Team:NUDT CHINA/Human Practices
Designed Protein Degradation Method Based on
Trim21 And Nanobody -- Human Practices
Silver HP
Overview
We investigeted several issues related to our project in order to make our work responsible and good for the world. Different measures were taken to engage with our relevant communities.
Click on words below for more info!
Expert Interviews
Part 1 Design
As the project design is the core of our work, we interviewed Professor Nie (from College of Chemical Engineering, Hunan University) about some details in the experiment.
The origin function of Trim21
Trim21 binds to the fc domain of the antibody, and the proteasome degrades the antigen, but the trim is endogenously expressed (intracellular), while the antibody is in the circular (extracellular). They are in two different places so that the meaning of existence of Trim21 protein should not be the degradation of antigen, so what is the main function of Trim21? Is it that the escaped virus carries antibodies outside the cell and is recognized and degraded after entering the cell?
TRIM21 is an intracellular antibody effector in the intracellular antibody-mediated proteolysis pathway. It recognizes Fc domain and binds to immunoglobulin G as well as immunoglobulin M on antibody marked non-enveloped virions which have infected the cell. Either by autoubiquitination or by ubiquitination of a cofactor, it is then responsible for directing the virions to the proteasome. TRIM21 itself is not degraded in the proteasome unlike both the viral capsid and the bound antibody.
TRIM21 is also part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus.
The delivery of Trim system
Method:
1) banding peptide
2) directly transfer proteins into cells
3) nanobody packaged
4) functional materials such as MOF
5) bring some ligands, and combine with the receptors on the cell to invade the cells, or directly into the cells
6) cationic modification
Design:
1) one for multiple targets with multiple nano body: multiple target screeninge
2) take DNA Aptamer for targeting
3) similar to glucagon, but with endocytosis exocytosis: estrogen receptor
The turnover of Trim21
How is Trim21's own turnover in the cell? Can it be self-ubiquitinated? Is it ubiquitinated with a complex?
Design:
1) fixation of GFP initial value
2) trim21 self-fusion gap
Trim21's oscillator circuit design
Enhancer expression degradation, with a simple double negative feedback loop design and optimize its oscillation time, combined with specific physiological phenomena, such as can be consistent with the clock time.
Part 2 Feasibility & Practicality
Although our project is just a small step forward on the way to specific protein degradation, if one day we find proteins that can induce cell carcinogenesis then we can use this technique to weed out these carcinogenic proteins in the human body, which will contribute a lot in the history of Medicine. However, we had no idea about the feasibility and practicality of the application of PR PREDATOR in actual disease, especially cancer. And to what extent could the new development of cancer treatment be promoted?
Based on the above questions, we contacted Professor Qiang Ma and Professor Yunshan Ning in School of Laboratory Medicine and Biotechnology, Southern Medical University by emails on the exchange and consultation.
Through the interviews, we had learned that the degradation of target protein is still a cutting-edge field in scientific research and clinical application. The environment of human cells is so complex we can’t exactly say which protein is absolute good or bad for humans. And the degree of degradation may affect the internal environment as well. Talking about the details in experiment, we still had some issues to consider, including cross-reaction, the affinity of single-domain antibodies and so on. Only when we solved these problems could we took further step in application and industrialization of our project.
Company Research
On 28th September, we visited Genetalks in hope of getting professional advice based on the current market and we were lucky enough to interview Dr. Zhuo Song. As the CTO, he provided us with very pertinent ideas about our project as well as comprehensive answers to our problems, which inspired us quite a lot.
Our interview questions fallen into three aspects as follows:
The first is about the limitations of existing methods of reducing gene expression such as gene knockout, RNAi and novel gene editing techniques. We hope that through having a clear view on what problems still exist, we can know which kind of improvements that we actually need to focus on.
Dr. Zhuo told us that all traditional methods of reducing gene expression had efficiency problems and moreover, they also yielded unknown effects.
The next one is safety issue. Although there are few clinical applications to degrade intracellular proteins at present, there is a general trend in market demand. The main problem is that the biological complexity leads to unknown practical application. Therefore, safety is the most essential topic we need to consider about.
Considering the safety issue about our project, Dr. Zhou raised two aspects.
1.Unknown effects in vivo environment
Dr. Zhou mentioned that the purpose of protein-specific degradation technology is to degrade abnormal or overexpressed proteins. The implementation of this method in vivo must ensure that it does not introduce other problems that are adverse to the organism. It should be noted that in vitro and in vivo environment are not equivalent.
2.Excessive protein degradation
From an evolutionary point of view, it is only the amount of protein present in an organism that determines its actual function. Therefore, the degree of protein degradation on the biological effects should also be taken into account. For example, the removal of certain overexpressed proteins may affect the normal physiological activities of the organism.
Last but not least, it is about the possibility of application. We hope based on his abundant market experience, Dr. Zhou can give us more detailed suggestions about the approaches to clinical application and marketization of our project. Here are some significant answers we get.
1. What difficulties do you think our project will meet in the result transformation?
Dr. Zhou: The first thing must be considered is security issues. Also, you have to look for clinical trials of medical institutions that are willing to cooperate. What’s more, you need to pay attention to the negative or positive result of the standard.
2. During the transformation of achievements in China, the situation that the intermediate process of commercialization and industrialization of scientific and technological achievements is missing, always occurs, which is related to both universities and enterprises. Can you give some suggestions to avoid the "blank" area?
Dr. Zhou: Much of the reason for the "pilot" area actually lies in the role of government. While protecting the intellectual property rights is to protect the benefits of universities, the cost of subsequent transformation is very high, which is related to the cost of patent and development. Foreign commercial interests are only granted to a company, and the risk of success or failure is shared. In our country, patents have not really transformed.
3. If the improved protein degradation method in our study is introduced, can it play a significant role in diseases such as malignant tumors in the future?
Dr. Zhou: If the improved protein degradation method in the subject is applied, the following problems should be considered:
a) Can protein degradation last functioning?
b) Are there any incidental effects of unknown after degradation?
c) To what extent can the degradation be? How do you control that?
d) Can the degradation cause cell idle?
e) Concerns about sensitivity and specificity
Try to think about the dynamics of the control network and choose a clear application point.
If these problems can be solved, this method will play an important role in the application of diseases such as tumors.
Speeches at Streets & Survey
To make our work responsible and good for the world, people’s attitude is an important factor. We did a survey of people’s cognition of traditional curing methods of tumor, cancer and Alzheimer’s disease and potential methods using PR PREDATOR. In addition to investigating this issue, we worked hard to engage with people by delivering speeches at streets. We inspired people by introducing our project in its future application and raised their awareness of preventing the fatal diseases mentioned above. The results of survey and details about speeches can be seen on our Education Page.
Through the survey and engagement with the public, we had a clear view of people’s attitude towards “target therapy” and the future application of PR PREDATOR. Since many of people involved had a positive attitude to our project, we would continue working on experiment and try to exert a greater influence in maintaining health.
Co-Research
Part 1 On Competition Experience
We have communicated a lot with CSU_CHINA. This is a video clip recording our exchange on participating in iGEM. During our first meetup, team leaders showed the situation and progress of projects. And the present members had a heated discussion. We hope more people can pay attention to and participate in iGEM, expanding influence of synthetic biology in society.
Part 2 On Fundamental Study
Knowing that there was a gap between scientific research and product development in China, with the proposal of team Tianjin team, six universities worked together to make research on how the laboratory results are converted to market products. We conducted market research on the products based on fundamental study and then investigated the evaluation mechanism. The final report of commercialization of research findings worked as a reference guide for foundational advance.
The body of the report is the analysis and research of this transformation process (with theory and case study) and our projects. The theoretical part was completed by iGEM Tianjin and our team. The case study was completed by OUC-China and Jilin_China. In our own project analysis section, ZJU-Chian and XJTU-China wrote a detailed application plan.
The theoretical analysis we were in charge of is as follows:
Finishing this part of the report, we discovered that the transformation of research findings was a comprehensive process related to many factors. Government, users, companies, and scientists should work together for the final goal. In view of our project, we should consider market demand if we wanted to convert our innovative protein degradation tools to a practical curing method.
CFDA Inquiry
As our project will finally apply to mammalian cells in curing protein-related diseases, we must take laws and policies into consideration from the time we conduct experiments in the laboratory. We have inquired China Food and Drug Administration about this issue and got a feedback of the interpretation related to Technical Guidelines for Research and Evaluation of Cell Therapy Products.
Cell therapy technology is currently the key development field of international medical frontiers, which provides new hopes for the treatment of some complicated diseases. This policy was released in 2015 in order to better create an environment for cell product research and development and provide technical support to relevant scientific research institutions and enterprises.
The contents helpful to our project are shown as follows.
According to the general principles and basic requirements, we designed and conducted our experiment and future work in a more effective and rational way. We had a deeper investigation of non-clinical research on cell therapy products.
Gold & Integrated HP
Overview
Initially, we interviewed professors in universities about the design, feasibility and application of our experiments. Based on the promoted lab work, we conducted company research for more market-based information, and then had the idea of degrading target proteins related to some diseases. Next, we investigated people's attitudes towards our innovative tool of curing methods with speeches at streets and got some positive feedbacks. Realizing most people were not so familiar with the degradation process, we educated them in different ways and the public engagement activities worked as expected. As for the transformation of scientific findings, we, together with five other teams, had research on how the laboratory work were converted into products. Our team mainly focused on theoretical analysis of the transformation procedure and got a deeper understanding of issues like evaluation assessment and intellectual property protection. In addition, we had an inquiry into laws and policies with CFDA and obtained guidelines in cell therapy products, which would help our further cell experiment.
Click on words below for more info!
Expert Interviews
Part 1 Design
As the project design is the core of our work, we interviewed Professor Nie (from College of Chemical Engineering, Hunan University) about some details n the experiment.
The origin function of Trim21
Trim21 binds to the fc domain of the antibody, and the proteasome degrades the antigen, but the trim is endogenously expressed (intracellular), while the antibody is in the circular (extracellular). They are in two different places so that the meaning of existence of Trim21 protein should not be the degradation of antigen, so what is the main function of Trim21? Is it that the escaped virus carries antibodies outside the cell and is recognized and degraded after entering the cell?
TRIM21 is an intracellular antibody effector in the intracellular antibody-mediated proteolysis pathway. It recognizes Fc domain and binds to immunoglobulin G as well as immunoglobulin M on antibody marked non-enveloped virions which have infected the cell. Either by autoubiquitination or by ubiquitination of a cofactor, it is then responsible for directing the virions to the proteasome. TRIM21 itself is not degraded in the proteasome unlike both the viral capsid and the bound antibody.
TRIM21 is also part of the RoSSA ribonucleoprotein, which includes a single polypeptide and one of four small RNA molecules. The RoSSA particle localizes to both the cytoplasm and the nucleus.
The delivery of Trim system
Method:
1) banding peptide
2) directly transfer proteins into cells
3) nanobody packaged
4) functional materials such as MOF
5) bring some ligands, and combine with the receptors on the cell to invade the cells, or directly into the cells
6) cationic modification
Design:
1) one for multiple targets with multiple nano body: multiple target screeninge
2) take DNA Aptamer for targeting
3) similar to glucagon, but with endocytosis exocytosis: estrogen receptor
The turnover of Trim21
How is Trim21's own turnover in the cell? Can it be self-ubiquitinated? Is it ubiquitinated with a complex?
Design:
1) fixation of GFP initial value
2) trim21 self-fusion gap
Trim21's oscillator circuit design
Enhancer expression degradation, with a simple double negative feedback loop design and optimize its oscillation time, combined with specific physiological phenomena, such as can be consistent with the clock time.
Part 2 Feasibility & Practicality
Although our project is just a small step forward on the way to specific protein degradation, if one day we find proteins that can induce cell carcinogenesis then we can use this technique to weed out these carcinogenic proteins in the human body, which will contribute a lot in the history of Medicine. However, we had no idea about the feasibility and practicality of the application of PR PREDATOR in actual disease, especially cancer. And to what extent could the new development of cancer treatment be promoted?
Based on the above questions, we contacted Professor Qiang Ma and Professor Yunshan Ning in School of Laboratory Medicine and Biotechnology, Southern Medical University by emails on the exchange and consultation.
Part 3 Further Arrangements
Through the interviews, we had learned that the degradation of target protein is still a cutting-edge field in scientific research and clinical application. The environment of human cells is so complex we can’t exactly say which protein is absolute good or bad for humans. And the degree of degradation may affect the internal environment as well. Talking about the details in experiment, we still had some issues to consider, including cross-reaction, the affinity of single-domain antibodies and so on. Only when we solved these problems could we took further step in application and industrialization of our project. We planned to communicate with group of Professor Nie once a month to obtain a mature scheme with proposal. Since Trim was a new field, we would pay more attention to delivery in chemistry.
Company Research
On 28th September, we visited Genetalks in hope of getting professional advice based on the current market and we were lucky enough to interview Dr. Zhuo Song. As the CTO, he provided us with very pertinent ideas about our project as well as comprehensive answers to our problems, which inspired us quite a lot.
Our interview questions fallen into three aspects as follows:
The first is about the limitations of existing methods of reducing gene expression such as gene knockout, RNAi and novel gene editing techniques. We hope that through having a clear view on what problems still exist, we can know which kind of improvements that we actually need to focus on.
Dr. Zhuo told us that all traditional methods of reducing gene expression had efficiency problems and moreover, they also yielded unknown effects.
The next one is safety issue. Although there are few clinical applications to degrade intracellular proteins at present, there is a general trend in market demand. The main problem is that the biological complexity leads to unknown practical application. Therefore, safety is the most essential topic we need to consider about.
Considering the safety issue about our project, Dr. Zhou raised two aspects.
1.Unknown effects in vivo environment
Dr. Zhou mentioned that the purpose of protein-specific degradation technology is to degrade abnormal or overexpressed proteins. The implementation of this method in vivo must ensure that it does not introduce other problems that are adverse to the organism. It should be noted that in vitro and in vivo environment are not equivalent.
2.Excessive protein degradation
From an evolutionary point of view, it is only the amount of protein present in an organism that determines its actual function. Therefore, the degree of protein degradation on the biological effects should also be taken into account. For example, the removal of certain overexpressed proteins may affect the normal physiological activities of the organism.
Last but not least, it is about the possibility of application. We hope based on his abundant market experience, Dr. Zhou can give us more detailed suggestions about the approaches to clinical application and marketization of our project. Here are some significant answers we get.
1. What difficulties do you think our project will meet in the result transformation?
Dr. Zhou: The first thing must be considered is security issues. Also, you have to look for clinical trials of medical institutions that are willing to cooperate. What’s more, you need to pay attention to the negative or positive result of the standard.
2. During the transformation of achievements in China, the situation that the intermediate process of commercialization and industrialization of scientific and technological achievements is missing, always occurs, which is related to both universities and enterprises. Can you give some suggestions to avoid the "blank" area?
Dr. Zhou: Much of the reason for the "pilot" area actually lies in the role of government. While protecting the intellectual property rights is to protect the benefits of universities, the cost of subsequent transformation is very high, which is related to the cost of patent and development. Foreign commercial interests are only granted to a company, and the risk of success or failure is shared. In our country, patents have not really transformed.
3. If the improved protein degradation method in our study is introduced, can it play a significant role in diseases such as malignant tumors in the future?
Dr. Zhou: If the improved protein degradation method in the subject is applied, the following problems should be considered:
a) Can protein degradation last functioning?
b) Are there any incidental effects of unknown after degradation?
c) To what extent can the degradation be? How do you control that?
d) Can the degradation cause cell idle?
e) Concerns about sensitivity and specificity
Try to think about the dynamics of the control network and choose a clear application point.
If these problems can be solved, this method will play an important role in the application of diseases such as tumors.
After collecting the market-based information above, we had the idea of degrading target proteins related to some diseases. The cell experiment would especially focused on the problems mentioned by Dr.Zhuo and we would work more on the possibility of transformation process.
Speeches at Streets & Survey
To make our work responsible and good for the world, people’s attitude is an important factor. We did a survey of people’s cognition of traditional curing methods of tumor, cancer and Alzheimer’s disease and potential methods using PR PREDATOR. In addition to investigating this issue, we worked hard to engage with people by delivering speeches at streets. We inspired people by introducing our project in its future application and raised their awareness of preventing the fatal diseases mentioned above. The results of survey and details about speeches can be seen on our Education Page.
Through the survey and engagement with the public, we had a clear view of people’s attitude towards “target therapy” and the future application of PR PREDATOR. Since many of people involved had a positive attitude to our project, we would continue working on experiment and try to exert a greater influence in maintaining health.
Knowing that most people were still not familiar with the degradation process, we made a Wechat questionnaire for publicity including basic knowledge of proteins in human cells and challenging questions related to our project, which could achieve greater publicity step by step.
Co-Research
Part 1 On Competition Experience
We have communicated a lot with CSU_CHINA. This is a video clip recording our exchange on participating in iGEM. During our first meetup, team leaders showed the situation and progress of projects. And the present members had a heated discussion. We hope more people can pay attention to and participate in iGEM, expanding influence of synthetic biology in society.
Part 2 On Fundamental Study
Knowing that there was a gap between scientific research and product development in China, with the proposal of team Tianjin team, six universities worked together to make research on how the laboratory results are converted to market products. We conducted market research on the products based on fundamental study and then investigated the evaluation mechanism. The final report of commercialization of research findings worked as a reference guide for foundational advance.
The body of the report is the analysis and research of this transformation process (with theory and case study) and our projects. The theoretical part was completed by iGEM Tianjin and our team. The case study was completed by OUC-China and Jilin_China. In our own project analysis section, ZJU-Chian and XJTU-China wrote a detailed application plan.
Finishing this part of the report, we discovered that the transformation of research findings was a comprehensive process related to many factors. Government, users, companies, and scientists should work together for the final goal. In view of our project, we should consider market demand if we wanted to convert our innovative protein degradation tools to a practical curing method.
As for the transformation of our project, PR PREDATOR, basically we have been following the mode of GCIUR. The laws and policies from the government work as the guidance and support. The potential users’ attitudes are mostly positive. There is a huge market demand in innovative tools of degrading proteins in practice so the industry part is willing to work. Plus, our research in the university has been going and will continue to be done.
Many of the diseases caused by abnormal proteins are fatal or can’t be easily cured. Even though there have been some effective cures for them, the disadvantages and limitations still exist. And at present, the application of medicines for degrading proteins is still a new field. It can be concluded that the transformation of our research has a huge market demand, so that the supply and demand market for collaborative innovation with enterprises can be formed.
As for patent application, we plan to add further experiment after iGEM and make our project better-rounded. Fortunately, bioresearch has been emphasized in education of our university, which will make the process go more smoothly. But we may not be able to apply the patent individually, but in the name of our school.
There is no denying that we need technological improvements in the further transformation process. For example, we must consider whether the system will work in vivo environment, the degree of degradation, which disease will be targeted to and other relative issues. After solving these questions, the commercialization will be possible, theoretically.
CFDA Inquiry
As our project will finally apply to mammalian cells in curing protein-related diseases, we must take laws and policies into consideration from the time we conduct experiments in the laboratory. We have inquired China Food and Drug Administration about this issue and got a feedback of the interpretation related to Technical Guidelines for Research and Evaluation of Cell Therapy Products.
Cell therapy technology is currently the key development field of international medical frontiers, which provides new hopes for the treatment of some complicated diseases. This policy was released in 2015 in order to better create an environment for cell product research and development and provide technical support to relevant scientific research institutions and enterprises.
The contents helpful to our project are shown as follows.
According to the general principles and basic requirements, we designed and conducted our experiment and future work in a more effective and rational way. We had a deeper investigation of non-clinical research on cell therapy products. Even though we haven't transformed our work into actual drugs or products, we still learn some details from the policy.