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How should we carry out human practice, how to use human practice to improve our experimental design? This is an urgent problem that has plagued our team. The core of human practice is to improve the experiment through continuous practice and work. Therefore, we must improve and optimize our experimental design and experimental program through continuous communication and discussion, social investigation and social work.
Selection of ACE2
Fig.1. Inhibition of ACE2 expression in hypoxic environment
At the beginning of the experiment design, we visited Professor Li Xiangkai, School of Life Sciences, Lanzhou University. Professor Li believed that inhibiting the expression level of ACE2 was an effective method for the treatment of novel coronavirus.
At the same time, we found that the decrease of ACE2 level caused by plateau environment did not cause harmful effects on human body, and with the increase of altitude, the mortality of COVID-19 gradually decreased, which may be related to the inhibition of ACE2 expression in plateau environment. Therefore, we chose to knock down the expression level of ACE2 receptor to achieve the purpose of treatment.
Fig.2. LZU-CHINA member discuss experimental design with Professor Weilin Jin
Interviews with Professor Weilin Jin of Lanzhou University helped us further shape and determine the experiment design. After the preliminary design of the experiment, we chose the Adeno-Associated virus (AAV), which has been widely used in gene therapy and vaccine research, as our therapeutic tool. The Adeno-Associated virus is also a safe gene therapy tool that the public prefer to accepting. In order to ensure the rationality of the experiment design, we visited Professor Weilin Jin, the Executive Director of the Medical Frontier Innovation Research Center of the University of Lanzhou. Professor Weilin Jin is committed to the basis of tumor molecular therapy and immunotherapy. After understanding our experiment design, Professor Weilin Jin made recommendations on the treatment tools we chose. Professor Jin suggested that we choose lentiviruses with high efficiency of exogenous gene expression and higher efficiency of in vitro infection experiments as our treatment tools.
Lentivirus replaces AAV
Fig.3. LZU-CHINA members discuss experimental design with Genechem's technicians
The LZU-CHINA team held discussions with the Genechem's technicians. According to the results of the questionnaire, we planned to transmit the Cas13d effector to SARS-Cov-2 patients with Adeno-Associated virus (AAV) as the carrier. We chose AAV because AAV is a safe gene therapy tool that can express foreign genes in vitro for a long time and is widely used in gene therapy and vaccine research worldwide. However, according to a large number of experimental practice, Genechem's technicians give some suggestions: because our current only involves cell experiments, and Adeno-Associated virus is not suitable for in vitro infection effect, in vitro transfection efficiency is low, it is not easy to transfer into the three plasmid system, it is recommended to replace other lentiviral vectors with higher transfection efficiency. Through consulting with Professor Weilin Jin and Genechem's technicians, we modified the experimental design and chose lentivirus as our experimental treatment tool. Our team also obtained experience and suggestions of many experimental design schemes through interviews, and further improved the experiment design.
Selection of cell lines
Fig.4. Interview with Associate Professor Yong Wang
After the initial completion of the experiment design, we visited associate professor Yong Wang, member of the College of Life Sciences, Lanzhou University. The main research directions were environmental microorganisms and insect microorganisms.
Associate Professor Yong Wang, after understanding the specific scheme of our experimental design, suggested that we choose to replace the normal cell line of our original experimental design with immortalized cell line when constructing the stable cell line, so as to ensure the cell activity of multiple passages, which will help us to carry out the experiment smoothly.
Fig.5. Discussion on experimental issues in online conference of CRISPR
At the end of the experiment, the LZU-CHINA team communicate with multiple teams through online meetings at the CRISPR conference. Our partner NEFU_China also answered some questions that have been plagued our experiments.
Our team originally planned to serially express RfxCas13d and sgRNA on lentivirus vector to target the cleavage of hACE2 mRNA in 293T stable transfer conductor. NEFU_China recommended that direct repeats of 30nt and 36nt be selected between the spacers when sgRNAs were expressed in tandem. The valuable suggestions of NEFU_China gave us great help.
How should we carry out human practice, how to use human practice to improve our experimental design?
This is an urgent problem that has plagued our team. The core of human practice is to improve the experiment through continuous practice and work. Therefore, we must improve and optimize our experimental design and experimental program through continuous communication and discussion, social investigation and social work.
Part.1 Survey of public intention
Facing the global novel coronavirus pandemic, LZU-CHINA team is determined to provide a novel coronavirustreatment for novel coronavirus pneumonia through this experimental project. At the beginning of our project design, according to the content of our project, we released a questionnaire to the public through online and offline channels to investigate the public ' s understanding of the novel coronavirus, the attitude of our project and the understanding of RNA editing technology.
Fig.6. LZU-CHINA Members are distributing questionnairs
Part.2 Source of questionnaire
Finally, We collected 590 questionnaires from people from all walks of life in all ages. From the results of our questionnaire, we know that nearly 70% of the answers are composed of people aged 18 to 55. Among them, 87% of respondents had at least received high school education, and their work was very different.
Part.3 Understanding of existing treatment methods
The survey showed that the public did not have enough confidence in the cure of novel coronavirus pneumonia based on the existing vaccine drugs, but had great confidence in the development of specific drugs to cure novel coronavirus pneumonia. Therefore, it is particularly important to develop a novel coronavirusanti-virus prevention method that is trusted by the public. The results also confirmed our determination to develop novel coronavirustreatments.
Part.4 Understanding of RNA Editing/CRISPR Technology
The survey data showed that only 20% of the sample population thought they knovel coronavirusenough RNA editing technology. However, when it comes to the treatment of diseases using RNA editing technology, 74% of the sample population is willing to accept this therapy. Among the 25% of the sample population who were unwilling to accept this technology, 55% were unwilling to accept the application of this technology in clinic because they did not understand RNA editing technology. At the same time, The biggest concern is the security involved in RNA editing, which is also one of the most concerned issues when any novel coronavirustechnology comes out.
From these reactions, we can infer that the public still lacks knowledge and education about RNA editing technology. These responses also encourage us to carry out relevant social education and popularize novel coronavirustherapeutic technologies such as synthetic biology to students of different ages, such as RNA editing technology. This is also the starting point for the educational activities of the LZU-CHINA team.
Part.5 Acceptance of therapy
In the questionnaire, we introduced that the LZU-CHINA team will treat novel coronavirus pneumonia(COVID-19) through CRISPR-Cas13d system technology. We designed some questions to investigate the acceptability of the sample population for CRISPR-Cas13d system technology in treating COVID-19. 85% of the sample population were willing to accept this system technology, while 15% of the sample population were unwilling to accept this technology, mainly because they were not familiar with the system technology and worried about its security. If they reached a critical high-risk state, 75% of the sample population were willing to accept CRISPR-Cas13 system technology as treatment. At the same time, we investigated the public’ s choices of the two therapeutic tools — Adeno-Associated Virus/Lentivirus. The survey results showed that 69% of the sample population were more willing to choose the Adeno-Associated virus with safety certified by the EU, so we chose the Adeno-Associated virus that the public was more willing to accept as our therapeutic vector. In the selection of our clinical treatment, we introduced to the public the strategy of atomization inhalation delivery of drugs (CRISPR-Cas13d system) to the lungs (drugs mainly act on the lung tissues mainly infected by novel coronavirus to improve its safety). 85% of the sample population expressed willingness to accept this clinical treatment.
Through extensive social surveys, we are fully aware of public awareness of the novel coronavirus and attitudes towards the direction of the LZU-CHINA project. This not only helps shape our experiment design, but also provides direction and content for other social activities in the future.
Zhang Ruifeng,Wu Yingli,Zhao Meng,Liu Chuanxu,Zhou Lin,Shen Shaoming,Liao Shihua,Yang Kun,Li Qingyun,Wan Huanying. Role of HIF-1alpha in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells.[J]. American journal of physiology. Lung cellular and molecular physiology,2009,297(4):