Abstract

In our journey of iGEM, it is crucial that our project does not become self-serving. We have a responsibility to the world to carefully consider both the positive and negative effects that Gassle may have. From the moment of initial inspiration, we identified key stakeholders such as researchers and patient advocacy groups. We engaged in discussions with them, seeking their feedback and insights. We also reached out to additional stakeholders that emerged during these conversations to further enhance our project.

Human Practices Stakeholders

• Academics (for deciding on substances, considering potential scenarios, and building models)
• Patient advocacy groups (to determine if our solution is truly effective from the patient's perspective)
• Medical professionals (to assess whether our approach addresses the challenges)
• Industry (involved in regulatory and practical aspects of capsule commercialization)

Fig. 1. Our Relationship with Stakeholders

The direction of the arrows is important: solid arrows indicate the strength of the relationship.

Through discussions with these stakeholders, we improved the project in four key areas:

1. The Rationale for Choosing Propionic Acid
2. Anticipated Use Cases for Gassle
3. Comparison with Fecal Calprotectin
4. Mechanism for Delivering the Drug to the Large Intestine
5. Challenges in Implementation

1. The Rationale for Choosing Propionic Acid

We also considered using a suppository to deliver the substance to the large intestine, but opted for an oral medication due to its simplicity.

Regarding detection through smell, we received feedback from IBD Plus that identifying the condition by the smell of gas could cause discomfort for patients. Some patients already struggle with flatulence, and increasing the unpleasant odor would not be ideal. In response, we are working on controlling the growth of E. coli using the Herpes Simplex Virus Thymidine Kinase/Ganciclovir system (HSV-TK/GCV system), which would allow us to eliminate Gassle at any desired moment before it produces an odor.

In the early stages of the project, we considered using a pigment to color stool instead of relying on the smell of gas, but we abandoned this idea due to concerns about the pigment adhering to the intestines. Similarly, we explored the idea of producing a pleasant odor, but ran into problems such as the high odor threshold, leading us to abandon that option as well.

Ultimately, we chose propionic acid because of its low olfactory threshold and anti-inflammatory properties, making it useful for diagnostics. However, in the future, we also hope to explore the possibility of generating pleasant odors.

2. Anticipated Use Cases

• For undiagnosed patients: To help them distinguish between IBD (particularly Ulcerative Colitis (UC)) and Irritable Bowel Syndrome (IBS) before seeking medical care.
• For patients already diagnosed with IBD: To detect the transition from the remission phase to the active phase of the disease.

Concerns Regarding Increased Burden on Patients with Early Detection

Feedback from IBD Plus (a patient support group) raised concerns about whether early detection might increase patients' financial burden if they do not qualify for rare disease designation. According to Financial Planner Ms. Kuroda, this is indeed a possible scenario in Japan, where some patients diagnosed with IBD do not meet the criteria for rare disease designation.

However, the reason for rare disease designation is that such conditions often lead to increased medical expenses and difficulties in maintaining employment, which can put financial strain on households. Early detection and appropriate treatment could lead to either a cure or symptom relief, reducing not only medical costs but also potential lost income, as patients would be more likely to maintain stable employment. When considering informal care costs (the unpaid care provided by family members), early detection clearly has significant value. It became evident that early detection does not increase the burden on patients.

2-1. For undiagnosed patients: To help them distinguish between IBD (UC) and IBS before seeking medical care.

We discussed with Professor Hisamatsu (Professor of Kyorin University) that since Crohn's disease can occur not only in the colon but also in the small intestine, our project is primarily capable of detecting ulcerative colitis. This project has great potential in distinguishing between IBD (inflammatory bowel disease) and IBS (irritable bowel syndrome). IBD and IBS are difficult to diagnose because their symptoms are similar, and they are often confused, especially in the early stages. Due to these reasons, IBD diagnosis can be delayed, which may cause the patient to miss the critical treatment window when medication is most effective. Through our interviews with IBD Plus, we learned that some patients are hesitant to visit the hospital, suspecting they may have stress-induced IBS.

Traditionally, IBS diagnosis is primarily conducted through diagnosis by exclusion (a method where other diseases are ruled out to confirm IBS)[1].

To investigate whether the distinction between IBD and IBS is recognized as significant in other countries, we reached out to the patient support community IBD India and conducted a survey. However, according to interviews with IBD India, it was found that the social significance of distinguishing between IBD and IBS in India is not as pronounced.

According to IBD India, this is because tuberculosis is endemic in India, and intestinal tuberculosis presents symptoms similar to Crohn's disease, leading to many patients being diagnosed with intestinal tuberculosis rather than IBD. While this distinction might be useful in Japan, there are cases where it may not hold the same importance in other regions.

2-2. For patients already diagnosed with IBD: To detect the transition from the remission phase to the active phase of the disease.

Our aim: To be used by patients who have already been diagnosed with IBD. To diagnose whether or not the IBD is in an active phase.

Through discussions with IBD Plus, we have learned that patients diagnosed with IBD are often concerned about the transition from remission phase to active phase and the return of pain.

For employees, the transition to the active phase would affect their work. So they want to know about the transition as soon as possible. In fact, the fear of coming out at work is not an uncommon consultation.

Some people hide the fact that they have IBD because they think it will affect their employment or job, but it is conceivable that patients may be anxious about when they will enter the active phase because they are hiding it. It is thought that this may help to reduce that anxiety.

2-3. Other opportunity of use.

We thought about using it for a normal person who doesn't have any symptoms. In that situation, we got feedback that it was kind of unrealistic to give it as a diagnostic drug to all people.

From this feedback, we have tried to limit the number of people diagnosed using the information we can get from the health check or interview.

Firstly, we researched the paper about BMI, which could be known by height and weight, which was information that was easily accessible. We also looked into various living habits, such as smoking and drinking. From this research, we found a strong correlation between BMI and IBD, but there was some data that could be considered that low BMI was caused by IBD. We found this casual relationship to be the problem.

On the other hand, smokers and co-smokers (within four years from quitting[4]) also had a strong correlation with IBD[2][3][4]. This data was found on a lot of different papers, so we decided it was trustworthy. As a result, the following recommended dosage targets have been set.

[Target]

• Smokers and former smokers.
• People who tend to have diarrhea.

3. Comparison with Fecal Calprotectin (FC)

It is said that the transition from remission phase to active phase could be diagnosed by Fecal Calprotectin (FC) with an accuracy of about 90%[5]. In the discussion with Professor Hisamatsu, it was considered that FC is the rivalry. On the other hand, we could point out some problems with FC.

Problem 1: High cost and the limit of insurance coverage

In Japan, diagnosis with FC is very expensive and insurance coverage is limited to once every three days. In developing countries, the situation is more difficult and in India, for example, it is only done if you can afford to do it in a private treatment facility with your own money.

Problem 2: The time it takes for the result to appear

In the case of a diagnosis with FC, it will take 1-2 weeks for the results to appear. This means that patients have to wait anxiously, and this is caused by a lack of immediacy. FC also has the weakness that it can only take data from one spot, so it is difficult to monitor patients continuously.

Gassle finds a solution to these problems in the results of the Dry Lab. (For more information, see the Dry Lab Model page.) According to the model, Gassle takes continuous data for a few days immediately, unlike FC. From this, we can resolve the anxiety patients have. This also makes it possible to start treatment at an earlier stage than the diagnosis of FC. This gives hope for cheaper treatment in the long term. If you think about Japan, with FC we would only be able to take data once every three months, but with Gassle, you can take a few days (or dozens of days) of data in a month. And this should be a big merit.

4. Mechanism for transporting drugs to the large intestine

Given that existing diagnostics take a lot of time, to ensure its immediacy and sensitivity, we thought about transporting Gassle alive in liquid medium to the large intestine to increase the amount of probiotic acid produced as a fart. After that, when we were discussing with Professor Nishikawa (Professor of Tokyo University of Science) who works for Drug Delivery System (DDS), we were taught about azo polymer as a colon disrupting preparation. This was based on the property of the azo bonds to be broken down in a specific way by the intestinal bacteria in the large intestine.

In this case we focus on “chitosan” which is already in a product. Chitosan can be graded by the degree of substitution, and as this substitution was nitrogen, it was found that if a drug also had nitrogen, an azo bond could be formed with the two nitrogens to create a prodrug in the large intestine. So we went to Aicello Co., Ltd. to talk about chitosan, but they pointed out critical production issues, such as temperature conditions, and we started looking at other substances. We also interviewed Nisshin Pharma Co., Ltd. about chitosan. And Nisshin Pharma Co., Ltd. also gave us a lot of hard opinions. So we thought it would be difficult to produce and wouldn't have a good effect on society, so we decided to use a different material.

Professor Nishiyama advised against drugs that inhibit propionate absorption or affect peristalsis, as they may have a negative effect on immunity and were not recommended. As a result, we got another idea about coating and cancelled using chitosan for it. But if we succeed to prodrug it in the future, it will be possible to produce a variety of effects depending on the type of medicines added and it will have a further range of applications. (For more information on the structure of the capsule, go to Hardware experiment.)

5. Challenges in Implementation

Genetically modified organisms in vivo

Social acceptance of GM technology is low and regulatory and social challenges remain. For this reason, a device was developed to control the growth of E. coli using the HSV-TK/GCV system. However, Professor Hisamatsu and others believe that genetic modification of odor-producing bacteria is a different approach to conventional methods and has been evaluated as a new diagnostic option.

Relationship between gut inflammation and NO

Professor Hisamatsu pointed out that it may be difficult to distinguish it from infectious enteritis, and a literature survey was conducted on this. However, little research has been done and it is unknown [6]. It was found that dysentery and gastrointestinal illnesses are serious in India, and that it is important for the test to be more accurate in the future.

False positives Possibility of switches other than NO

Professor Kuchitsu (Professor of Tokyo University of Science), an expert on reactive oxygen species, pointed out that it is not possible to function by them.

We measured the nitric oxide specificity of our biosensor to see if there was anything other than NO to react with, especially hydrogen peroxide generated in the gut and OONO- produced by the reaction of nitric oxide and hydrogen peroxide. (see wet results, Measurement).

Time Line

Inspiration

In order to make it continuously effective as a diagnostic agent, we thought that the only way was to maintain its activity for a long period of time with the help of microorganisms, rather than taking it temporarily. We focused on IBD, a disease whose number of patients is increasing worldwide, and wanted to improve it with synthetic biology. We also wanted to devise a simple and unique diagnostic method, so we focused on “smell” as a method to determine the disease qualitatively rather than quantitatively.Our project may be described as a study of the simplicity of diagnostic methods.

iGEM Japan Meetup 2024 1st (Collaboration)

Summary

• The team interacted with the Japanese iGEM team.
• After interacting with each team, the entire team was enlisted.
• We also discussed with each other how to gather team members.

Prof. Makiya Nishikawa (Academicians)

Summary

• He taught me about the drug delivery system (DDS) in the colon.
• This led me to investigate the chitosan coat, an early idea for Hardware.

Prof. Kosuke Kusamori (Academicians)

Summary

• Ganciclovir is already used as a pharmaceutical, particularly for the treatment of herpes virus infections.
• It is unclear whether it is equally effective against E. coli, requiring further investigation.
• Excessive doses can cause side effects like bone marrow suppression.
• Shifted focus towards using Dry Lab modeling to assess its effects on E. coli.

About Ganciclovir

• Overview: Administered for RNA virus infections to suppress viral replication.
• Safety: Side effects include bone marrow suppression and decreased blood cell counts.
• Dosage: Standard dosage is 200 mg, five times daily.
• Structure and Mechanism: Inhibits viral DNA replication by blocking polymerase.
• Prodrug: Valganciclovir is converted into ganciclovir in the gastrointestinal tract.

Consideration of Suicide Genes

• Ganciclovir’s effectiveness in E. colirequires further confirmation.
• Examples include Acyclovir, CD, iCasp9 Ap20187.
• Caspase activation induces apoptosis.
• IC9 has high binding specificity but E. coli may lack caspase-9.

Prof. Atsushi Kambayashi (Academicians)

Summary

• He suggested a compartmentalized model.
• His advice helped Dry Lab make significant progress.

Prof. Chiharu Nishiyama (Academicians)

Summary

• The decrease in propionic acid is suggested to have a negative impact on gut health, leading to a shift in the hardware approach.
• Propionic acid, due to its anti-inflammatory properties, was considered useful as a substance to be produced by Gassle, influencing compound selection.

Anti-inflammatory effects of propionic acid

• SCFAs act on intestinal epithelial cells via G-protein-coupled receptors, suppressing inflammation.
• Propionic acid reduces inflammation by inducing gene expression in the nucleus.
• Decreased propionic acid levels may lead to dysbiosis and enteritis.
• Excess propionate increases cancer risk, but the appropriate amount varies between individuals.

Detection

• Propionic acid concentration is not detectable by smell but can be measured by gas chromatography.
• Differences between nitric oxide and propionic acid effects need to be calculated.

Absorption mechanism

• The absorption mechanism of propionic acid is unclear; studies on inhibitors may provide insight.
• Transporters may control propionate absorption, but pH changes are not considered significant.

Others

• We were allowed to smell propionic acid, which had a characteristic odor.

Prof. Takashi Kamakura (Academicians)

Summary

• He advised on codon optimization of the UL23 gene in the Nissle 1917 strain.
• He suggested introducing the Herpes Simplex Virus Thymidine Kinase/Ganciclovir system (HSV-TK/GCV system).

Aicello Co., Ltd. (Industry)

Summary

• There are two main methods for applying chitosan to capsules: spraying onto hard capsules and pre-forming the capsule with chitosan.
• Chitosan dissolves in acidic solutions, so removing the acid after encapsulation is crucial to prevent premature dissolution in the body.
• The product is contraindicated for patients with diarrhea, and the disintegration time in the large intestine varies among individuals.

Chitosan Application Methods

• Spraying on hard capsules: Chitosan is sprayed onto the capsules containing E. coli and dried at high temperatures, posing a risk to *E. coli*.
• Pre-forming the capsule with chitosan: This method avoids the high temperature issue but requires careful attention to drying to minimize the impact of heat.

Solubility of Chitosan

• Chitosan is insoluble in water but dissolves in weak acidic solutions. Removing acid after encapsulation is critical to prevent premature dissolution in the body.

Contraindications for Patients with Diarrhea

• The product cannot be used by individuals with diarrhea.

Variability in Time to Disintegration in the Large Intestine

• The disintegration time of the capsule in the large intestine varies significantly among individuals.

Nisshin Pharma Co., Ltd. (Industry)

Summary

• We were taught that cellulose derivatives such as HPLC are more reliable for colonic DDS than chitosan because they dissolve at different pH levels, which influenced the Hardware protocol decision.

Prof. Takeshi Hanawa (Academicians, Industry)

Summary

• He was kind enough to lend us experimental equipment and supervised the collapse tests.
• He consulted deeply with us on the project's significance and was indispensable to its implementation.

Prof. Tadakazu Hisamatsu (Medical Professionals)

Summary

• He provided clinical advice and discussed the challenges of obtaining approval in Japan.
• He highlighted the potential of odor-based diagnosis and the social acceptability of genetic modification technology.

Probiotics and the Influence of Gut Microbiota

• The modified **Gassle** is expected to colonize the gut semi-permanently unless antibiotics are used.
• Concerns were raised about unforeseen effects on other gut microbiota.

Odor and Diagnosis of Inflammatory Bowel Disease

• The odor-based diagnostic system was seen as non-invasive and simple, but concerns about strong odors were raised.

Competition with Fecal Calprotectin

• The system must demonstrate superiority over fecal calprotectin in convenience and cost-effectiveness.

Challenges of Genetic Modification Technology

• Social acceptance of genetic modification remains low, and regulatory challenges exist.

Targeting Crohn's Disease and Ulcerative Colitis

• Clarifying the target disease (Crohn's or ulcerative colitis) is essential for accurate diagnosis.

Differentiation Between IBD and IBS

• NO levels may be used to distinguish between IBD and IBS, though early-stage detection remains challenging.

Future Challenges and Improvements

• Improving diagnostic accuracy and patient adherence is critical to the system’s success.

Prof. Kazuyuki Kuchitsu (Academicians)

Summary

• We asked him about reactive oxygen species and their interaction with NO.
• His advice prompted us to conduct experiments to improve accuracy and consider societal implementation.

QLife IBD plus (Patient Advocacy Group)

Summary

• Distinguishing between remission and active phases in IBD is challenging, and fecal calprotectin variability adds difficulty.

Difficulty in Distinguishing Between Remission and Active Phases

• Determining remission or active phases using fecal calprotectin depends on the doctor's discretion.

Distinguishing Between IBS and IBD

• Early-stage differentiation between IBD and IBS is often challenging.

Feedback from an IBD Patient Perspective

• Once diagnosed, patients may face insurance challenges for certain tests or treatments.

Patients' Social and Psychological Issues

• IBD patients may avoid disclosing their condition at work due to social anxiety.

iGEM Japan Meetup 2024 2nd (Collaboration)

Summary

• Each iGEM team presented their projects and gave each other feedback, which was a valuable experience.

iGEM Japan Bio-risk management workshop (Safety)

Summary

• A training course for laboratory workers at the National Institute of Infectious Diseases increased awareness of safety management.
• We learned again the significance of our HSV-TK/GCV system.

IBD India (Patient Advocacy Group)

Summary

• In India, diagnosis methods and financial burdens pose significant challenges for IBD patients.
• Distinguishing between IBD and IBS is common, with additional confusion from tuberculosis.

Diagnosis methods and management of IBD in India

• Diagnosis involves blood tests, stool tests, endoscopy, and biopsy, but endoscopy poses challenges in public hospitals.
• The fecal calprotectin test is expensive and not widely used in public hospitals.

Issues with distinguishing between IBD and IBS

• Awareness about the differences between IBD and IBS is low among healthcare professionals.
• Intestinal tuberculosis is often confused with Crohn’s disease.

Main inquiries from patients

• Financial assistance, access to medical care, insurance coverage, and employment prospects are common concerns.

Toneunga Koryuukan (Public Engagement)

Summary

• They provided a place where we could introduce our project to children, and they played BotchanLab-Tokyo's board game.

Financial Planner Naoko Kuroda (Patient Advocacy Group)

Summary

• The intractable disease designation system impacts medical costs and employment for IBD patients in Japan.
• Improvements in early diagnosis and treatment can reduce costs and alleviate patient burdens.

Status of Medical Expense Subsidies for IBD in Japan

• Patients with moderate or higher ulcerative colitis and Crohn’s disease are eligible for subsidies.

Financial burden of patients without intractable disease designation

• Patients without the designation face significantly higher out-of-pocket costs for treatment.

Importance of Early Detection and Appropriate Treatment

• Early diagnosis reduces costs, improves patient employment prospects, and lessens the burden on informal caregivers.

Prof. Misaki Sakashita (Academicians)

Summary

• Simulation results were presented on Dry Lab modeling, and feedback was received to enhance accuracy.

In the end...

We feel that the involvement of a wide range of people has helped us to bring more resolution to our project and our challenge to use Gassle for the early detection of IBD will continue.

References

[1] Chey, W. D., Drossman, D. A., Chang, L., et al. (2023). Diagnosis and Management of Irritable Bowel Syndrome (IBS) With Diarrhea or Mixed Bowel Habits: A Clinical Practice Guideline. Clinical Gastroenterology and Hepatology.

[2] Cosnes, J. Smoking, physical activity, nutrition and lifestyle: environmental factors and their impact on IBD.

[3] Xu, F., Park, S., Liu, Y., Greenlund, K. J. Dietary intake patterns among adults with inflammatory bowel disease in the United States, 2015.

[4] Qualqili, T. R., Rayyan, Y. M., Tayyezchvm, R. F. Lifestyle and Dietary Factors Associated with Inflammatory Bowel Disease among Jordanian Patients.

[5] Jang, H. W., Kim, H. S., Park, S. J., Hong, S. P., Kim, T. I., Kim, W. H., Cheon, J. H. Accuracy of three different fecal calprotectin tests in the diagnosis of inflammatory bowel disease.

[6] Janoff, E. N., Hayakawa, H., Taylor, D. N., Fasching, C. E., Kenner, J. R., Jaimes, E., Raij, L. Nitric oxide production during Vibrio cholerae infection.