maintain remission [6].

The main approaches are drug therapy, dietary therapy, and surgical therapy. Drug therapy suppresses inflammation through oral medication, intravenous injection, or suppositories to induce remission. Dietary therapy avoids irritants and focuses on low-fat diets to prevent worsening of inflammation. Surgical therapy treats the disease by resecting inflamed intestinal segments.

Currently, drug therapy is the mainstay, while dietary therapy is supplementary [7]. Surgical therapy has been decreasing due to the advancement of drug treatments [8].

The currently common drugs are as follows:

The main focus of current drug treatment is to suppress immune activity and reduce inflammation. However, IBD varies greatly among individuals. Thus, it is important to choose and combine drugs with different mechanisms tailored to each patient.

However, even with these modern treatments, it remains difficult to induce and maintain remission. Even in mild cases, one out of three UC patients and two out of three CD patients progress to moderate or severe disease [11]. For moderate to severe cases, only about half of the patients can maintain remission [12].

Therefore, in addition to current immunosuppressive therapies, treatments with different mechanisms and those that promote mucosal healing are expected for the future [13]. At the same time, drugs that directly promote mucosal healing are still far from practical application and face problems such as side effects and high cost [13].

Vicious Cycle of Inflammation

One of the reasons that remission in IBD is difficult is the “Vicious Cycle of Inflammation.” When epithelial tissue is damaged, damage-associated molecular patterns (DAMPs) invade the mucosa [14]. Immune cells inside and outside the intestine sense these factors, leading to inflammation, which further destroys epithelial tissue, thus creating a “Vicious Cycle of Inflammation.” Because of this, even if the symptoms are temporarily suppressed, they are likely to relapse once the drug effect wears off. This makes remission difficult.

Current treatments mainly suppress inflammation and can “weaken” the cycle, but treatments that “repair” epithelial tissue are limited [15]. Due to the diverse pathology of IBD, current therapies cannot fully address mucosa, and only about 50% of moderate to severe patients achieve sustained mucosal healing [12].

Therefore, there is a demand for therapies that can not only suppress inflammation but also heal the mucosa, thereby breaking the cycle from both sides [16][15].

Medication Adherence

In addition, poor medication adherence hinders remission. In diseases requiring long-term medication such as IBD, it is difficult for patients to follow prescriptions by doctors since it often happen that they forget doses or stop taking medication by their own judgement. For example, 5-ASA, which is widely used from mild to severe cases, requires up to three doses per day, which causes a great psychological burden on patients [9]. To achieve effective treatment and reduce patient burden, approaches that reduce the number of doses are needed.

Our Solution: “Xylego”

Our developed “Xylego” solves the vicious cycle and the adherence problem, providing a “low-burden therapy that can be combined with existing treatments” and aims to improve remission rates. Since Xylego acts through a mechanism different from existing anti-inflammatory therapies, it can be used together with them, achieving higher remission rates than current treatments alone. Furthermore, reducing the frequency of administration reduces the psychological burden on patients.

Xylego is an engineered Escherichia coli Nissle 1917 that incorporates three systems:

1.Colonization support

Uses xylitol, which other intestinal bacteria cannot use, as a carbon source to promote colonization, thereby reducing the required administration frequency.

2. Mucosal healing

Heals damaged epithelial cells, inducing remission when combined with anti-inflammatory treatments.

3. Kill-switch

Commits suicide after treatment is completed to minimize side effects.

Details of each design can be found in the Design section.

Usage and Effects

Xylego is used in conjunction with existing medications such as 5-ASA, biologics, and steroids. Xylego is taken orally together with xylitol. While normal probiotics require daily intake[17], Xylego can maintain its effectiveness with reduced frequency of intake thanks to the colonization assistance system, thereby decreasing the burden on patients. The EGF produced by Xylego heals epithelial cell wounds and induces symptom remission. After treatment completion, Xylego commits suicide via the Kill Switch, thus reducing side effects and ensuring high safety.

1. Colonization Support

Escherichia coli Nissle 1917 (EcN) is a probiotic microorganism known to exert beneficial effects on human health and has been reported to be effective in the treatment of inflammatory bowel disease (IBD) [18]. The colonization of Nissle in the intestinal tract enhances its therapeutic efficacy [18]. However, EcN is generally unable to establish long-term colonization, and daily administration is required to maintain its abundance [19].

There are two major factors that make intestinal colonization difficult:

In this study, we aim to promote the colonization of the probiotic EcN by engineering a nutrient competition system and applying the concept of prebiotics. Such an approach is referred to as synbiotics, which is an actively developing field. Synbiotic therapy involves co-administration of probiotics with their corresponding prebiotics, allowing targeted colonization while preventing off-target growth of other intestinal bacteria. Previous studies on synbiotic therapy have shown that co-administration of probiotics with components of human breast milk enabled high-level colonization during the intake period [25], after cessation of bacterial ingestion and continuing only with the breast milk component intake, high levels of colonization persisted for as long as 30 days [26]. These findings demonstrate that synbiotic strategies are effective for promoting probiotic colonization.

However, conventional synbiotic therapy cannot be directly applied to EcN. This is because most sugars and nutrients metabolizable by E. coli are also utilized by many other intestinal bacteria [23], leading to intense competition. Therefore, prebiotics that can be specifically utilized by E. coli are virtually absent.

To overcome this limitation, we aim to enable E. coli to use xylitol, a sugar alcohol that most intestinal bacteria cannot metabolize, as a prebiotic. Our engineered strain, Xylego, expresses xylitol dehydrogenase(xdh) and xylulokinase (xylB) , allowing xylitol to be converted into intermediates of the pentose phosphate pathway, which E. coli natively possesses. This modification enables Xylego to utilize xylitol as a carbon source. This functionality has never been realized before and has a very high level of novelty. If this function can be implemented, we can achieve symbiotic therapy using xylitol with Xylego. By consuming xylitol simultaneously with Xylego, Xyylego can grow without competing with other intestinal bacteria, thereby extending its colonization period in the intestine.

2. Mucosal Healing

In approaches using E. coli for IBD treatment, many projects including 2022 UZURICH [27] have engineered E. coli to produce immunosuppressive molecules. However, this method faces challenges: it is difficult to combine with conventional anti-inflammatory therapies, and production itself is technically demanding, so successful examples are limited [27][28].

Other teams have attempted to produce lactic acid or butyric acid [29], but these metabolites can be partly mimicked by commercial probiotics. We sought a molecule that could overcome these limitations.

Based on expert interviews, we decided to engineer Xylego to secrete epidermal growth factor (EGF), which induces the regeneration of damaged epithelial cells. EGF promotes proliferation and repair of epithelial cells [30]. In a clinical trial with human subjects, a remarkable result showed that 83% of people achieved remission after daily EGF enemas (5 mg in 100 mL)[31]. However, orally administered EGF is easily degraded in the digestive tract and cannot reach epithelial cells effectively. To solve this, Xylego produces EGF directly in the gut, thereby healing damaged epithelial cells.

Other teams have attempted to produce lactic acid or butyric acid [29], but these metabolites can be partly mimicked by commercial probiotics. We sought a molecule that could overcome these limitations. Based on expert interviews, we decided to engineer Xylego to secrete epidermal growth factor (EGF), which induces the regeneration of damaged epithelial cells. EGF promotes proliferation and repair of epithelial cells [30]. However, orally administered EGF is easily degraded in the digestive tract and cannot reach epithelial cells effectively. To solve this, Xylego produces EGF directly in the gut, thereby healing damaged epithelial cells.

By combining Xylego with conventional anti-inflammatory treatments, we break the vicious cycle through dual actions: suppressing inflammation and repairing epithelial tissue, which together promote remission.

3. Kill-Switch

EGF administration during inflammation promotes epithelial healing and reduces colitis-associated tumors. However, continuing EGF administration after wound healing and remission may carry a risk of promoting tumor growth [32]. Furthermore, engineered E. coli must comply with the Cartagena Protocol to prevent environmental spread.

EGF administration after wound healing and remission may carry a risk of promoting tumor growth [32]. Furthermore, engineered E. coli must comply with the Cartagena Protocol to prevent environmental spread.

To solve these problems, we designed an inflammation-dependent Kill Switch. Xylego senses tetrathionate, which increases in inflamed intestines, via the TtrSR sensor [33] and activates a restriction–modification (R-M) system [34]. During inflammation, DNA is protected by methyltransferase, preventing cleavage by restriction enzymes. When inflammation resolves and tetrathionate disappears, production of methyltransferase and restriction enzymes stops, leaving DNA unmethylated. This results in DNA cleavage by the restriction enzyme and death of Xylego.

In addition, intestinal bacteria can undergo horizontal gene transfer or uptake of foreign DNA [35].

The restriction enzyme we selected cuts the ampicillin resistance gene, ensuring that dead Xylego’s antibiotic resistance gene cannot be transferred to other gut bacteria.

Future

Xylego offers advantages such as long-term colonization that reduces dosing frequency and psychological burden for patients, and built-in safety through self-elimination once the disease is cured. It can be used alongside existing anti-inflammatory drugs, potentially improving remission rates.
We have verified that Xylego can acquire a nutritional niche by assimilating xylitol even in the presence of native microbiota. However, future work needs to determine to what extent the dosing frequency can be reduced.
In studies of similar synbiotic therapies, it has been reported that bacterial cells can still be detected for one week after discontinuation of probiotic and prebiotic intake [25]. Therefore, although individual differences may exist, it is expected that administration of Xylego and xylitol every few days will be sufficient to maintain bacterial populations. However, further validation using model organisms such as mice is necessary.
There is also concern about excessive EGF production caused by overgrowth of Xylego. In human clinical trials using EGF-containing suppositories for IBD treatment, concentrations up to 50 ng/ml have been confirmed to be safe. However, there are no safety data for concentrations above this level. If there is a risk of excessive EGF production, it could be controlled by adjusting the intake amount of Xylego or xylitol. If overproduction causes severe adverse effects, it is expected that the majority of the bacterial cells will detach within more than one week after stopping the intake of Xylego and xylitol [25]. If it becomes necessary to immediately eliminate the side effects, taking antibiotics to which Xylego does not have resistance can solve the problem. Professor Tuchiya mentioned that probiotics administered to the host are likely to disappear upon antibiotic treatment.
Potential adverse effects of xylitol on the intestinal environment are also a concern. Intake exceeding 20 g at once can cause bloating, gas, or diarrhea [36]. In similar synbiotic therapy studies, the amount of prebiotic administered at one time was 18 g, and since our project uses a carbon source as a prebiotic, it is considered that our values will not differ significantly from this range [25]. Furthermore, because Xylego utilizes xylitol as a prebiotic, the amount of xylitol remaining in the intestine is expected to be smaller. Therefore, adverse effects caused by xylitol are considered unlikely to occur.
Currently, Xylego cannot express all three systems simultaneously. Therefore, further optimization is required to integrate them into a single bacterial strain.
The optimized Xylego system functions on a plasmid. Colonization support and Kill Switch systems require little change in gene sets across species and can be flexibly adjusted depending on the strain. The EGF production system has so far been limited to Gram-negative bacteria, but there are reports of successful EGF production in Gram-positive bacteria well. Thus, EGF production can be applied to many probiotics. Since patients’ gut environments differ and different probiotic species colonize better in different individuals [21], our system can be implemented across multiple species and administered as a bacterial cocktail, making it applicable to a larger number of patients.
Through the development of Xylego, we aim to bring new hope to IBD treatment.

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