Team:WHU-China/Human Practices
Prof. Liu Tiangang
Prof. Zhang Xiaolian
Prof. Zhang Qiuping
Dr. Peng Zhiyong
Prof. Chen Pu
Prof. Yang Daichang
Dr. Ma Zhaotang
In a nutshell, integrated human practices demand for thoughtful and reciprocal interactions with the community, with the diversity of involved groups especially highlighted. This year, we designed and implemented the interconnected work of human practices, which integrated our project with outside world, while strictly based on principles presented by iGEM.
In the face of COVID-19 pandemic, human practices this year differ greatly from normal iGEM seasons. To accomplish the goal of integration, we developed varied human practices strategies for difficulties in divergent periods, i.e. social distancing period (from May to August)-online face-to-face communications aided by Tencent Meeting and Zoom, back-to-campus period (from September to October)-offline meetings with sufficient self-protection. The online human practices, in a degree, displayed the advantages of convenience over offline human practices, which bypassed the obstacles of long-distance travelling and unnecessary time loss. It’s admitted that the flexibility of social media tools enabled more meaningful integrated human practices events with higher efficiency than our expectation, and successfully kept the continuity of our evolving project interacting with stakeholders in real time. Most importantly, the integrated human practices works did motivate our project from different aspects in distinct stages.
Despite more efforts needed to improve our project (mainly the part of Sensing Module concerning human immunity), we very much appreciate the advantageous instructions offered by the top experts, and hold the confidence that our two-phase project ‘The Negotiator’ will eventually find the solutions to these existing technical barriers and benefit the world with a novel and achievable therapeutic methodology.
Prof. Liu Tiangang is the professor of Wuhan University School of Pharmaceutical Sciences and the director of Hubei Engineering Laboratory for Synthetic Microbiology, and his group mainly focuses on the discovery and overproduction of natural products guided by in vitro platforms. During the coronavirus outbreak and later city lockdown, his laboratory collaborated with Renmin Hospital of Wuhan University, and made critical contributions in pathogen diagnosis and drug use. To have a better understanding of nosocomial infections and demonstrate the necessity of our project in the epidemic, we established collaboration with Prof. Liu. Moreover, he accepted our invitation to join the team and serve as secondary PI.
As one of the leading experts fighting the first wave of COVID-19 epidemic, Prof. Liu noted that nosocomial infections in the epidemic were way more pervasive than usual, given the crowded hospital facilities, and became a major death cause in Wuhan, comparable with another well-known factor, cytokine storm caused by over-activated immune system. He also addressed the necessity of our aim, i.e. ventilator-associated pneumonia (VAP) that our project highlights included a large proportion of nosocomial infections in intensive care units.
Prof. Liu’s group and co-workers developed an advanced diagnostic method named nanopore targeted sequencing (NTS) that highlights the implementation of MinION and targeted PCR amplification to rapidly characterize hidden pathogens, including virus, bacteria and fungi, with higher sensitivity than regular methods. The massive sequencing data and real-time analysis could lead to insights into the species of pathogen bacteria and fungi, as well as their antibiotic resistance phenotypes, and ultimately guide precise drug use to eliminate the pathogens. Prof. Liu provided us with relevant results and especially mentioned that gram negative bacteria are troublesome, due to their complicated cell wall structures. Currently, to cope with nosocomial infections caused by gram negative bacteria, polymyxin could be the potent cure in most cases. Our target, Pseudomonas aeruginosa, is indeed an important gram negative pathogen, however, Acinetobacter baumannii, a common opportunistic gram negative bacteria hard to tackle in clinical practices, cannot be handled by our current strategy. Prof. Liu suggested us to re-evaluate our project and propose a practical strategy versatille to all gram negative bacteria in Phase II.
It’s known that COVID-19 infection also renders deteriorative and dysfuctional human microbiota in digestive tract, which is considered another dangerous factor amongst immunocompromised patients. As introduced by Prof. Liu, traditional combinatorial probiotic therapies have been implemented to improve the disordered gut microbiota of endangered patients, Clostridium difficile proliferation and accumulation in particular. He approved of we choosing Escherichia coli Nissle 1917 as the chassis, yet considering the uncomfortable smell of E. coli, a more acceptable chassis is recommended in Phase II, if we want to turn ‘The Negotiator’ to a real product. In addition, as a successful industrial practitioner, Prof. Liu briefly introduced the prospects of engineered probiotic and micro-ecological therapy to date, and recommended us to search for entrepreneurship information from well-known companies, such as Synlogic and Seres Therapeutics.
Prof. Liu’s laboratory has been working on cell-free platforms over the past decade. Having known our plan to reconstitute quorum sensing pathways in vitro for the discovery of quorum sensing inhibitors and prototype genetic parts of interest, he offered us valuable suggestions and experimental facilities including a natural product library to enable the screening. More importantly, Dr. Liu Ran and Mu Xin of Liu Group, whose interests include in vitro reconstituted natural product biosynthesis, became the instructors of our team to give us concrete guidance in cell-free methodology.
Prof. Zhang Xiaolian is the executive deputy director of Department of Immunology, Basic Medical College. Her group mainly specializes in
infection immunity of pathogenic microorganisms and glycobiology, which involves chemotaxis and relevant mechanisms.
Immunity is amazingly intricate and unpredictable. The excessive expression of chemokines may cause cytokine release syndrome(CRS), asthma and many other problems, while the compromised immune systems will provide the intruders with increasing opportunities. To avoid detrimental reactions and meanwhile implement Sensing Module, Prof. Zhang noted that the delicately controlled and tissue-specific gene expression can make us better control the expression level of chemokines and reduce the possibility of those syndromes.
Additionally, the leakage of Sensing Module might lead to unpredictable results-we never want human microbiota to randomly obtain the ability to activate our immune system. Prof. Zhang nodded to our idea of developing a toxin-antitoxin system in case of horizontal gene transfer.
For respiratory tract infections, Prof. Zhang suggested intaking the engineered probiotics by aerosols, a delivery pattern similar to asthma inhalation. Yet whether the probiotics could reach the right spot in this way still needs further animal experiments for the proof-of-concept.
Prof. Zhang Qiuping is the deputy director of Department of Immunology, School of Basic Medical Sciences, Wuhan University, and the director of Hubei Society For Immunology, the vice president of Wuhan Society For Immunology, and the vice chairman of Hubei Society of Medical Bio-Immunology’s Basic Medical Sciences chapter. Her main research directions include chemokines and receptors, drug resistance mechanisms, and metastasis in acute lymphoblastic leukemia.
Theoretically, Prof. Zhang thought the design of Sensing module is reasonable. However, while applying our engineered probiotics in the real-world situation, the results will be highly unpredictable: The responses to chemokines vary greatly among individuals, so the appropriate expression level will not have a exact number, but also vary among individuals. As the cytokines with no direct path to induce inflammation, chemokines are, however, involved in the complex immune regulatory networks, which means chemokine expression might indirectly upregulate the expression of several key inflammatory factors. Thus, more efforts should be considered for preventing potential inflammations arising from chemokine expression, which can easily lead to airway obstruction.
Existing strategies for dealing with pulmonary bacterial infections include antiobiotics and immunity enhancement, and the latter include injection of interferon, thymosin, colony stimulating factors and so on to improve overall immunity. Prof. Zhang explained that there is limited clinical example of applying chemokines to address pathogen infections in COVID-19 epidemic according to her knowledge, and she suggested us to contact Dr. Peng Zhiyong, the leading expert familiar with ICU situations in Wuhan.
To estimate the appropriate dosage of chemokines, it is necessary to find relevant literature and conduct preliminary tests by ourselves. Transwell experiments are suggested to illustrate the effects of differing chemokine varieties, as well as to determine the appropriate concentration. Culturing immune cells is hard, so it is recommended to try to culture monocytes first, and neutrophils later. Importantly, neutrophils induced to differentiation are less difficult to culture than neutrophils isolated from peripheral blood. Prof. Zhang generously offered us the monocyte cell line THP-1.
Dr. Peng Zhiyong is the director of Department of Critical Care Medicine in Zhongnan Hospital of Wuhan University, who possesses a comprehensive understanding of
intensive care units during the epidemic. His team began to participate in fighting the COVID-19 pandemic in early January, played a decisive role in fighting coronavirus as well as nosocomial infections, and didn’t quit until the pandemic was under control. We visited his office and interviewed this devoted and experienced expert face to face, and received constructive advice for improving our projects from all perspectives.
As implied by Dr. Peng, many patients with severe diseases need to put on a ventilator as soon as they are sent to the hospitals, which leads to quite a high rate of the nosocomial infections (including ventilator-associated pneumonia). However, Dr. Peng said, while nosocomial infections do correlate with the physical conditions of the patients and therefore with the severity of the disease, they also have a lot to do with the environment where the patients stay, that is, they relate to the quality of the medical workers including the awareness of aseptic technique and hand hygiene. Moreover, the overuse of antibiotics may have an influence as well. This shows the seriousness of the problem and the importance of our work. And he mentioned that in spite of Pseudomonas aeruginosa, there was another pathogen called Klebsiella pneumoniae which was becoming more and more dominant in the area of ventilator-associated pneumonia. This kind of bacteria, which also belongs to Gram-negative family, can be our future target as we want to choose more quenching enzymes aiming at different kinds of AHLs. Dr. Peng told us that current solutions to nosocomial infections mainly focus on the prevention, like cleaning the wards frequently, using aseptic technique and so on. If effective, our project may contribute a lot to the prevention of such diseases.
As suggested by Dr. Peng, usually the biofilm forms at the inserted branch tube of a blood vessel. The blood vessel considers the tube as foreign matters and will wrap it up gradually to remove it, and finally the biofilm forms. However, it’s difficult for most antibiotics to pass through the biofilm, which makes pathogenic bacteria easier to grow in it. Dr.Peng told us that there are no effective methods to solve this problem currently, and what doctors can do is to use some special antibiotics that can pass through the biofilm and kill the pathogens in it. Thus since our probiotics can prevent the formation of biofilms, it may help decrease the amount of pathogens and the frequency of nosocomial diseases.
As mentioned by Dr. Peng, there have long been studies on chemokines recruiting immune cells, but none of them have completely succeeded mainly because of the complex network of different chemokines, each of which cannot be targeted as a single pathway. Besides, the potential risk of inflammatory factors storm makes it harder to utilize this method in clinic. In spite of such a tough situation, Dr. Peng still provided us some new ideas of solving the problems: by using a method similar to hemodialysis, their team has worked on letting the blood of the patients pass through special materials with the ability of non-specific adsorption of cytokines to reduce the peak of the cytokines, which can prevent the inflammatory factors storm. In the pandemic this year, their team had successfully reduced the concentrations of cytokines of some patients and kept them from inflammatory factors storm in this way. As for our project, it may be a nice try to use this method locally for patients’ lungs after using our probiotics to prevent inflammatory factors storm. But more factors should be taken into consideration then.
Dr. Peng has approved of our choice of using mist spray to deliver the drugs (our engineered probiotics) and the possibility of mixing anti-inflammatory drugs with it to reduce the risk of inflammation. And he also suggests that we should ask experts of drug manufacture to get more advice on how to choose the proper type of spray. This may lead to our further HP activities in the future.
Although Dr. Peng was not very familiar with engineered probiotics, he still showed great interest on this idea. He thought that our engineered probiotics could make up several disadvantages of the traditional antibiotics therapy as the probiotics could break down the biofilm which could greatly weaken the function of antibiotics. But he also pointed out that to convince the public of the safety of our probiotics, we should focus more on explaining what “fate” the probiotics will finally end up with (Will it disrupt the normal microbiome in our bodies? How is it cleared from our bodies?). This directly manifested the importance of our design of several parts to prevent the probiotics from doing harm to the human bodies. It also made us decide to explain the safety of our probiotics more carefully.
As Dr. Peng has much clinical experience, we also asked him to give us some advice on our popular science comics. He provided us many useful suggestions. For example, he noticed that the expression “using buccal intubation instead of nasal intubation can prevent ventilator-associated pneumonia” is not rigorous as buccal intubation is also an invasive method which cannot prevent ventilator-associated pneumonia actually. So he made us change that into “using noninvasive method instead of invasive method can prevent ventilator-associated pneumonia”. Thanks to Dr. Peng, our popular science comics is much more rigorous now so that it won’t lead the public to a wrong direction.
Prof. Chen Pu is from Wuhan University School of Basic Medical Sciences, whose group focuses on chip-based biomedical engineering. To demonstrate the feasibility of using a
microfluidic chip to help illustrate our strategy, we established long-time connection with Prof. Chen and aimed to have further collaboration (i.e. build the chip by 3D printing, optimize the chip by data-driven fluid dynamics models and perform experiments upon the chip) in Phase II of our project.
Prof. Chen approved our design to immitate human respiratory tract and establish in vitro disease models, for circumventing the use of animal models. However, to simulate different organs, the design of microfluidic chips varies significantly. Unfortunately, Prof. Chen’s group didn’t utilize respiratory tract-mimicking microfluidic chips before. Thus, Prof. Chen suggested us to seek help from companies that have similar products, and attentively evaluate the challenges that are described below.
The design of a microfludic chip sometimes relies on matched equipment, such as pumps and robotic liquid handlers. Choosing software-assisted pump, gravity flow or capillary flow to control the fluid is a key step, which is in accordance with the specific experimental needs. Importantly, fluid dynamics models help to construct a chip that can be applied into actual use.
Culturing cells on the chip requires much more experiences and skills than on the plate, thus adequate preliminary experiments are greatly needed. Prof. Chen offered us beneficial suggestions for protocol optimization, e.g. the choice of cell lines and the appropriate density of cells cultured on the chip.
Prof. Yang Daichang, an academic leader in the State Key Laboratory of Hybrid Rice, is well-known for his experiences in entrepreneurship. He founded Oryzogen (Wuhan Healthgen Biotechnology Corp.), and endeavoured to manufacture plant-origin recombinant protein products, mainly using rice endosperm cells as the factory. Recently, their state-of-the-art drug, OsrHSA (recombinant human serum albumin), has been examined and approved by both FDA and CFDA to perform clinical trials. To further understand the difficult route from lab to fab, we interviewed Prof. Yang and obtained useful suggestions to improve our
ongoing business plan.
We were worried about the public attitudes towards genetic engineering and released a questionnaire about engineered probiotics. In response to our concerns, Prof. Yang noted that genetic engineering is a technique but not a product, and if the engineered product outperforms currently-used competitive goods in safety and effectiveness, especially in the fields of medicine, the market and public will know and respond first-hand.
For freshmen in entrepreneurship, Prof. Yang offered us valuable suggestions on business plan. Importantly, to address the ‘pain spot’ of a fluctuating market, he told us to comprehensively analyze competitive products to date and critically weigh advantages and disadvantages. Besides, long-term perspective is encouraged in a business plan that has the potential for real-world applications. To evaluate biotechnology start-ups, no less than 7 years are needed. Based on sustaining investment and improvable techniques, extraordinary perseverance might lead to success in stages.
Prof. Yang introduced the remarkable examples of Oryzogen’s plant-origin recombinant human serum albumin. The idea of producing human serum albumin in the plant cell factories originated in 2005, and researchers took six years to enable the proof-of-concept. They then optimized the manufacturing technique from four steps to three steps with enhanced product purity, and finally met the clinical standard in recent years.
Most importantly, Prof. Yang recapitulated the general demands for biomedical products with four words, i.e. safety, effectiveness, controllability, availability. He explained that a novel drug should be at least safe and effective to patients, and controllability means the manufacturing streamline is strictly controlled in order to guarantee the quality of products. After the demands for ‘safety’, ‘effectiveness’ and ‘controllability’ are fulfilled, the appropriate and reasonable price should be proposed to enable the ‘availability’ towards the products amongst consumers, whilst considering maximizing the profits.
Dr. Ma Zhaotang, deputy director of new drug application service center of Taizhou pharmaceutical high tech Industrial Park, is good at the registration and application business of enterprise new drug development process. The new drug application service center has a long-term connection with the Chinese Food and Drug Administration(CFDA), providing professional services in the whole process of new drug registration and application for enterprises, and has rich relevant experience. This interview mainly focused on the
procedures and policies of product transformation in China.
Engineering probiotics belong to therapeutic biological products. The product development process includes:
1)First, it is necessary to carry out sufficient basic pharmacology research and safety and effectiveness evaluation (preclinical research of new drugs, including animal experiments, etc.), and these data will be submitted to the FDA for communication (ind application, new drug clinical research application).
2)The center for drug evaluation (CDE) of the food and Drug Administration (CFDA) will send a special team to conduct the evaluation and issue a clinical trial notice (license) if there is no problem.
3)Based on this notice, clinical trials (including phase I, II, III) will be carried out, and the data of clinical trial results (including the change and optimization of process and quality standards, and the stability of manufacturing process quality) will be collected and sorted out for the declaration. The drugs can be put into the market after being successfully declared.
It is suggested that we should cooperate with the professional development team of the reliable enterprise in the form of technology transfer or authorization, and the enterprise should carry out the second half of the work starting from the clinical research. Clinical trials and even later construction of the production line need a lot of funds and hundreds of millions of yuan of investment. (our team) if we can finish the preliminary work, it will be good enough.
For reference, the policy and regulation documents include the administrative measures for drug registration (biological products) and some technical guidelines for the development of biological products (refer to the official website of FDA for details).
If there are technical details involved, it is hard to say anything. We need to ask the evaluation experts of CDE (Drug Evaluation Center) or refer to the technical guidelines (documents) about genetically modified biological products on the official website of the FDA.
First of all, patent application is needed. Patent protection is very important for scientific research team. Cooperation involves specific terms of business cooperation, which requires mature operation experience of someone in the team. Last but not least, the subject of technology holder needs to be clear.
