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In order to better understand the real demands of patients and optimize our projects, we had the honor of interviewing Zhang Fanren, a popular Bilibili vlogger, and went to the Affiliated Hospital of Nantong University to interview two IBD patients offline to explore their inner world. This process is far more meaningful than just the surface in promoting the progress of medical research and the optimization of clinical practice. The personal experience and sincere feedback of patients, like a precious beacon, illuminate the way for doctors and researchers to understand the complexity of the disease, and enable them to have a deeper understanding of the diversity of IBD symptoms, treatment response and psychological fluctuations of patients.
During the interview, we consulted patients about their diagnosis experience, treatment options and satisfaction, life impact and balance, public awareness and education needs, and discussed gene editing technology, ethical risks and treatment selection preferences for gene therapy. The patient's perspective is like a mirror, reflecting the comprehensive impact of the disease on their lives-from trivial inconveniences in daily life to subtle changes in work and family relationships. These meticulous observations have helped us build a comprehensive and realistic picture of the lives of IBD patients. Based on their descriptions, we learned that IBD is insidious and difficult to diagnose. At the same time, the treatment cycle is long, it is difficult to cure, and long-term medication is required. Therefore, we chose to use oxidative stress promoters as the switch of the circuit to ensure that our engineered bacteria are only open in the IBD environment. At the same time, we intend to use live bacteria to treat the problem of long-term medication.
In addition, the spread of patient stories is an important bridge to break prejudice and enhance social understanding. It allows the public to feel the tenacity and difficulty of IBD patients up close, inspire sympathy and support from all walks of life, and jointly create a more inclusive and warm environment. This not only reduces the psychological burden of patients, but also inspires positive energy throughout society, encouraging each patient to bravely face challenges and work together on the road to health.
In order to understand what clinical indicators are used to determine whether a patient has IBD? What are the requirements for probiotic colonization and the duration of medication? What are the traditional treatments for IBD? We interviewed Director Cui Bota of The Second Affiliated Hospital of Nanjing Medical University.
According to the interview with Director Cui, we learned that IBD mainly affects young people, and its diagnosis depends on multiple evaluations such as clinical symptoms, laboratory tests, endoscopy, imaging and pathology. Traditional treatments include drug therapy, but there are disadvantages such as high side effects and limited efficacy. Probiotics and flora transplantation are emerging therapies. The former is difficult to colonize and requires long-term supplementation, while the latter replaces the disordered flora by transplanting healthy flora, mostly using endoscopic injection, and requires long-term periodic intervention. At the same time, doctors are open to biological therapies based on gene editing technology, believing that it is a general trend, but safety must be ensured. Finally, after comprehensive consideration, we carefully constructed five modules with unique functions based on the probiotic Escherichia coli Nissle 1917 (EcN), using the exquisite principles of synthetic biology, in an attempt to build a solid fortress against IBD.
The treatment of IBD still faces many problems, and we still need to conduct more in-depth research and connect with reality. Next, according to the guidance of experts, we will visit more patients, including those who have been cured and those who are being treated, hoping to help our project positively. At the same time, we will continue to visit more experts and professors to escort the theory of our project. We will also continue to promote knowledge about enteritis to the public.
In order to better explore how our engineered bacteria can better deliver drugs and understand the difference between traditional biological drugs and chemical drugs, we interviewed Wang Zhengjun, director of the R&D center of Suzhong Pharmaceutical Group Co., Ltd., and discussed with him the safety and public acceptance of genetically engineered drugs.
Director Wang Zhengjun mentioned that the pathogenesis of IBD is not clear at present, and biological agents are mainly used for treatment. In terms of pharmaceuticals, Director Wang proposed that the characteristics of different pH in different parts of the intestine can be used to achieve drug dissolution in the intestine, and affirmed our experimental design using the SoxR promoter. Director Wang suggested using oral tablets as a method of administration, which will greatly reduce costs and the economic burden on patients.
Director Wang is very optimistic about our project. He believes that the project we made can achieve long-term treatment and improve the quality of life of patients.
Through interviews, we learned about the main drugs for the treatment of IBD on the market and their advantages and disadvantages, and learned about the relevant regulations of national team drugs. We learned that biological agents are now the main ones on the market, and there are also traditional chemical agents. The main method of administration is oral, and there are also suppositories, granular agents, etc. This time we received valuable advice on the method of administration, and the experimental design of engineered bacteria was recognized.
When we crushed and purified the membrane protein and did WB, the protein molecular weight was offset, and the band was still incorrect after changing the temperature of the protein. In order to solve this problem, we actively sought help and interviewed Professor You Shuai of Jiangsu University of Science and Technology. Professor You Shuai suggested that we try to extend the gel running distance. If the effect is not ideal, we can consider using mass spectrometry. At the same time, the professor also pointed out that it is acceptable for there to be a certain error between the expected molecular weight and the actual gel running result, and that the expression and modification of the mouse protein in Escherichia coli may have changed in the case of two bands. Based on Professor You Shuai's suggestions, we re-optimized our experiments and obtained some good results.
After that, we encountered protein expression-related problems many times, such as the inclusion body phenomenon, which also caused us great trouble. The protein is present in large quantities in the precipitate, and there is less supernatant, which hinders the progress of the experiment. Professor You Shuai once again provided us with a solution and suggested denaturation and renaturation, or controlling the induction speed and slowing down the expression rate. Based on the professor's suggestions, the team members patiently adjusted the experimental parameters and finally made the protein correctly expressed, laying a solid foundation for subsequent research.
When expressing adhesin (mussel foot silk protein), we encountered the problem of heterologous expression. Professor You Shuai suggested codon optimization, which provided us with new ideas. We conducted in-depth research on the characteristics of codons, optimized the gene sequence specifically, and finally achieved effective expression of adhesin.
In order to solve the problem that PD-L1 is difficult to express using E. coli, we tried to use the solution of truncating PD-L1 to a certain functional domain, and analyzed the site after truncation and the impact on PD-1 binding, and interviewed Dr. Tian, an expert in molecular docking.
Dr. Tian pointed out that it is necessary to test the binding sites of PD-L1 and PD-1, so that after knowing the binding site, a filter can be performed, and it is very likely that its site can not be changed, but in the end, it still needs to look at the experimental results of wet-lab. At the same time, it is recommended that we use Alpha-Fold2 and Alpha-Fold3 for relevant predictions.
Through continuous experiments and analysis, we gradually clarified the direction and strived to ensure the effective expression of PD-L1 without changing its function, taking a key step in the construction of the treatment module.
In order to understand the current clinical treatment and detection methods, the impact of IBD on patients, and topics related to artificial intelligence detection, synthetic biology, probiotic treatment, and gene editing technology, we invited Wang Yun, chief physician of Jiangsu Province Hospital, to conduct an online interview.
In this interview, Director Wang Yun discussed with us the existing clinical diagnosis of IBD and listed relevant indicators, telling us that neural network technology is still in a blank stage in the relevant diagnosis of IBD. We learned about the current clinical practices and side effects of treating IBD. And for today's IBD patients, they are under tremendous physiological, psychological and economic pressure in their lives.
In the design and treatment of engineered bacteria, Director Wang pointed out that our current experimental direction has certain feasibility, but it is necessary to pay special attention to its safety in the human body, and also to study specific symptoms.
Through this interview, we learned about the frustration of today's IBD patients with long-term treatment, side effects and high prices of clinically related therapeutic drugs, and realized that research on synthetic biology needs to pay special attention to its safety and specificity in the human body, which inspired us to design engineered bacteria with a more rigorous solution.
In order to better understand the clinical manifestations of IBD, the use of biological agents, and the impact of enteritis on patients' lives, we conducted an online interview with Wu Ying, a doctoral supervisor at the Affiliated Hospital of Jiangsu University, and a member of the "IBD Study Group" of Jiangsu Province.
Director Wu Ying told us: It is difficult to diagnose IBD through indicators, and it mainly relies on multidisciplinary comprehensive diagnosis. The patient's symptoms are generally abdominal pain and diarrhea. At the same time, the existing treatments for IBD. Alkyl salicylic acid, whole-gut nutrition, only nutrient treatment, and glucocorticoids. Although glucocorticoids are effective in the short term, they have many side effects. There is also immunosuppressant treatment, which is only effective for some patients. Biological agent treatment is now being introduced.
She also emphasized that biological agents are a revolution, which reduces the recurrence rate of patients and reduces the degree of inflammation. But as a drug, it is ultimately a protein. Whether it will cause allergic reactions, reduce immunity, cause cancer, and affect fertility is still unknown. Now there are many small molecule reagents that may reduce the possibility of side effects while ensuring the effect.
These contents have provided great help for us to better understand IBD and treat IBD.
As the project progressed, we interviewed relevant personnel from the NMPA. When discussing the regulatory context and technological innovation of synthetic biological bacterial drugs, we deeply analyzed the uniqueness of drug classification, special policies, and approval processes under the current regulatory framework, as well as multi-dimensional challenges and opportunities such as clinical trials, safety assessments, production and quality control, market access, follow-up monitoring, and ethical considerations. We paid special attention to the fact that the NMPA's supervision of such cutting-edge drugs is not only reflected in the meticulous approval process, but also incorporates profound considerations of safety, bioethics, and social impact.
In the design of clinical trials, the NMPA emphasized special requirements for the characteristics of synthetic biology to ensure the rigor of the trial and patient safety. At the same time, safety assessments focus on key indicators and advocate the use of advanced testing methods to escort the safety of drugs. In terms of production and quality control, the NMPA sets high standards, emphasizing strict control of batch consistency and purity, as well as comprehensive assessment of environmental risks, to ensure the excellent quality of products from source to terminal.
When entering the market, clear labeling requirements and special markings not only protect consumers' right to know, but also promote the healthy development of the drug market. After the launch, strict monitoring measures and the effective operation of the drug vigilance system have built a solid line of defense for public safety. At the ethical level, the NMPA carefully examines every step of the progress of synthetic biology drugs to ensure that scientific and technological progress is consistent with ethical norms.
For specific technical modules such as SoxR/SoxS sensor system, PD-L1 immune regulation, Mfp intestinal repair and SOD antioxidant, the NMPA has put forward regulatory guidance opinions, which reflects the deep understanding of innovative technologies and the wisdom of scientific supervision. Especially in terms of automatic lysis module, multi-module system integration and environmental impact assessment, the suggestions of the NMPA have pointed out the direction of compliance for us.
Facing the future, the NMPA encourages us to explore more safety measures, such as enhancing the reliability of the self-destruction mechanism, long-term impact monitoring, genetic stability guarantee, etc., to ensure the absolute safety of drugs in long-term use. At the same time, the NMPA has also paid great attention to social issues such as environmental release risks, drug resistance and public acceptance, advocating close cooperation between scientific research and supervision to jointly promote the healthy development of synthetic biology drugs.
Based on the suggestions and opinions of the Drug Administration, we found that the existing safety lysis module has security loopholes, so we plan to design another safety module dedicated to biosafety and in vitro safety, namely the blue light induced suicide system, to better meet the requirements of the Drug Administration and doctors.
In recent years, advances in biotechnology have enabled the development of directed evolution, which has introduced random mutations into enzyme engineering. However, it is basically impossible to explore the huge sequence space using random mutagenesis. Therefore, in order to strategically introduce mutations to enhance enzyme activity before designing directed evolution, we asked Senior Sister Zhang Ting for advice on how to make reasonable amino acid mutations and the direction and methods of improving enzyme activity.
During the exchange, we learned about the great significance of computer simulation and prediction for protein modification, which reduces blindness while increasing the success rate. It is a valuable method worthy of reference for our team. This exchange gave us guidance on how to improve enzyme activity. We humbly learned from the reference materials provided by Senior Sister Zhang Ting, and learned the principle of virtual amino acid prediction by consulting the literature on protein structure simulation and prediction. With the understanding of the principle, we further learned from Senior Sister how to use foldx software to perform virtual amino acid mutations on a protein-ligand complex based on interaction forces, and determined the key amino acids in the active site and the amino acid mutation targets that can improve affinity to improve SOD enzyme activity.
