Abstract

Celiac Disease (CD) is a lifelong autoimmune condition that affects about 1 in 100 people worldwide. It’s triggered by gluten: a protein found in wheat, barley, and rye which causes the immune system to attack the small intestine. Even with strict gluten-free diets, accidental exposure to tiny amounts of gluten can cause serious symptoms.

And it is not just CD, people with wheat allergy or non-celiac gluten sensitivity (NCGS) also face similar life challenges. Most people tend to dismiss gluten as something unimportant, but for people with celiac disease, it’s something they need to watch out for.

Our project focuses on addressing the unmet needs of people with NCGS and those who suffer from accidental gluten exposure due to cross-contamination. For these groups, complete avoidance of gluten is nearly impossible and current solutions are limited.

That’s why we developed GluClear: an oral supplement containing gluten-degrading enzymes. It is a site-specific mutated and optimized version of Bga1903, a gluten-specific serine endopeptidase. When taken before meals, we aim GluClear to act directly in the digestive tract to help break down gluten, aiming to reduce symptoms and improve comfort. By offering a practical, science-based tool, GluClear supports people in managing their condition and enjoying more freedom in their daily life without drastic dietary restrictions.

Why?

There were many reasons why we initially chose Celiac Disease (CD) as the starting point of our project. The iGEM motto, “Local people solving local problems,” inspired us to examine challenges within our own community. In Türkiye, where wheat-based foods form the cornerstone of daily diets, avoiding gluten is not only a dietary issue but also a cultural and social challenge. Despite this, Celiac Disease remains significantly underdiagnosed, leaving many affected individuals without timely treatment or support.

Furthermore, there are many instances where restaurants and food suppliers cannot guarantee no cross-contamination in their supposedly gluten-free products. This makes the already hard life of a gluten-intolerant individual exponentially harder. Therefore, we want to make these individuals feel a bit safer and have a bit less of a doubt each time they have to dine out.
During our research, we explored different gluten-related disorders: Celiac Disease (CD), Non-Celiac Gluten Sensitivity (NCGS), and wheat allergy. Although they are often confused, they differ in both their underlying mechanisms and their severity.

Initially, our aim was to design a solution for Celiac Disease. However, we soon realized the complexity of its autoimmune nature. Even trace amounts of gluten such as 20 parts per million (ppm) which is approximately twenty drops of water in a full Olympic-sized swimming pool, can trigger severe inflammation. This can also happen sometimes even before gluten reaches the gut. Considering that a typical Western diet contains 5 to 20 grams of gluten daily, breaking down gluten enzymatically to safe levels for CD patients proved to be practically unfeasible.

Recognizing this limitation, we pivoted our focus toward Non-Celiac Gluten Sensitivity (NCGS) and the issue of accidental gluten exposure due to cross-contamination. Unlike CD, the immune response in NCGS is milder and more manageable, making enzyme-based strategies a realistic solution.

Also in Türkiye, CD has long been defined as a childhood disorder, which is why pediatric gastroenterologists in particular have greater expertise in this field. However, the number of research groups remains quite limited. The main reasons include low economic returns and the restricted outcomes of existing studies. Enzyme-based approaches have not proven effective for celiac disease, yet in cases of gluten sensitivity they have been shown to reduce symptoms by up to 55%. Since these studies are often evaluated based on the number of symptoms reported, it is thought that psychological factors may also have influenced some of the results. Nevertheless, the existing literature and ongoing research remain highly encouraging and motivating.

With this new focus, we set out to develop GluClear. While not a cure, it aims to reduce gluten-related symptoms and improve quality of life, particularly in situations where cross-contamination may occur. By combining synthetic biology and enzyme engineering, we hope to provide a practical, accessible, and science-based tool for safer eating.

Disadvantages of Current Products on the Market

While many enzyme-based supplements on the market claim to protect against celiac disease, scientific research has shown that these products have significant biochemical and clinical limitations. These supplements do not provide reliable protection for celiac patients. They appear to be more suitable for individuals prioritizing a healthy diet or those with mild gluten sensitivity. On the contrary, GluClear offers an innovative solution by using the most recently discovered enzyme which also happens to be harmless to consume. Our project aims to take a new approach by enhancing the enzymes’ capabilities of degradation.

Research indicates that enzymes such as DPP-IV and Aspergillopepsin (ASP), when used alone, cannot fully degrade harmful gluten peptides (Krishnareddy et al., 2017). Another enzyme, ANPEP (Alanyl aminopeptidase), has also been studied for its gluten-degrading potential (Collela et al., 2024; Elkenawi et al., 2022). However, while ANPEP can break down certain immunogenic gluten epitopes, it has been associated with increased expression in some tumor types, such as colorectal and pancreatic cancers. Overexpression of ANPEP has been linked to tumor progression, angiogenesis, and metastasis in various studies. Therefore, the use of ANPEP in enzyme-based supplements may carry oncological risks, limiting its safety for chronic oral administration. Furthermore, because these enzymes are vulnerable to stomach acid, they often require additional medications such as antacids to remain effective, which can further complicate their clinical utility.

While Caricain has more specific degradative activity among alternative proteases, clinical studies show that this enzyme can only reduce symptoms and not provide complete protection (Tanner et al., 2021). Most products on the market offer limited protection against low levels of gluten exposure, and many of these products are not supported by robust scientific evidence.

Our project aims to disrupt trace gluten particles early and effectively in the digestive system and directly address these scientific shortcomings. This will prevent immune system activation and provide a safe solution for people with gluten sensitivity. Our goal is not only to alleviate symptoms but also to provide patients with a scientifically based, reliable, and protective alternative for situations such as cross-contamination or trace gluten exposure that may occur in daily life.

Current Diagnosis Process

Non-Celiac Gluten Sensitivity (NCGS) shows symptoms similar to many other digestive system problems, so there is no definite diagnosis method yet. That’s why a person suspected of having gluten-related issues first goes through tests for celiac disease and wheat allergy. If these possibilities are ruled out, then NCGS is considered. In other words, the patient first enters the diagnosis process for celiac disease, and NCGS is considered only after other possibilities are excluded. In order to diagnose CD (Celiac Disease), the patient must undergo a series of tests while still consuming gluten. These tests include blood work and, in many cases, an intestinal biopsy (NIDDK, n.d.; Celiac Disease Foundation, n.d.).

1. Serologic Tests
   The process begins with blood tests that check for celiac-specific antibodies. The most common test is tTG-IgA (Tissue Transglutaminase IgA). If the patient has an IgA deficiency, additional tests such as tTG-IgG or DGP-IgG may be used (NIDDK, n.d.; Mayo Clinic, n.d.; NHS, n.d.).
2. Genetic Testing (Optional)
   Doctors may check for HLA-DQ2 or HLA-DQ8 genes. If the patient does not carry these genes, CD can be ruled out with high confidence. Having these genes alone is not enough to confirm CD (Mayo Clinic, n.d.; Coeliac UK, n.d.).
3. Upper Endoscopy with Biopsy
   If blood tests are positive, a duodenal biopsy is usually performed. Doctors examine tissue samples for signs of small intestine damage, including: Villous atrophy, crypt hyperplasia, increased intraepithelial lymphocytes (Celiac Disease Foundation, n.d.; Hopkins Medicine, n.d.; NIH, 2018).
4. Follow-up Testing
   After diagnosis, doctors may recommend: Repeat serologic tests to monitor recovery and/or nutrient level assessments (iron, vitamin D, etc.) All diagnostic tests must be done while the patient is still eating gluten. Starting a gluten-free diet too early may lead to false-negative results (Mayo Clinic, n.d.; NIDDK, n.d.).

Data and information adapted from the Canadian Digestive Health Foundation (2022).

Statistics

The prevalence of celiac disease in Türkiye ranges from 0.3% to 1%. Based on these figures, it is estimated that there are between 250,000 and 750,000 celiac patients in the country. However, it is believed that only approximately 10% of cases are diagnosed. According to data from the Ministry of Health's Health Information Systems, the number of diagnosed celiac patients in Türkiye as of the end of 2022 was reported as 154,027.

There is no definitive epidemiological data on non-celiac gluten sensitivity (NCGS) and wheat allergy in Türkiye. The main reasons for this include the lack of specific biological markers for NCGS, the disease's highly variable clinical manifestations, and the potential for these symptoms to be confused with other conditions. For these reasons, while reliable statistics on celiac disease in Türkiye exist, clear data on gluten sensitivity are lacking.

History of CD

Celiac Disease (CD) is an autoimmune disorder that is triggered by gluten in people who are genetically predisposed. Symptoms and effects of the disease have been observed since ancient times. The first known case may date back to the 1st century AD, when archaeologists discovered the skeleton of a young woman in Italy showing signs of malnutrition and the presence of the HLA-DQ2.5 gene, which is now known to be linked with CD (Beyond Celiac, n.d.-a; Losowsky, 2008).

The first medical description of the disease came in the 2nd century AD by a Greek doctor called Aretaeus of Cappadocia. He named the condition koiliakos, from the Greek word koilia (abdomen), and described people who had chronic digestive problems (Beyond Celiac, n.d.-a; Losowsky, 2008).

In the 19th century, a physician named Matthew Baillie noted that patients with ongoing diarrhea got better on a rice-based diet, without realizing he had eliminated gluten. Later in 1887, Dr. Samuel Gee gave the first modern explanation of what he called the “celiac affection” and suggested that diet plays a key role, although the exact cause was still unknown at the time (Beyond Celiac, n.d.-b; Burkitt, 2019).

A big step forward came during the 1940s when Dutch pediatrician Dr. Willem Dicke noticed that children with CD got better during bread shortages in World War II. He later confirmed wheat as a main cause of the disease. In 1952, British studies supported this theory and also connected rye to the disease (Beyond Celiac, n.d.-a; Caio et al., 2019).

In 1956, Dr. Margot Shiner developed intestinal biopsy as a new diagnostic method, allowing doctors to detect villi damage in the small intestine more reliably (Beyond Celiac, n.d.-a; Celiac Disease Foundation, n.d.).

Between the 1970s and 1990s, CD became officially known as an autoimmune disease. Scientists discovered the genetic markers HLA-DQ2 and DQ8, and in 1997, tissue transglutaminase (tTG) was found to be the major autoantigen. This discovery helped create blood tests that are still used for diagnosis today (Caio et al., 2019; Ludvigsson et al., 2013; NIH, 2018).

In the past few decades, awareness of CD has grown significantly. Organizations like Beyond Celiac (established in 2003) helped to spread information and support scientific research. Clinical trials on drugs started in the early 2000s, showing that a gluten-free diet might not be enough for all patients (Beyond Celiac, n.d.-a; Burkitt, 2019).

Today, CD is recognized by the World Health Organization (ICD-10 code: K90.0) and affects around 1 in 100 people globally (Hopkins Medicine, n.d.; Caio et al., 2019).

References

• Asmaa Elkenawi, et al. “ANPEP: A Potential Regulator of Tumor Cell—Immune Metabolic Interactions in Prostate Cancer of Men of African Descent.” JCO Global Oncology, vol. 8, 2022, pp. 52–52. https://doi.org/10.1200/GO.22.58000.
• Beyond Celiac. “From Mussels to Bananas to Gluten: Celebrating Samuel Gee & Advances in Celiac Disease Research.” Beyond Celiac, 27 Aug. 2018. https://www.beyondceliac.org/celiac-news/from-mussels-to-bananas-to-gluten-celebrating-samuel-gee-advances-in-celiac-disease-research/.
• Beyond Celiac. “History of Celiac Disease.” Beyond Celiac, n.d. Accessed 7 Oct. 2025. https://www.beyondceliac.org/celiac-disease/celiac-history/.
• Burkitt, D. “The History of Coeliac Disease.” The Lancet Gastroenterology & Hepatology, vol. 4, no. 4, 2019, p. 281. https://www.thelancet.com/journals/langas/article/PIIS2468-1253(18)30424-2/abstract.
• Caio, Giacomo, et al. “Celiac Disease: A Comprehensive Current Review.” BMC Medicine, vol. 17, no. 1, 23 July 2019, p. 142. https://doi.org/10.1186/s12916-019-1380-z.
• Celiac Disease Foundation. “What Is Celiac Disease?” Celiac Disease Foundation, n.d. Accessed 7 Oct. 2025. https://celiac.org/about-celiac-disease/what-is-celiac-disease/.
• Coeliac UK. “About Coeliac Disease.” Coeliac UK, n.d. Accessed 7 Oct. 2025. https://www.coeliac.org.uk/information-and-support/coeliac-disease/about-coeliac-disease/.
• Colella, M., C. Cafiero, and R. Palmirotta. “Aspergillus niger Prolyl Endopeptidase in Celiac Disease.” World Journal of Gastroenterology, vol. 30, no. 24, 28 June 2024, pp. 3044–3047. https://doi.org/10.3748/wjg.v30.i24.3044.
• Halk Sağlığı Genel Müdürlüğü. “Çölyak Hastalığı Görülme Sıklığı ve İllere Dağılımı.” T.C. Sağlık Bakanlığı, 30 Nov. 2023. https://hsgm.saglik.gov.tr/tr/metabolizma-ve-colyak/colyak-gorulme-sikligi.html.
• Johns Hopkins Medicine. “Celiac Disease.” Johns Hopkins Medicine, n.d. Accessed 7 Oct. 2025. https://www.hopkinsmedicine.org/health/conditions-and-diseases/celiac-disease.
• Krishnareddy, S., K. Stier, M. Recanati, B. Lebwohl, and P. H. Green. “Commercially Available Glutenases: A Potential Hazard in Coeliac Disease.” Therapeutic Advances in Gastroenterology, vol. 10, no. 6, 2017, pp. 473–481. https://doi.org/10.1177/1756283X17690991.
• Losowsky, M. S. “A History of Coeliac Disease.” Digestive Diseases, vol. 26, no. 2, 2008, pp. 112–120. https://doi.org/10.1159/000116768.
• Ludvigsson, Jonas F., et al. “The Oslo Definitions for Coeliac Disease and Related Terms.” Gut, vol. 62, no. 1, Jan. 2013, pp. 43–52. https://doi.org/10.1136/gutjnl-2011-301346.
• Mayo Clinic Staff. “Celiac Disease: Diagnosis & Treatment.” Mayo Clinic, 30 July 2025. https://www.mayoclinic.org/diseases-conditions/celiac-disease/diagnosis-treatment/drc-20352225.
• National Center for Biotechnology Information. “Celiac Disease.” StatPearls, 3 Dec. 2019, StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK441900/.
• National Institute of Diabetes and Digestive and Kidney Diseases. Celiac Disease. Oct. 2020. https://www.niddk.nih.gov/health-information/digestive-diseases/celiac-disease.
• NHS. “Coeliac Disease: Diagnosis.” NHS, 31 Mar. 2023. https://www.nhs.uk/conditions/coeliac-disease/diagnosis/.
• Tanner, M. K., Z. Tang, and C. A. Thornton. “Targeted Splice Sequencing Reveals RNA Toxicity and Therapeutic Response in Myotonic Dystrophy.” Nucleic Acids Research, vol. 49, no. 4, 26 Feb. 2021, pp. 2240–2254. https://doi.org/10.1093/nar/gkab022.
• The Lancet Gastroenterology & Hepatology. https://doi.org/10.1016/S2468-1253(24)00248-6.