- Human Pratices
Collection of Interviews
of Shenzhen Second People's Hospital
Ms. Qi is a thin, middle-aged woman who has a steady job. She loves a vegetarian diet and sports, and has never had anything unusual in her annual physical examination. So before this illness, she thought she was perfectly healthy.
She told us the course of her illness. One night in March, she was disturbed by sudden abdominal pain in the middle of the night. The next day, she went to the community health service and was diagnosed with stomach trouble. After taking stomach medicine for stomach trouble, her pain did not ease. So she went to the hospital again, but the doctor saw the drug prescribed by the community health service center, though there was no problem and advised Ms. Qi to continue taking it. Since then, Ms. Qi has suffered from frequent diarrhea "More than ten times a day, which greatly affected my life." It tormented Qi, but due to the hospital's misdiagnosis and her ignorance of IBD, she mistakenly believed it was caused by medication. Ms. Qi appeared again with the symptom of blood in stool after a few days. After coming to a hospital to explain a situation to the doctor again, the doctor suggested she get a gastroscope. However, she was diagnosed with erosive gastritis and reintroduced new drugs. After diarrhea did not stop, Ms. Qi did a colonoscope again and was finally diagnosed with ulcerative colitis.
Missed diagnosis and misdiagnosis for many times, really made us feel worried for Ms. Qi at that time. This confirmed our determination to educate the public about IBD and intestinal health in our HumanPractice and Education campaigns.
She admitted to us that she had never heard of IBD before. After the attack, she was often in a mood of anxiety, worry and confusion, so that she often reflected on her past life habits. How to live a healthy life in the future? She had no answer. At the same time, the choice of food often troubled her. We spoke to doctors and nurses, who also confirmed that the hospital produced insufficient materials for patient education, thus making it difficult to address the lack of patient knowledge.
Ms. Qi is the first IBD patient we have come into contact with. She helped us to have a more concrete understanding of IBD than just literature. Being in touch with patients has also made me more hopeful and appreciative of what we are going to do. We think what we are doing is very meaningful!
Shenzhen Second People's Hospitall
Fig.1 The interview with Dr. Feng Juerong.
Shenzhen Second People's Hospital is the medical unit with the most important departments in Shenzhen. Dr. Feng Juerong is the physician-in-charge of gastroenterology here. Having treated many patients, he has very rich clinical experience!
Dr. Feng told us that inflammatory bowel disease (IBD) is an autoimmune disease. There are two subtypes of ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a disease with abdominal pain, diarrhea. The occurrence of mucus blood stool is the primary performance, mainly involving the large intestine. Crohn's disease can affect the entire digestive tract, from our esophagus to the rectum and anus. In China, both diseases mainly affect teenagers, that is, our young workforce. And it's a lifelong disease with no cure. The lifelong disease is a disease of physical injury, so this kind of disease has a greater impact on the whole patient's family and society.
IBD is often characterized by recurrent diarrhea with blood and pus and blood stools. However, clinical diagnosis can be made only by combining colonoscopy, chemical examination, pathological examination and other auxiliary examinations. Clinical use of calprotectin to help diagnosis, but the diagnostic indicators are not detailed enough, still need a variety of factors.
As for the treatment methods and treatment plans of IBD, Dr. Feng told us that the current first-line clinical treatment drugs mainly include glucocorticoid, 5-aminosalicylic acid, mesalazine, and biological agents, such as Entyvio, Humira and infliximab. Interleukin-13 antagonists and immunosuppressants. Such as azathioprine, methotrexate, etc.
Mesalazine is administered in the acute phase in large doses and halved in the chronic phase for nonspecific inflammation suppression. Its price is relatively low and generally preferred. In addition to mesalazine, the biological agent infliximab can also be used as the primary treatment. According to the clinical treatment plan, an infliximab injection is generally used three doses per course of treatment, and the cost of a treatment period is about 6000 RMB.
Dr. Feng told us that the social cognition of IBD in China is low. Many IBD patients have mild symptoms or no specific symptoms for an extended period. The appearance of these typical symptoms of irritable bowel syndrome is often difficult to attract patients' attention, and they regard it as a common digestive system disease. Even if they come to the hospital with severe symptoms, they are likely diagnosed with irritable bowel syndrome. In addition, the diagnosis procedure of IBD is complicated, which often requires the combination of clinical manifestations, endoscopy, histopathological examination and imaging examination to make a comprehensive judgment. In addition, the recurrence and remission of IBD is also an essential factor that makes it difficult to be diagnosed. The longer the diagnosis is delayed, the greater the risk of intestinal stenosis and intestinal surgery. According to Dr. Feng, the current trend of IBD management is to be based on specialized care and individualized treatment according to the degree and location of the patient's disease, as well as a multidisciplinary response aimed at induction therapy and maintenance of remission. Current drug therapies include glucocorticoids, immunomodulators, biotherapy, and mesalazine used in ulcerative colitis. The use of mesalazine requires individualization, but each has large or small limitations. It is worth noting that Dr. Feng told us that intestinal probiotics such as bifidobacteria and yeast would be used in the clinic to regulate intestinal flora. At present, how to use intestinal flora in the treatment of IBD is also a hot clinical concern.
Shenzhen Second People's Hospital
Fig.2 The Interview with Nurse Chen Fuyan.
Nurse Chen mentioned that IBD patients have an urgent pursuit of quality of life and high requirements for living habits and emotions. This requires society to be able to timely understand this disease and give the unfortunate patients adequate care. At the same time, because IBD is difficult to diagnose, the phenomenon of missed diagnosis is severe, so it is urgent for public education, so that suspected patients are aware of their potential disease.
The remission stage of IBD is worth paying attention to. Most IBD patients have a long and chronic course of the disease, often alternating between the onset and remission stages. Unlike hospital care during the onset, the care of patients in remission is critical to improving the patient's quality of life, but is more likely to be overlooked. Dietary care is of great significance because patients are at risk of malnutrition due to chronic diarrhea and malabsorption. This needs to guide the patient to eat soft, easy to digest, less fiber, enough calories of food. Thus conducive to absorption, reduce the stimulation of intestinal mucosa, while maintaining the needs of body metabolism. But because the patient is not familiar with the knowledge and concept of nutrition, they often can not choose the right food, leading to the recurrence of the disease. Food diary recording is a good solution, but patients' compliance is not high due to the lack of convenient recording tools.
Q: As far as we know, inflammatory bowel disease (IBD) is a lifelong disease. So if a patient is diagnosed with inflammatory bowel disease (IBD), how should he be treated and handled correctly during the whole process of his nursing?
A: Speaking of this, because the course of IBD is very long, we first need a national organization of doctors to carry out professional diagnosis and treatment of this disease. At present, there are academic groups under the National Gastroenterology Society, and many IBD diagnosis and treatment centers have been established in various places. This is an excellent organization. So patients should have a relatively clear understanding of the disease, and don't worry about it. Because it is a disease that cannot be cured temporarily, but can be well controlled with the proper treatment. Then it is essential to cooperate with the professional treatment of the doctor. Can't say that treatment is not to take medicine after the symptoms are alleviated, we often encounter many patients, when their stomach stops to pain, not have loose bowels and they will stop taking the drug, and then result in repeated recurrence, each recurrence of the disease is aggravated once, which is very bad. Therefore, patients should understand the disease from doctors, nurses, and various popular science books, treat it correctly and cooperate with the doctor's treatment, so that the disease can be well controlled, and they can also live a normal life and return to work.
Prof. You Conghui is a Ph.D. and master's advisor in biology. She received her Ph.D. in genetic cell and molecular biology from Institut Pasteur in France and the University of Versailles in France. She also worked as a postdoctoral fellow in the Department of Biology and Physics at the University of California, San Diego in the United States. Currently, she is an associate professor of Biology at the School of Life Sciences, Shenzhen University. She has long been engaged in microbiology, molecular biology, quantitative biology and other research and has a deep understanding of biological knowledge such as new research methods of microbial physiology combined with quantitative biology and systems biology.
Professor You thinks our project is meaningful. She says that she has worked in the neighborhood of intestinal microbes and has also done the characterization of SOD. "We need to choose the right promoter strength," she said. "We can't just pursue the long-term expression of compositional type and ignore the strength." She also stresses that we need to consider plasmid loss, especially when it comes to our bacteria colonizing the gut and passing generations. It is suggested that we determine the relationship between the transfer algebra and the proportion of plasmids, so as to confirm the effective colonization of our engineered bacteria. She also gave us advice on plasmid design.
Professor You Cong-hui put forward the problem of the colonization of the flora to us. The colonization effect of probiotics itself needs to be designed since the treatment effect of directly adding probiotics is limited. At the same time, she reminded us that we need to pay attention to the influence of engineered bacteria on healthy people. We subsequently designed relevant acid-sensitive promoters to achieve control, achieving low expression in normal people and high expression in patients. Meanwhile, we designed a suicide switch considering the problem of bacterial escape.
At the same time, Miss You also gave us some suggestions on feasibility. She told us to look for examples and products of probiotics in the market and compare them with our own projects to find areas for improvement.
In order to avoid the continuous reproduction of bacteria and the loss of plasmids, a killing switch can be considered to make the bacteria without plasmids die, or plasmids can be successfully characterized in vitro and then transferred to genes through bacteriophages and CRISPR.
As for the specific target, the teacher reminded us that SOD is easy to form inclusion bodies. Reducing agents that eliminate ROS can be added, and it is easier for bacteria to produce metabolites than proteins.
As for bioethical issues, the teacher told us that the public is sensitive to gene-editing microbes, and that the project should be designed with the acceptance of the public as to whether editing tools are still in bacteria and whether they will run into human bodies and edit human genes. If the effect of engineered bacteria is unlikely to be different from that of drugs, the public will not accept it very well. Taking bacterial products doesn't cause much panic, but eating engineered bacteria directly can be a bit more difficult. People worry about whether the number and function of bacteria can be accurately controlled, but it is difficult for us to accurately control them at present.
Fig.3 The interview with Prof. You Conghui.
We are concerned that the engineered bacteria metabolize differently in vitro and in vivo, and the final treatment effect can have difference from our ideal experimental data Prof. You told us that SIAT has a simulated intestinal instrument, we can try the in vitro effect of test engineering bacteria, the anaerobic conditions have to be considered while the specific nutritional environment may not have much effect on the persistent expression of bacteria.
Ms. You also provided us with the method of bacterial interaction experiments. She told us that we could use fluorescence. Firstly culture one by one, then track it or use PCR to see the abundance of specific genes (only existing in a certain type of bacteria). First, do bacterial interaction, then do cell co-culture, and then make mouse models. Considering the limited project period, she suggested that cell interaction, co-culture and mice experience could be done in parallel.
Zhu Ning is a Ph.D. in molecular biology and head of the molecular department at Shenzhen Xbiome Biotechnology Co., LTD. She graduated from the Chinese University of Hong Kong with a Ph.D. and is currently responsible for genomics and gene editing-related work, responsible for the company's second-generation sequencing and gene editing-related technology, and building a platform for innovative drug development.
Fig.4 The interview with Dr. Zhu Ning.
Q. How to ensure the enzyme activity of lactobacillus protein after high-intensity cell wall breaking work.
A. Try to do it at a low temperature, and avoid adding substances that affect protein activity, such as buffer. You can get some patent information and see what factors affect enzyme activity.
Q. How to concentrate and improve plasmid concentration to improve the electric conversion efficiency of lactic acid bacteria.
A: 1. Large plasmid extraction; 2. More plasmid extraction and precipitation concentration
Q. Do we need to consider the degradation of strains during the transfer of our engineered bacteria from generation to generation, thus affecting the activity of expressed products? How can degradation be prevented without a cumbersome conservation process?
A: Yes, strain stability is one of the factors that must be considered in the development of the LBP drug.
Q. Can the IBD-related targets we selected to be supported?
A: It's theoretically possible because it's all about improving the symptoms/mechanisms of IBD, but ultimately, you have to look at the data, and for a drug, ultimately, you have to look at the clinical data. Phase I and Phase II have been successful, and there is still the possibility of failure in Phase III.
Q: It is observed that the strain degrades seriously. There is no good way to preserve the species. Is there any way to prevent degradation? The criterion for degradation is the difference in the concentration of plasmids. Right?
A: Yeah, it's not entirely clear why it degrades under selection pressure. It is possible that low copy plasmids (for optimal expression) lead to plasmid extraction concentration angles.
Q: pUC is a high copy plasmid, but the copy number is still limited. The bacteria sent by the company were directly preserved, but the preserved bacteria were still degraded after recovery (25% glycerol) and were cultured in Ampicillin positive LB medium at 37°C and 220 RPM. What could other possible reasons be behind this situation?
A: Consider strictly testing the pH of the medium, and obtain the culture conditions from the relevant patents of E. coli Nissle 1917 to understand the preference of the culture conditions of E. coli Nissle 1917.
Q: The E. coli Nissle 1917 we obtained had two original plasmids, which led to the difficulty of plasmid transfer. The current use of electric transfer resulted in better enzyme activity. E. coli Nissle 1917 had a particular plasmid load but had difficulty in transformation.
A: Have you ever tried transferring four plasmids together? Theoretically, the quantity of plasmid transfer has little effect.
Q: At present, only plasmid PCR results are available, indicating that the plasmid concentration is too low, about 17ng, and running electrophoresis bands at the same time is not ideal. The fluid was cloudy, but no plasmid could be extracted.
A: What about the number of plasmids? You can try 100ml bacterial solution to extract plasmid. (As a rule of thumb, it should be at least 10ml of bacterial extract plasmid) or try to enrich plasmid. 17ng may be the genomic DNA, and the correct bands in PCR indicate that genomic DNA may be extracted. It is best to add the protein solution according to the instructions of the kit. Consider first using PCR sequencing products to confirm that it is not an electrophoretic band, enrich protein by expanding the volume, and try plasmid extraction with another kit. (At present, the most worrying thing extracted is not our target plasmid, bacteria may have genomic DNA extracted) there is a kit that can remove genomic DNA. You can look for it, in theory, electrophoresis can quickly distinguish genomic DNA.
Q: The plasmid size extracted is 5-7k. Why does this happen?
A: You can check the range of plasmids extracted by the kit, whether it is possible to be caused by the column, or try to extract plasmids by SDS lysis.
Q: Currently, we have E.coli Nissle 1917, which can grow on ampicillin medium, so we rebuild the pet plasmid, remove the double sieve resistance, and use kanamycin as screening pressure. Only the target gene is expressed and IPTG induction is reserved as a backup plan. What's your opinion?
A: Is there a ready-made skeleton that expresses our target gene? Look at the expression of the protein.
Q: Constituent promoters are more regulated by bacteria, but the amount of expressed protein is low, and they are prone to revert mutations. SDS-PAGE and WB result in poor effect. How to solve it?
A: Constituent promoters need to be purified, generally 100-250ml of bacteria are crushed together and then concentrated for detection. Milipole has protein pillars and does not need to be purified by ultrafiltration tubes. It would help if you used multiple low-temperature centrifuges to concentrate more proteins.
Q: Any other suggestions?
A: The point being considered is that the plasmid quantity is too low and mixed with genomic DNA. The biggest possibility is that the plasmid is leaked out of the cell but still in the bacterial fluid, but will not be replicated and amplified. It can also be considered to increase the selection pressure of plasmids (2 times) to prevent other bacteria from rising too fast and inhibit the target bacteria.
Dr. Hao Huaijie is the senior research and development manager of the microbiome in Xbiome's R&D department. He is also the leader of the microbiome and has extensive experience in cell and microbial experiments.
In order to further verify whether our engineered bacteria and their protein products can effectively inhibit the inflammatory response, we hope to perform cell-level experiment. Cell experiments are unfamiliar to us, and the required culture environment for cells is also harsher. Relying on the resources of the Xbiome company, we have the opportunity to carry out higher-level verification.
The cell experiment period is relatively long (requires from half a month to a month), and in general,it is time-consuming and labour-intensive to explore the optimal condition to better carry out the experiment. Before the interview, we learned that Xbiome company had conducted cell interaction experiments with anaerobic bacteria in previous experiments.
First of all, we learned about the cell interaction experiments currently being conducted by the Xbiome, focusing on the interaction experiments between THP-1 cell line and PBMC primary cells with live bacteria, inactivated bacteria, and metabolites. The purpose is to screen for baterial strains with anti-inflammatory or pro-inflammatory effects.Dr.Hao also shared with us the main difficulties encountered during the experiment.,such as the lack of reproducibility of experimental results and differences between them. He recommended that we should explore the conditions in advance before conducting formal cell interactions，develop a plan and then verify it.
Since the chassis Nissle 1917 we use is a kind of aerobic bacteria, it grows and reproduces rapidly, which is very different from the company’s previous anaerobic bacteria interactions experiments. So the experimental program needs to be redesigned. In addition to preventing cross-contamination of different strains,we need to think about how to effectively remove live bacteria after the interaction is over, and at the same time, it can't have a greater impact on cell viability. This also prompted us to choose appropriate antibiotics and explore the concentration gradients when we formally conduct cell experiments.
For the setting of MOI, Mr.Hao also gave us many suggestions. MOI is the multiplicity of infection, which is used to measure the ratio of bacteria to cells. During the interaction experiment, the size of the MOI has a great influence on the experimental results. Before the cell interaction experiment is formally carried out, it is necessary to explore the cell viability under different MOIs in order to select the best viability MOI for the formal experiment. We later decide to use CCK-8 reagent to detect. In the future , we may try to use flow cytometry to calculate the rate of apoptosis or necrosis.
For the detection of cytokines, we expect to use the ELISA single factor detection kit (IL-6/IL-10) for testing, and this method is only for a single pro-inflammatory or anti-inflammatory factor experiment that takes a lot of time. Mr.Hao suggested that we can try multi-factor detection in the later stage. This method may detect changes in the level of multiple factors under the same time-consuming situation, which has high efficiency and comprehensive experimental results. This detection method is based on microspheres. In the immunological analysis method, each type of microsphere is respectively coupled with the capture antibody corresponding to the indicator, and the target protein content in the sample can be detected.
Finally, we talked about the in vitro intestinal epithelial model that the company is trying to establish. The purpose of constructing this model is to verify the influence of the strain on the intestinal barrier function, so as to preliminarily evaluate the potential safety issues of the strain. By using Caco-2 cells- Human colon cancer cell line, the mature Caco-2 can spontaneously undergo intestinal epithelial differentiation under in vitro culture conditions, and the differentiated cells form tight junction structures that can express some marker enzymes, so this cell line can be used to construct Intestinal epithelial model to simulate the intestinal environment in the body.
We benefited a lot from this interview with Dr.Hao. and we had a deeper understanding of the design and attention points of the cell experiment. In the formal cell experiment plan, we also adopted many suggestions from Dr.Hao, which greatly promoted the experiment.
Dr. Zhang Yulong is a doctor of microbiome in the research department of Xbiome, with rich experience in animal experiments.
In order to complete the relevant experiments of our drug development according to the IND application standard of FDA, we plan to complete animal experiments including mice experiments within the competition cycle to further verify the efficacy of our drug.
During the interview, we first asked Dr. Zhang about the selection of animal models. There are many different induction models for IBD mice, and what kind of induction model should be selected to complete the experiment. Dr. Zhang introduced that different mouse models have differences in specific indicators. In the actual experiment process, we need to consider the purpose of the experiment and the possible impression on mice, and choose a more mature model or a model with obvious relevant indicators. In summary, no single model can be entirely consistent with the clinical practice, but different disease models can be used to study specific disease mechanisms.
We then asked Dr. Zhang about the choice of animal models for studying gut microbes. Dr. Zhang mentioned that mice with intestinal flora related experiments are generally selected as GF mice or SPF mice. GF mice do not have all living organisms detected in their bodies, while SPF mice do not carry major potential infections or conditioned diseases and pathogens that interfere with scientific research. Generally speaking, only SPF mice are required for the study of intestinal microbiota related animal models. SPF mice do not have too many pathogenic sources, and the composition of intestinal flora is simple, which is convenient for scientific research analysis. If you want to conduct further models, you can also consider primate models. However, the current IBD research is mainly limited to mice, and there are not many mature primate models.
Fig.6 The interview with Dr. Zhang Yulong.
In order to determine the necessity of conducting experiments on mice according to the requirements of the iGEM competition, we raised questions again about whether animal experiments are needed and the ethical approval required for conducting experiments on mice. "If it is to formally develop a new drug, the animal model (mouse model) experiment is very necessary before IND application. The effect of the drug should be reflected in the animal model, and a series of drug toxicology and pharmacokinetic experiments must be considered before IND application." Dr. Zhang said. He pointed out that ethical approval of animal experiments is usually done by contacting the local ethical approval committee, and if animal experiments are submitted to CRO companies, only the relevant experimental protocol and drugs are submitted.
So far, we have demonstrated the necessity of conducting mouse experiments in our project. In order to further improve the mouse experiment plan, we asked Dr. Zhang for some specific questions about the experiment. First of all, the demonstration of drug effectiveness needs to follow the approval guidelines in the IND application. A large number of molecular, cellular and animal level experiments are required to demonstrate the efficacy, principle and elimination of half-life and other indicators of the drug. Dr. Zhang also introduced that for safety, it is necessary to calculate the safety factor, reliable safety factor and therapeutic index through the half effective dose (ED50), half lethal dose (LD50) and other indicators to measure the safety of drugs. Finally, the adverse reactions of drugs, including side reactions and toxic reactions, are also needed to comprehensively and effectively analyze the safety of drugs.
Finally, Dr. Zhang mentioned that if we focus on IBD disease experiments, we should also pay attention to some common indicators to evaluate the severity of IBD diseases, such as body weight, fecal viscosity, blood in the stool and other indicators that can be directly observed. In addition, it can be evaluated by histopathology, including mucosal layer damage, inflammatory cell infiltration, colon length, etc., which can be evaluated by anatomic and pathological experiments. Finally, the above indicators can be integrated and the more authoritative evaluation criteria such as the DAI evaluation system can be used for evaluation.
Thus, through communication with Dr. Zhang, we learned some details of the IBD mouse experiment and laid a foundation for our subsequent writing of the protocol for the mouse experiment. This provides us with more and more accurate means to demonstrate the effectiveness of drugs.
1.According to our literature survey, IBD mouse models can be divided into acetic acid induced, TNBS induced, DSS induced, and OXZ induced models. What induction models should be selected for our animal model validation, and what are the differences between different induction models?
Different models have different indicators, the purpose of the experiment and the possible impact of the experiment on mice need to be taken into account in the process of index determination. Try to choose animal models with obvious indicators or those with mature experience. In summary, although no single model is clinically consistent, different models can be used to study specific disease mechanisms. By understanding the differences, strengths and weaknesses among currently available IBD models, researchers can select the most appropriate mouse model for different purposes.
2. What animal model do we need to use for intestinal microbiota-related animal model experiments? What are the advantages of these animal models? What are the specific requirements for raising these model animals?
Animal models related to intestinal flora can be used in mice, especially in GF mice or SPF mice. GF mice are mice without all living organisms detected in vivo. In contrast, SPF mice are mice without major potential infections or conditioned diseases and pathogens that significantly interfere with scientific research. Generally speaking, only SPF mice are required for the study of intestinal microbiota-related animal models. SPF mice do not have too many pathogenic sources, and the composition of intestinal flora is simple, which is convenient for scientific research analysis. Other models include primate models, such as rhesus monkeys, but the company currently has no primate models, and the research on IBD primarily focuses on mouse models. Specific model animal feeding programs are available with relevant standards.
3.How to judge whether a project is necessary to conduct mouse experiments (combined with iGEM requirements)? What is the process of ethical approval for animal experiments? What details do we need to focus on?
If it is to develop a new drug formally, the animal model (mouse model) experiment is necessary before applying IND. The drug's effect needs to be reflected in the animal model, and a series of drug toxicology and pharmacokinetic experiments can be considered before IND application. Ethical approval for animal experiments is usually done by contacting the local ethical approval committee. If animal experiments are commissioned by professional CRO companies, only relevant experimental protocols and drugs are needed to be submitted, which can save time for ethical approval and subsequent operations.
4.For drug development and validation, what degree of animal experiments is sufficient to verify the efficacy and safety of drugs? How to evaluate the effectiveness and safety of drugs?
The verification of drug effectiveness needs to follow the approval guidelines in the IND application. At the same time, to promote the approval of new drugs through IND, many experiments at molecular, cellular and animal levels are needed to demonstrate the efficacy of drugs, the principle of efficacy, and the elimination of half-life and other indicators and all aspects. For safety, half effective dose (ED50), half lethal dose (LD50) and other indicators need to be passed. The safety factor, reliable safety factor and therapeutic index were calculated to measure the safety of drugs. At the same time, the adverse reactions of drugs need to be counted, including side reactions, toxic reactions, residual reactions, drug withdrawal reactions, specific reactions, allergic reactions and so on. Statistical drug safety as comprehensively and effectively as possible.
5.In IBD-related animal experiments, what are the common indicators to evaluate the severity of IBD disease? How are these indicators detected?
Common indicators that can be easily detected include weight, fecal viscosity, hematochezia and other indicators that can be directly observed. In addition, histopathological evaluation, including mucosal layer damage, inflammatory cell infiltration, colon length, etc., can be revealed by anatomical and pathological experiments. At the same time, in the scientific research community, a relatively mature evaluation criteria and disease activity index (DAI) evaluation system has been established for IBD mice, which only needs to refer to relevant literature for verification.
6.Can horizontal plasmid transfer of intestinal engineered bacteria be verified through mouse experiments?
Theoretically, there is no problem, and the existence of plasmid horizontal transfer can be determined by isolating the form of intestinal epithelial cells in mice or sequencing the whole genome of fecal intestinal flora. However, the current related studies are not sufficiently disclosed, so we may need to explore by ourselves.
Dr. Huang song is a Ph.D. graduate of the French National Institute for Agricultural Research (INRA). Currently, he serves as the chief preparation scientist of Shenzhen Xbiome Biotechnology Co., LTD., leading the team to build the preparation process innovation platform and responsible for the development of various types of new microecological preparations.
He has been committed to the research and development of probiotics and other microbial preparation processes. During his Ph.D. study, he developed a new live bacteria drying technology aiming at probiotics' cell stress response mechanism in the drying process. The technique was featured as a major news item at the 2016 International Probiotics Science Conference. Dr. Huang also created a new high-throughput strain screening platform for the Chinese Strain Research Center, which is used by DuPont's Us and European Strain research centers. In recent years, Dr. Huang song has published a number of papers, book chapters, and invention patents in the field of microbial technology. He knows a lot about microbiological agents!
In order to determine the form of our drug preparation, out of humanistic care, fully considering the needs of patients as well as the effectiveness and stability of drugs, we learned from Dr. Huang about the types and related details of a series of viable bacteria preparations.
First, Dr. Huang introduced that vacuum freeze-drying and microcapsule technologies are expected in living microbial preparations. Among them, vacuum freeze-drying technology is relatively mature, which has an excellent protective effect on microorganisms and a strong recovery rate. Microcapsule technology is relatively new and can be made into jelly, yogurt and other forms. However, mass production requires fluidized beds and other related equipment, so in our project, the freeze-dried powder is a better choice.
In order to understand the needs of patients, we asked Dr. Huang about the demands and expectations of the population, especially patients, for living bacteria preparations. Dr. Huang said that for patients who take drugs for a long time, most want them to be small， portable and have good privacy.
Then, Dr. Huang introduced the principle and simple operation process of vacuum freeze-drying. We learned that vacuum freeze-drying could work because of the existence of a protective agent. There are many different hypotheses about the effect of vacuum freeze-drying protectants, including the preferential theory of protective proteins, the water substitution hypothesis, the effect of protectants on the glass transition temperature and the effect of protectants on the membrane phase transition temperature. Generally speaking, lyophilized powder has specific enteric solubility, but in order to get better results, enteric encapsulation is also indispensable. Dr. Huang mentioned that the performance of freeze-dried powder could be verified from the recovery rate of freeze-dried bacteria, the gene expression and function of the recovered bacteria and the stability of the powder to judge the advantages and disadvantages of the protective agent. As for the performance verification of enteric-soluble capsules, the stability and intestinal solubility targeting can generally be confirmed by instruments such as disintegrating apparatus. Generally speaking, for those who are familiar with freeze-drying technology, they can judge the type and proportion of protective agents through experience or design orthogonal tests to get the best proportion of protective agents through relevant regression analysis.
Fig.7 The interview with Dr. Huang Song.
On this basis, we refer to the literature and determine the formulation of the first generation of lyophilized protectants. At the same time, according to the test method mentioned by Dr. Huang, the related performance of the protective agent was evaluated. After completing the first generation of products, we met with Dr. Huang again to ask questions and communicate with him about the problems arising in the experiment.
First, we asked about the poor recovery rate of the flora immediately after freeze-drying, which Dr. Huang said usually occurs because the flora is in a poor metabolic state. Generally speaking, the gradient dilution counting method will have a quantity deviation of ±20% due to the specific operation of the operator, and there is a possibility of deviation from our data results. Another possibility is that after freeze-drying, the engineered bacteria develop a living and non-cultivable state (VBNC), in which the bacteria are biologically active but cannot be cultured to multiply and form colonies.
We then consulted Dr. Huang on the formulation optimization of lyophilized protectants. Dr. Huang pointed out that the general optimization method of lyophilized protectants can increase the value of lyophilized powders by adding prebiotics such as dietary fiber. Prebiotics are organic substances that are not digested or absorbed by the host but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thus improving the host's health. General production of commonly used substances have oligosaccharides, such as oligosaccharides, galactose oligosaccharides, inulin and so on. In addition, polysaccharides and protein hydrolysates can be used as prebiotics. At the same time, oligosaccharides such as fructose-oligosaccharides can bring a sweet taste to the freeze-dried powder, and at the same time, increase the selling point of the product and improve the acceptance of patients.
In addition, Dr. Huang noted that different freeze-drying processes (including the time and temperature of freeze-drying) might affect the recovery rate of bacteria. Secondly, the control of humidity is also very important. For general laboratory, humidity can not be controlled. While in the actual production process, we need to control the relative humidity of the workshop below 50% to prevent the growth of bacteria in the powder. So it's acceptable to have these kinds of problems.
In order to put forward more specific ideas for our project, Dr. Huang proposed to directly replace the sucrose in our protective agent formula with oligosaccharides， or directly add the same amount of oligosaccharides as sucrose into the lyophilized bacterial powder, and perform resuscitation experiments and verify the enzyme activity. In fact, this advice has greatly helped us in optimizing the formulation of lyophilized protectants. Experiments showed that adding fructose-oligosaccharides equal to sucrose directly to the lyophilized powder had higher enzyme activity per unit of recovery rate than the unoptimized formula. This shows the effectiveness of formula iteration and embodies the theory of engineering iteration.
Finally, Dr. Huang put forward some personal opinions for our project. First, the determination criteria for the ratio of lyophilized protective agent formula should be determined by orthogonal test. Then consider adding functions, adding prebiotics and other additives to optimize the product. On the premise of mature technology, of course, we also can consider using microcapsules to explore the living bacteria preparation application scenarios further, but the application of microcapsule technology premise is still freeze-dried bacteria powder production, in order to make us a better understanding of microcapsule technology, Dr. Huang also provides us with some microcapsule samples for reference.
At the same time, according to the latest literature, there is still a little controversy in the academic community about the safety of E.coli Nissle 1917. Although Nissle 1917 is the safest Escherichia coli strain in general, it is necessary to consider L.lactis.
Based on the above interview, we learned about the principle, form and performance of living bacteria drug preparation, and on this basis, we really produced the lyophilized bacteria powder that belonged to our project and verified its performance with enteric-soluble capsules. It can be seen that such interviews have far-reaching significance for the promotion of the project.
1.What are the forms of living microbial agents, and what are their advantages and disadvantages?
Vacuum freeze-drying and microcapsule techniques are more common in living microbial preparations. Vacuum freeze-drying technology is relatively mature, which has better protection for microorganisms and can achieve a high recovery rate. However, the form of medicine is powder, which has the problem of a single form of preparation. The microcapsule technology is relatively new, and mass production needs fluidized bed technology. At the same time, the microcapsule can be made into yogurt, jelly and other forms due to the independence of the capsule.
2.What expectations do people have for living microbial agents? What form of preparation can improve patient compliance?
For patients taking live microbiological agents for a long time, they want them to be small and convenient, and easy to carry and swallow. At the same time, most people still want some privacy when they take their medication.
3.What is the principle and process of vacuum freeze-drying of bacteria? What is the principle of protectant? What is the difference between different protectants?
The main process is to enrich and cultivate engineering bacteria, then centrifuge and isolate the bacteria. On this basis, the protective agent was added to the bacteria, and the bacteria were quickly frozen for more than one hour in the -80°C refrigerator, and then it was processed in the vacuum freeze dryer. There are many different theories on the principle of vacuum freeze-drying protectants, including the preferential theory of protective proteins, the hypothesis of water substitution, the influence of protectants on the vitrification transition temperature and the influence of protectants on the membrane phase transition temperature. Different microbes need different proportions of protectants to work best.
4.From what angle can we evaluate the advantages and disadvantages of a protective agent?
For example, the recovery rate of the bacteria after vacuum freeze-drying, the gene expression and function of the recovered bacteria, and the stability of the bacteria powder after freeze-drying (considering temperature and humidity) can be used as evaluation factors.
5.How to adjust the type and proportion of protective agents to find the most suitable one?
People familiar with freeze-drying technology can generally judge the type and proportion of protective agents through experience. In scientific research, there are many methods to determine the type and ratio of protective agents. The most common way is to design an orthogonal experiment to assess the recovery rate of freeze-dried powder and obtain the best analysis results through relevant regression analysis.
6.How to optimize the protective agent? Can some prebiotics and dietary fiber be added to improve the nutritional value of lyophilized powder?
Protectants can be added with prebiotics, such as dietary fiber to enhance the value of freeze-dried powders. Prebiotics are defined as organic substances that are not digested or absorbed by the host but selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the host's health. Oligosaccharides are commonly used in production, such as fructose-oligosaccharides, galactose-oligosaccharides, etc. In addition, polysaccharides and protein hydrolysates can be used as prebiotics. Also, oligosaccharides such as fructose-oligosaccharides can bring a sweet taste to the freeze-dried powder, and at the same time, increase the selling point of the product and improve the acceptance of patients.
7.How to verify the characteristics of enteric-soluble capsules?
Disintegration instruments are commonly used to simulate the physical dissolution of pharmaceutical preparations before absorption.
8.Why did the newly freeze-dried flora recovery rate only reach 20-30% in our experiment?
This is usually due to a poor metabolic state of the flora. Generally speaking, the gradient dilution counting method will have a quantity deviation of ±20% due to the specific operation of the operator. Another possibility is that engineered bacteria develop a living but non-culturable state (VBNC). The bacteria are biologically active but cannot be cultured to multiply and form colonies.
9.What other factors might influence the recovery rate?
Different freeze-drying processes of bacteria (including time and temperature of freeze-drying) may affect the recovery rate of bacteria. Secondly, the control of humidity is also essential. In general, we cannot control the humidity in the laboratory, but in the actual production process, it is necessary to maintain the relative humidity of the workshop below 50% to prevent the growth of bacteria in the powder.
10. Do you have more advice for us?
Currently, the commonly used prebiotics are mainly applied to bifidobacteria. For our engineered bacteria, it may be necessary to explore the formula to determine the type and proportion of specific prebiotics.
The latest literature shows that E.coli Nissle 1917 may have some potential safety risks, which is controversial in academic circles. But taken together, it is already the safest strain of E. coli.
Generally speaking, the optimization sequence of lyophilized powder is mainly to first design orthogonal test to determine the protective agent formula and strain recovery rate, stability. Then consider adding features, adding prebiotics and other additives to optimize the product.
Will (Wang Jiang) is the Senior Manager of Talent Development in the People Administration Group of Xbiome Operations department. Will gave us full guidance in Business Plan writing, Education and other activities.
Knowing that we will be doing something to help "raise awareness about intestinal health," Will suggested that we further subdivide the target group, and just define it as adults, which is not enough. By subdividing the crowd into "science and technology white-collar workers" and so on, it can contain more available information, which is more conducive to precision marketing. In the process of setting up activities, it is necessary to fully analyze the target's life trajectory, living habits and other information to help outline the portrait of the audience. He also suggested that we can use live broadcast, campus KOL, linkage with official media and other ways to help carry out health communication activities.
About writing the business plan, he suggested that we refer to the operation mode and characteristics of mature health management platforms, such as Ali Health Platform, and pay attention to the operation of the community. He reminded us that the family members of patients are also a part of our target group.
After identifying the pain points of the target population, the proposed solution is best presented in the form of visualization, which can attract the attention of investors, and the development of the project will be more concise.
He offered a theme of "caring for all". There is an old saying in Chinese, "No filial son before a long hospital bed". Will believes that we can expand our attention to psychological problems from "patients" to "patients' families". Expanding from patients to "amily" -- the basic unit of the society will make the pattern of our project bigger and more meaningful! We think what he says is quite reasonable. He also suggested that we pay attention to "the happiness of out-of-hospital treatment" of patients. He hoped that people could treat patients as equals and not discriminate against them.This also requires education of the patient's family.
Finally, in terms of business model, he suggested that we should consider the platform as a portal to attract all parties to enter, so as to effectively monetize the daily active volume of users and other traffic.
"Don't make the whole family sick."
of Science and Technology
Tingting Yu is an attending physician in Gastroenterology Department of Southern University of Science and Technology Hospital. She has been a doctor for ten years and has rich experience in diagnosis and treatment of common diseases in gastroenterology. The purpose of this communication with Dr. Yu is to listen to the clinician's comments and suggestions on our project.
For the first time we meet with Dr. Yu. She evaluated our program comprehensively. She agreed with the information we had learned and the efforts we had made. She gave us a clinician's point of view on the determination of drug forms. She completely overturned our previous belief that medicine as a snack would be more popular with patients. In fact, ordinary forms of medicine can give patients more privacy protection and care. In addition, she was so enthusiastic that she also helped us to review the materials we made -- picture books and three folding brochures. After finding that there was no problem, she agreed to use our materials as publicity materials in her department for patients to read. Her approval was so inspiring!
She also showed us the scale being used by the clinical psychology department, and introduced to us the relevant situation of patients' psychological problems, which is very helpful for us to understand the psychological needs of IBD patients.
Inspired by her, we also conceived the idea of establishing patient groups in Shenzhen and even Guangdong to strengthen communication between patient communities. However, due to practical conditions, we have to document the idea on our health management platform, hoping that it will become part of the platform 's functionality someday.
In addition, Dr. Yu also reminded us that we need to pay attention to the product name selection, do not use "colonization", "gene editing" and other unfamiliar terms for patients, which may trigger their doubts on safety. It is better to focus on the expression of "supplement probiotics" and "regulation of intestinal flora", which reflect the care of patients.
In short, Dr. Yu patiently listened to our problems and gave us a lot of good ideas. We are very grateful to her!
After our hardware was finished, we went to visit Dr. Yu in Southern University of Science and Technology Hospital again. Show her the hardware package we made. She praised our finished product and thought it was really practical. Especially for our manure collector, which she thought was very creative! Finally, we also officially placed our materials such as three folds brochures and picture books in every corner of the hospital, hoping to make a little contribution to patient education.
1. Q: According to your own impression and experience, what is the cognition level of IBD among people?
A: The level of knowledge about IBD is very low, and I think it is very meaningful to educate those with early symptoms. However, we don't know for sure who will show early symptoms of IBD. So it is necessary to promote awareness of the disease throughout society.
2. Q: What is the mentality of IBD patients you have contacted?
A: They all know there's no cure, especially CD's patients. For the children of 14 or 15 years old, parents will be very worried, far more than the children's worry about the disease. So, it is often the mentality of parents that brings bad influences to children. For adults, especially the middle-aged, the mood is even more broken.
3. Q: (After a brief introduction of the project) After listening to our introduction of the project, from the perspective of clinicians, how feasible is our project?
A: You should consider the comparison with existing treatment options to maintain remission. The current project is only in the experimental stage, so it is really difficult to do so much analysis. However, Dr. Yu still believes that our project has clinical value, and if our drug action pathway is similar to existing biological agents, it will be more feasible. The low diversity and abundance of IBD intestinal flora is indeed a starting point to consider.
4. Q: For patients with enteric-related diseases, do they have the need for dietary records, and how detailed is the record?
A: At present, there are no special dietary records made in clinic.
5. Q: In the process of clinical treatment, how do doctors interpret the patient's diet records, what specific diet information is involved, and whether the current recording method is inadequate?
A: During the hospitalization, the medical staff did not record the patient's diet. They just gave patients some simple dietary instructions, and dietary guidelines are not comprehensive enough, it is all in the form of Q&A.
6. Q: What are the specific clinic methods for the regulation of intestinal flora in patients? Are changes in intestinal flora related to the patient's diet? Can a patient's diet influence his gut flora to some extent?
A: Long-term dietary habits may affect the intestinal tract. Bifidobacterium triplex lives enteric-soluble capsules are commonly used in clinical practice to regulate the intestinal flora of patients. Patients should have regular daily work and rest, and their diet should not be too high in oil and salt.
7. Q: If this platform can be rolled out, can it effectively address the pain points existing in patients' dietary records? Do you have any other comments?
A: In theory, it can solve certain pain points. In clinical practice, pancreatitis and other diseases need to pay attention to diet, and often the realization of this concern requires the nutrition department to tell patients the specific diet content. Therefore, the platform can also be applied to other diseases to facilitate clinical services.
8. Q: Do you have IBD patients? Our team made a picture book on popularizing IBD disease. If possible, could it be used in your department?
A: Yes, picture books or brochures can be printed and placed in the nurse's station, publicity department or outpatient department of the hospital for patients to read. (Eventually we placed our picture books and brochures in the nurse station of the Hospital of Southern University of Science and Technology!)
9. Q: Do you have any recommendations for drug forms?
A: I think the points to consider include: ① The key is portability and storage conditions, capsules and other forms do not have too much influence. ② Targeted packaging design should also be carried out for drug concealment. Now clinically, we intentionally prescribe powder or pellet drugs for children.
10. Q: Do you have any other suggestions for our project?
A: I think it's important to educate the public about the microbiota in the gut and, in turn, about healthy eating. The idea that “human health is as important as microbial health” should be promoted. They should be told that they should work regularly, watch their diet, avoid high oil and salt, and maintain a normal lifestyle
At the same time, for the existing pain points, that is, the uncertainty of patients' inaccurate judgment of symptoms during the recovery period and lack of professional advice will lead to panic.
Therefore, attention should be paid to patient education, which has been mature at present (such as CCCF), but its role is limited. Secondly, we can help establish patient groups in Shenzhen, Guangdong, and strengthen communication between patient communities.
which is responsible for drug filings.
In order to understand the application approaches and methods of synthetic biology engineering bacteria drugs, we contacted the clinical department of Xbiome in charge of drug application, who has rich experience in applying for FDA certification. Through their sharing, we have gained a particular understanding of the procedures of FDA certification application, the procedures of Live Biotherapeutics (LBP) therapeutics application, and the requirements for clinical data. They also shared with us some domestic pharmaceutical enterprises successful declaration of ideas and models. This helps us with more preparations for drug approval in the accumulation of experimental data, confirms the premise that our products have commercial prospects, and provides a reference for implementing our products.
1.Q: What is the difference between the FDA approval process for synthetic biology-based engineered bacteria drugs and the normal LBP therapeutics approval process? What is the relationship between the FDA approval process for LBP therapeutics and orphan drug designation?
A: No difference. Engineering bacteria drugs based on synthetic biology belong to the category of LBP drugs, except for the related LBP drugs guideline (Early Clinical Trials with Live Biotherapeutic Products: Chemistry, Manufacturing, and Control Information), with some additional special requirements. However, these other requirements are not explicitly stipulated, these are only suggestions and considerations for safety. For each drug, we can negotiate with the FDA separately.
LBP therapy and orphan drugs are two different processes. LBP is the drug form, and orphan drugs are targeted for rare disease indications. LBP will be stricter, while orphan drugs have a fast track for declaration and policy advantages in some cases.
2.Q: What kind of experimental data are required for IND application of engineered bacterial drug (LBP therapeutics) based on synthetic biology? Do different types of drugs require additional experimental data according to the context in which they are used and the indications for using them? By which agency are these experiments designed?
A: There is no specific data required, but there is a general framework based on Guideline. The adequacy of the data is up to the FDA to determine.
3.Q: How long does it take to get an LBP treatment medicine to market?
A: At least five years. (guideline was published in 2016, but there are no mature LBP drugs on the market, no matter ordinary LBP or genetically engineered bacteria drugs) Even after an IND application, a new drug must undergo phase I to Phase III clinical trials and so on, and only after these trials are successfully completed, and the efficacy is verified can it enter the market stage.
At present, Evelo/ Seres /4D Pharma is leading the way in generic LBP drugs. One of the drugs from Zhiyi Biotech in China received IND approval from the FDA. But these are still in the clinical stage. Synlogic's LBP drug, based on genetically engineered bacteria, is ahead of the curve, and its PKU product is currently in phase IIa. They also have a pipeline for cancer, and in July, they partnered with Roche to start laying out a pipeline for IBD.
4.Q: What are the international and domestic regulatory and review institutions related to synthetic biology-based engineered bacteria and drugs? What is the specific review and supervision process?
A: The international authority is FDA, and the domestic authority in China is The National Medical Products Administration. The IND application process is roughly shown in figure below. Clinical trials can only be carried out after obtaining IND approval, and possible side effects should be disclosed at any time after clinical trials are carried out.
5.Q: Is there any precedent for the application of synthetic biology-based engineered bacterial drugs in China? Are there any cases where LBP drugs have been approved by domestic food and Drug Administration?
A: There is no precedent for genetically engineered bacteria drugs in China, which is also the difficulty and challenge. At present, there are two directions for reference, one of the directions is gene therapy, the other is LBP drugs. The LBP drug such as SK08 from Zhiyi Biotech in China is currently in clinical phase II.
6.Q: From the perspective of professionals in regulatory approval, how do you evaluate the possibility of a drug being approved? Is it possible for our team's project to pass regulatory approval?
A: It is based on data security. (Refer to PPT for research on related issues of genetic engineering drug development) Due to the current lack of data, it cannot be evaluated.
7.Q: In order to pass the relevant examination and approval and meet the standards of audit, from what angles can our team's project be modified or improved?
A: In addition to considering the safety of the drug itself, the production process also needs to meet relevant requirements. Generally speaking, it is a relatively complicated issue, according to the guideline.
FDA has an open attitude in this area, and there are no specific rules. However, once our drugs are effective, or we reach the stage where we think we can further open up, we can have a Interact meeting with FDA about what we do and seek opinions/suggestions from each other.