Type 2 diabetes mellitus is a serious chronic metabolic disease with a large number of patients worldwide. The current treatment methods mainly include insulin injection and oral hypoglycemic drugs. The existing treatments have some limitations, such as the need for frequent administration and the possibility of side effects such as hypoglycemia. Therefore, it is of great practical significance to develop a more effective and safer drug for the treatment of diabetes. Our team's goal is to develop a novel glucose-lowering Lactobacillus based on synthetic biology, which can effectively regulate blood glucose level, reduce side effects, and improve the quality of life of patients by releasing fibroblast growth factor 21 and P9 proteins. Using genetic engineering technology, the new lactic acid bacteria will design and construct specific biological systems, and eventually synthesize edible yogurt. To achieve long-term therapeutic effects on diabetes.
The long-term treatment of diabetes with synthetic lactic acid bacteria yogurt was determined as the iGEM project topic.
Members with different professional backgrounds, including medical engineering, pharmacy, clinical medicine, stomatology, etc., were recruited to form an interdisciplinary team.
The literature in the field of diabetes treatment was extensively reviewed to understand the advantages and disadvantages of existing treatments and research progress.
To evaluate the potential of various synthetic biology technologies, such as gene editing, metabolic engineering, and protein engineering, in the treatment of diabetes.
Based on the pathophysiology of diabetes, design biological systems that can regulate blood glucose levels. We designed an engineered bacterial strain that can secrete fibroblast growth factor FGF21 and P9 proteins, and constructed a genetic circuit that can sense bile acid concentration, indirectly reflect blood glucose changes and regulate insulin secretion.
Computer simulation and modeling tools were used to optimize the design of biological systems to improve their performance and stability.
Build and test the designed biological system. It includes experimental steps such as gene synthesis, vector construction, and cell transformation.
Cell culture and biomimetic human small intestine model were used to verify the effectiveness and safety of the biological system. An integrated biomimetic array (iBAC) was used to establish a human small intestine model, and the intestinal secretion efficiency and therapeutic effect of the engineered bacteria were tested on the model.
According to the experimental results, the biological system was optimized and improved. These include adjusting gene expression levels, improving vector design, optimizing culture conditions, etc.
Experiments were repeated to ensure that the optimized biological system had better performance and stability.
Conduct a comprehensive safety assessment of the developed product. This includes the evaluation of toxicity, immunogenicity, genetic stability and other aspects of the engineered lactic acid bacteria.
Environmental safety assessment was carried out, and the engineered Lactobacillus suicide plasmid was constructed to ensure that the engineered Lactobacillus would not cause harm to the environment.
(1) Select the appropriate animal model
(2) Determine the number and grouping of experimental animals
(3) The experimental protocol and detection indexes were formulated
(1) Acute toxicity test
(2) Long-term toxicity tests
(3) Special toxicity test
(1) Pharmacodynamic test
(2) Pharmacokinetic test
(1) Determine the type and purpose of the clinical trial
(2) Selection of appropriate clinical trial institutions and investigators
(3) development of clinical trial protocols and informed consent forms
(1) Phase I clinical trial: preliminary safety and tolerability evaluation
(2) Phase II clinical trial: preliminary efficacy evaluation
(3) Phase III clinical trial: confirmatory efficacy and safety evaluation
(4) Phase IV clinical trial: post-marketing monitoring and evaluation
(1) Data collection and entry
(2) Data quality control
(3) Selection of statistical analysis methods
Understand and comply with food and drug regulations and policies, ensure that product development and application comply with legal requirements.
Conduct ethical review to ensure that the development and application of products comply with ethical standards.
Prepare project reports, posters and demonstration videos to show project results.
Participate in iGEM competitions and other academic exchange events to exchange experiences with other teams and get feedback and advice.
In the future, the team will design validation experiments for the perforin peptide. Intestinal perforin peptides can carry bioactive molecules secreted by synthetic lactic acid bacteria across the intestinal barrier and enter the blood circulation or tissues and organs. The integrated biomimetic array (iBAC) human small intestine model will be constructed to study the intestinal transit efficiency and therapeutic effect of perforin peptide coated FGF21 and P9. At the same time, ELISA and other detection kits were used to detect the concentration of perforin in tissues to quantitatively analyze its penetration effect and penetration rate. The effectiveness of perforating peptides was verified by experiments to provide a basis for the development of lactic acid bacteria delivery system, improve the bioavailability of products, reduce side effects, and improve the therapeutic effect.
With the continuous progress of technology and the continuous expansion of application scenarios, our project will show a broader development prospects in the future, such as improving the treatment pressure of diabetes patients, increasing probiotics, reducing injection, and providing new ideas for the treatment of diabetes.
We will continue to uphold the spirit of innovation, pragmatic and enterprising, and continue to promote the deepening application and development of the project, to provide patients with more high-quality and efficient services.
Defined as surface modification of individual bacteria, achieved through nanomembranes or surface decoration.
Example: Utilizing microencapsulation techniques to enhance the in vivo survival rate of engineered bacteria, resisting the acidic environment of the gastrointestinal tract, and avoiding clearance by the immune system(Lyu et al., 2024).
Exploiting materials and devices ranging from millimeters to centimeters in size to encapsulate groups of bacteria, offering advantages such as extended in vivo lifespan, enhanced immunoisolation, and integration with wireless electronic technology.
Example: Employing hydrogel matrices, microneedles, and capsules as carriers for macroencapsulated bacteria, assisting in bacterial therapy through oral administration and other delivery routes.(Lyu et al., 2024).
Enhancing the activity and adhesion of bacteria within the body through genetic editing.
Example: Controlling the mucus-binding properties of probiotics by heterologously expressing or altering surface proteins and cellular components.(Lyu et al., 2024)
Selecting strains that already reside within the host as the starting bacterial strains to enhance the engraftment of engineered bacteria.
Example: Designing a commensal E. coli strain for mice that can maintain the ability to respond to inflammation in the mouse gut for at least six months(Lyu et al., 2024).
Achieving "synthetic engraftment" of engineered bacteria in the gastrointestinal tract through diversified methods such as mechanical immobilization and magnetic field control.
Example: Using magnetic hydrogels and wearable magnets to achieve intestinal localization, retention, and diagnosis.(Lyu et al,. 2024)
In the treatment of type 2 diabetes, we have employed an innovative microencapsulation technique to enhance the delivery efficiency and viability of probiotics within the human body. This technology encapsulates probiotics within protective microcapsules, enabling them to withstand the corrosive effects of stomach acid and digestive enzymes, safely reaching the intestines to exert their function. The probiotic we selected is L.lactis, which is not only beneficial to human health but has also been demonstrated to survive and provide therapeutic effects within the gut(Klojdová et al,. 2023).
The microcapsules are made from sodium alginate, a biocompatible material that remains stable in the acidic environment of the stomach and rapidly releases probiotics in the neutral environment of the small intestine. This pH-sensitive release mechanism ensures that the probiotics are released at the optimal location and time, thereby maximizing their therapeutic effect on type 2 diabetes(Lyu et al,. 2024).
To improve patient acceptance and compliance, we have combined the microencapsulated probiotics with yogurt. As a widely accepted food, yogurt not only provides an ideal environment for the growth and survival of probiotics but also, with its natural sweetness and cold food characteristics, makes the treatment more palatable for patients. Compared to traditional drug therapies, this method of delivering probiotics through daily diet is more natural and convenient, helping to reduce the psychological burden on patients.
Furthermore, our delivery strategy allows patients to manage their health conditions through simple dietary changes without relying on complex drug intake or medical procedures. This approach not only enhances treatment compliance but may also improve patients' overall quality of life, as it offers a more natural and intuitive way to manage type 2 diabetes.
In summary, the application of microencapsulation technology ensures the effective delivery and activity of probiotics and, by integrating treatment into daily diet, improves patient acceptance and compliance. This method provides a safe, effective, and user-friendly alternative therapy for individuals with type 2 diabetes.
Targeted delivery of L.lactis was achieved by encapsulating it in pH-sensitive sodium alginate microspheres that are targeted to the small intestine. Specifically, the sodium alginate microspheres exhibit pH responsiveness; when the environmental pH is lower than the pKa of sodium alginate, the -COO- groups gain protons to form -COOH, resulting in the formation of a solid hydrogel that encapsulates L.lactis. As the pH increases, the electrostatic repulsion between -COO- groups causes the hydrogel to swell, leading to the disruption of the hydrogel and the release of L.lactis. Furthermore, the application of a chitosan shell and small intestine-targeting antibody proton-dependent transporter 1 (PepT1) on the outer surface of the sodium alginate microspheres enables the microspheres to adhere more effectively to the intestinal wall and assists the microsystem in better targeting the small intestine. This approach achieves targeted delivery of L.lactis and enhances its colonization efficiency in the small intestine(Pan H et al., 2022).
Cells in the intestinal crypts, such as enterocytes, often express specific markers. For instance, intestinal stem cells frequently exhibit Lgr5, Bmi1, and CD133, while Paneth cells may express SOX9(Barker et al., 2007), among others. The L cell, a type of endocrine cell in the gut, primarily secretes glucagon-like peptide-1 (GLP-1). The surface of L cells often expresses specific markers, including the GLP-1 receptor (GLP-1R) and tyrosine hydroxylase (TH)(Drucker et al., 2006). Therefore, we can utilize protein engineering and chemical synthesis techniques to design ligands (such as peptide sequences, nucleic acid aptamers) or antibodies that can specifically bind to these markers. pH-sensitive PLGA nanoparticles are designed using PLGA-PEG copolymers containing pH-sensitive linkages, preparing nanoparticles that disassemble under acidic conditions. Subsequently, chemical conjugation (such as EDC/NHS coupling) is used to covalently attach targeting ligands or antibodies to the surface of the nanocarriers or microspheres. This modification allows for specific recognition and binding to GLP-1R, TH-expressing L cells in the intestinal crypts, facilitating drug release in the acidic environment, such as the slightly acidic surroundings of the intestinal crypts.
E-mail: pengyuoms@fmmu.edu.cn
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