Rome was not built in a day. Many people, including researchers, clinicians, ethicists, entrepreneurs, and the general public, have profoundly influenced our projects at some point. Their suggestions made us realize the shortcomings of the project and make improvements in time. Next, we will state which content on the wiki was made under their influence to show how we approached our design step by step to their desire at different stages.
Overview about our integrated Human Practices
Proposed implementation1: target patients
Situation: Since the novel coronavirus appeared in Hubei, Wuhan at the end of 2019, more than 200 million people have been confirmed (2021/10/13/23:00) and more than 4 million people have died. According to the "China-World Health Organization Novel Coronavirus Pneumonia (COVID-19) Joint Investigation Report", "Most patients infected with novel coronavirus are mild and recoverable", but there are still "13.8% of patients with severe illness." As medical students, we deeply feel the great responsibility and hope to contribute to the health and epidemic prevention of the novel coronavirus in the world. Therefore, at the initial stage of our project, we established our project service targets as patients with novel coronavirus pneumonia.
Question: Novel coronavirus pneumonia is a complex disease, and it is difficult to find a treatment that is suitable for all patients. We need to further limit our target patients.
Feedback: In order to further clarify the target patients, we interviewed Li Chengzhong, director of the Department of Infectious Diseases, Shanghai Changhai Hospital. He once went to the front line of Wuhan to fight the epidemic. He said that according to the " Novel Coronavirus Pneumonia Diagnosis and Treatment Plan (Trial Eighth Edition Revised Edition)", critically ill patients with COVID-19 mainly rely on supportive therapies, and there is currently no specific therapy for them. The occurrence of cytokine storm is an important reason for the transition of patients from mild to critically ill and even death.
Improvement: Professor Li's clinical experience made us feel the importance of providing specific treatments for critically ill patients with COVID-19. We further considered critically ill patients as target patients to fill the gap. (Proposed implementation: target patients for details.) In addition, cytokine storm is our key consideration. Solving the cytokine storm of patients with COVID-19 may provide a solution for the treatment of patients with COVID-19.
(Interview with Dr.Li in ChangHai Hospital)
Engineering success（Chassis & Ms）
Situation: We have decided to use chimeric antigen receptors（CAR）currently applied to lymphadenoma to explore our project.
Question: It is clinically proven that CAR-T cells have serious side effects that trigger a cytokine storm, and this is what we need to avoid in the process of treating patients with COVID-19, so we explore other cell types that can be applied.
Feedback: After interviewing with immunology expert Cheng Qian, I learned that M1 macrophages are macrophages that can produce pro-inflammatory cytokines and are called classical macrophages. It has strong killing properties of microorganisms, but these characteristics are also prone to cause tissue damage. M2 macrophages are also known as Alternatively Activated Macrophage. Stimulants such as CSF-1, IL-4, IL-10, TGF-β and IL-13, fungal and helminth infections have been found to facilitate the differentiation of M2 subsets. It can be considered that M2 has certain anti-inflammatory properties and can secrete inhibitory cytokines such as IL-10 or TGF-B to down-regulate the immune response. In the human body, macrophages are stimulated in different ways and differentiate in different directions, which is according to the dynamic change of the internal microenvironment.
Improvement: In the conversation with Professor Qian, the anti-inflammatory properties of M2 attracted us, and we began to consider using macrophages as the chassis. Later, we chose the THP1 cell line and consulted Professor Qian about safety issues. She said that it was an absolutely safe cell line, and no safety issues have occurred in his experience.
For COVID-19 patients with cytokine storms, it is best to use CAR to stimulate the transformation of macrophages into M2 with anti-inflammatory properties. We constructed CARMERTK that can reduce inflammation and activate macrophages to engulf virus. (More details in Design of CARMERTK )(CARMERTK BBa_K4040018) In subsequent experiments, we successfully induced macrophages to exhibit anti-inflammatory properties while reducing viral load by designing the intracellular domain of CAR. (More details in Engineering Success: Testing the CAR receptors )
Situation: In our experiment, four CAR intracellular domains have constructed, and the other three CARs（including CARγ）have the properties of promoting inflammation.
Question: In the early stages of infection, the body's natural immune response helps to clear the virus due to a low viral load. Imagine if clinicians misjudge the use of CAR-M therapy, it will suppress the immune response of the body, resulting in a worsening of the disease, and the body can not properly perform an effective immune response.
Feedback: In the second phase of the project, we visited Professor Qian, an immunologist. We had a clear and thorough grasp of the infection mechanism of SARS-CoV-2. Professor Qian highlighted that SARA-CoV-2 has many mutated strains, such as the Alpha variant, which could cause a secondary infection, the Beta variant, which makes it harder for the vaccines to stay effective, the Gamma variant, which is more virulent, and the Delta variant, which is highly infectious. It has been reported that some strains invade the body in a dormant and a mitigatory way which result in more transmissible variant. At the beginning of the infection, SARS-CoV-2 can not only suppress the innate immune response by inhibition of interferon Ⅰ mediated inflammatory cells recruitment, but it can also escape the murder of acquired immune cells by lowering the expression of surface antigen. And the virus can consequently get mass propagation in alveolar epithelia cells without the worry of being destructed by the immune response. This is also the cunning of the novel coronavirus, the magic of ecstasy. In addition, Professor Qian also reminded us that it has a relationship with each person's constitution. Older people with underlying diseases have fewer white blood cells that can be mobilized, and are less able to detect new germs. Therefore, they may not experience severe inflammation.
Improvement: We constructed the proinflammatory CAR-γ macrophages (Mγ), to address the problem of low immunity in the early stage of COVID-19 infection. (More details in Design of CARγ )(CARγ BBa_K4040016) And in vitro experiments have verified that M γ promotes the secretion of cytokines and strengthens the body's elimination of viruses. (More details in Engineering Success: Testing the CAR receptors )
(Interview with Prof.Qian in the national key laboratory of immunology)
Situation: We have constructed pro-inflammatory Mγ and anti-inflammatory Ms, both of which have the ability to reduce viral load.
Question: After talking with Professor Dong Yuchao, who went to the front line of the fight against COVID-19, we learned that the symptoms and cytokine levels of COVID-19 patients did not exactly match during the clinical diagnosis process, and they could not accurately judge the timing of the application of the therapy. Even when serum cytokine concentrations are measured at any time, transient changes in inflammation levels cannot be obtained.
Feedback: We are inspired by the William_and_Mary team's project that we can achieve dynamic, automatic conversion of two cell phenotypes by designing CAR macrophages to sense cytokine levels in the environment.
Improvement: We decided to integrate CARMERTK and CARγ, and design a Smart macrophage, which regulates the phenotypic transformation between Mγ and Ms through a cytokine sensor, so as to realize virus clearance and bidirectional regulation of immunity. (More details in Circuit Design )
(Interview with Prof.Dong at ChangHai Hospital)
Situation: We are determined to design a Cytokine Sensor.
Question: There are a variety of cytokines in COVID-19 patients. Which cytokines should be selected as indicators of CRS?
Feedback: We asked immunological Professor Cheng, who pointed out that the intelligent transformation process of macrophages is a process of multi-factor interaction and is regulated by many regulatory molecules. This mechanism is complicated and difficult to determine a single cytokine. In general, IL-6 can be regarded as the core factor triggering the cytokine storm, but the influence of other cytokines on macrophages cannot be ignored.
Moreover, the application of drugs that block IL-6 activity gives us inspiration to choose IL-6 sensor as the button to initiate transformation.
Improvement: We consulted the literature on IL-6 signaling pathway and designed the IL-6 cytokine sensor after several rounds of brainstorming. We hope that by designing IL-6 receptor, the influence of IL-6 on macrophages will be expanded far beyond that of other cytokines.
Through gene circuit design, we successfully verified in vitro experiments that IL-6 Sensor activated GFP (representing CARγ expression) and mCherry (representing CARMERTK expression) respectively according to the concentration of IL-6 in the environment. (Details in the Proof of Concept: Testing Cytokine the Sensor). (More details in Proof of Concept: Testing Cytokine the Sensor )
Proposed implementation2：Safety Aspect
Situation: After finishing the design of Toggle Macrophage, for safety problems may happen in clinical application implementation. We consulted Professor Guo Meng from the National Key Laboratory of Immunology who engaged in the study of macrophages.
Question: If Toggle Macrophage is to be used in clinic, what should we consider about safety?
Feedback: Professor Guo Meng believes that although Toggle Macrophage has been proved to regulate the level of cytokines, the human body is a complex environment and we still need to carefully consider its safety. At least, a final "gate" is needed to prevent unexpected immune failure or overactivation. He recommended to us the chemical drugs which are widely used to precisely induce cell apoptosis.
Improvements: By referring to literature and consulting PI, we designed kill switch. A certain concentration of AP1903 can start the expression of Caspase9, so as to activate the rapid apoptosis of Toggle Macrophages. (More details in Safety )
(Interview with Prof.Qian in the national key laboratory of immunology)
Proposed implementation3：using method
Situation：The need to design the Using Envision of Toggle Macrophage.
Question：As clinicians, what are their expectations for the use of Toggle Macrophage? What are the key messages you want to learn?
Feedback: Professor Dong said, the primary concern for clinicians when using a drug is its dose. Cell therapy focuses on the number of engineered cells, so we should first tell the clinician the appropriate amount of macrophage. In addition, the ability of patients of different ages to tolerate the cytokine storm is certainly different, we should ensure that our product can control the cytokine within a certain level.
Improvement: Using method was improved to clarify the key operations that doctors need to perform: collect patients' peripheral blood, detect serum IL-6 concentration (I0), evaluate patients' physical condition, and determine the range of IL-6 control ([l, u]). (Details in the Proposed Implementation: Using Envision).
In addition, we built a model which can output the most suitable number of macrophages “n” after doctors input I0, l and u. Here, the “most suitable” refers to 1. The level of inflammation can be controlled to [l,u] promptly. 2. There is less fluctuation in control process. (More details in Model) In addition, the model also solved the problem of cumbersome procedures and high cost of therapy proposed by Professor Du. (See below)
Situation: Our project is still in the early stage of laboratory research, and the application of this therapy is not closely related to the reality of the public. Therefore, we need to build a bridge between synthetic immunology and the public, strengthen their understanding of our project content, and expect to get suggestions for improvement of the project. So we did a cell therapy based questionnaires to find out what they thought of our program.
Question: The survey found that most people did not know much about cell therapy at present. As for toggle macrophage, most of them thought it was far from what they knew, failing to understand its working principle and doubting about the efficacy.
Improvement: Through the questionnaire survey, we finally learned that the public was most concerned about the convenience, safety and cost of this therapy. At the same time, we also noticed that there was still a large group of people who didn't know much about synthetic immunology. In order to publicize the application value of our project under realistic conditions, we integrated the introduction of the project into the education package and picture book collection, so that more people can realize the practical significance of our project. The results of the questionnaire survey also provide guidance for the design of other contents and methods of our publicity and education, including freshman publicity, elective course opening, new media publicity, and the compilation of synthetic biology manuals. At the same time, we have established long-term cooperation with a number of colleges and universities and volunteer teaching teams to update the content of education package, hoping to promote the popularization of cutting-edge knowledge of biological science education for primary and secondary school students in backward education areas in China. (More details in Education)
(LZU-China used our Education Package and completed our users’ feedbak)
Academia→Proposed implementation4：Other Challenges
Situation: After the initial completion of the project, we asked Dr. Du Juan, Hematology Department of Shanghai Changzheng Hospital, to evaluate our project again.
Feedback: She pointed out that in the early stage of coronavirus infection, the body mainly starts the nonspecific immune response and the phagocytes play a main role. But as the viral load gradually rises, the body activates the humoral immune response by producing antibodies to neutralize the virus. At this point, macrophages are no longer the main force in eliminating viruses, and their anti-inflammatory effect through secreting inhibitory cytokines is limited.
Furthermore, the polarization state of macrophages is elusive and heterogeneous with various factors affecting polarization. So M2/Ms may differentiate into different types due to the complex local tissue microenvironment (TME), unable to ensure its continuous performance of inhibiting inflammation.
At the same time, she pointed out that Silent Macrophage can not only inhibit inflammation, but also promote the repair of scar tissue. The generation of lung scar will lead to the obstruction of lung ventilation, resulting in the damage of lung function. These questions will continue to guide our thinking on how to improve the project. We expect that these problems will be further improved and solved with further research in related fields in the future.
She pointed out that although cell therapy still had some problems to solve, it was now more and more accepted by patients in clinic. She suggested that our treatment idea could be extended to other inflammatory cytokine storm diseases, which really gives us a broader range of applications.
Finally, when we proposed the possibility of commercialization, she thought that considering the cost of disease treatment, if we induce macrophages to commit suicide or add drugs to accurately induce their apoptosis, we need to constantly detect the condition and timely inject monocytes, which will make the treatment complicated and greatly increase the cost. She suggested that we can start from metabolic pathways to regulate macrophage activity and resting state. She also gave concrete suggestions: consider using the macrophage itself in accordance with ecanoic acid pathway metabolic regulation as an anti-inflammatory role. Resting metabolism pathway should be designed so that when the cytokines in the body trigger a dangerous threshold, macrophages keep a resting state.
Improvement: We have not considered the three problems raised by Professor Du and we have not had the energy to solve these three problems this year. These three questions are undoubtedly proposed by Professor Du on the basis of years of clinical experience and extensive literature reading, and are the key issues that we cannot bypass. These three questions are so wonderful that the content of our “Proposed implementation: Other Challenges” becomes very substantial.
(Interview with Dr.Du at Changzheng Hospital)
Industry→Excellence in Another Area
In order to explore the possibility of practical application and industrialization of our project, we investigated the industry lines and operations of CAR-T cell therapy companies in Shanghai that have conducted clinical trials. We learned that there are currently two mature CAR-T therapy products on the market in China, dozens of domestic companies have already arranged CAR-T therapy. The Frost Sullivan research report shows that the market size of analytical cell therapy will grow from 200 million yuan to 5.3 billion yuan in 2021, and grow to 28.9 billion yuan in 2030. However, the domestic homogenization competition is serious and the targets are clustered. Currently, the global CAR-T therapy layout is still targeted at hematoma. And in the future, CAR-T therapy which have a significant effect on solid tumors will stand out in the market of hundreds of billions of dollars.
In China, CAR-M therapy is still in the laboratory stage and has not yet begun clinical trials. As of November 2020, two clinical trials based on CAR-M strategies have received FDA approval. CARISMA Therapeutics' candidate cT-0508 (Phase I clinical trial), along with Maxyte's McY-m11, is currently recruiting volunteers for phase I clinical trials. In China, CAR-M therapy is still in the laboratory stage and has not yet begun clinical trials.
After market research, we drafted a business plan and presented it to Ms. Li in the biomedical research group of investment banking department of Guosen Securities Company to evaluate the possibility of our commercialization. (More details in Entrepreneurship)
（We invited Investment Bank Department of Guosen Securities to evaluate our business plan）
Cell therapy has been clearly assigned to drug management in China. Some suggestions were put forward to regulate the production and quality control of cell therapy products according to the contents in the Criterions for the Quality Control of Drug Manufacturing released by National Medical products Administration (NMPA).
（We put forward suggestions to NMPA about quality control of cell therapy）
Our products are mainly used to treat critical COVID-19 patients, of whom the major group are old people. Therefore, we also put forward corresponding opinions to the Ministry of Human Resources and Social Security of the People's Republic of China on the protection of rural elderly people in the prevention and control of the epidemic, hoping to provide them with more humanized services and conveniences from the perspective of prevention, control and prognosis.
(Suggestions about the basic living and medical care for the rural elderly people in China under the epidemic situation )
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