At the frontier of exploration science, every step is accompanied by a fear of the unknown and a commitment to safety. iGEM not only brings together the world's top biotech wisdom, but also regards safety as the cornerstone of project success. This paper aims to comprehensively describe DUT-China team's persistent efforts in ensuring the safety of all aspects of the project through the construction of safety module, covering four modules: project design ethics and technical safety, laboratory safety and future application safety of project results. We firmly believe that only under the premise of ensuring safety, the light of science and technology can illuminate the road of mankind.
From the initial stage of the project design, DUT-China has been deeply aware of the importance of observing ethical principles, and regards ethical principles as the light that guides our actions. At the beginning of the idea of using gene editing to treat hepatitis B, DUT-China set up an ethics committee and designated the principal responsible person to brainstorm the ethical risks of the project.
By investigating how HBV infects cells, we determined that the CRISPR-Cas12a nuclease used in the project targets HBV gene fragments designed according to the HBsAg gene, and avoids most human genes with similar sequences by sequence design. The selection of this target not only ensures the effectiveness of removing HBV DNA by gene editing, but also ensures that human genes are not threatened by gene editing. The selection of this site meets the requirement that CRISPR-Cas does not target human genes in iGEM and received the approval of technical experts.
In view of the particularity of the treatment of human diseases, our proposed therapy must pass human cell experiments, live animal experiments and clinical experiments to prove the reliability of the technology. In view of the possible risks, we actively deleted the human cell and animal experiments in the iGEM project design. The design eliminates possible ethical safety risks in human cell experiments and animal experiments. We studied the White List carefully and carefully checked all the microorganisms, proteins, nucleic acids and small molecules involved in the project. DUT-China guarantees that all tools, molecules and microorganisms used are sourced from authorized sources of purchase and synthesis and are included in the White List. We also selected non-pathogenic E.coliK-12 by project design to minimize ecological risks and threats to human health.
The original intention of DUT-China's project is to provide a cure for people infected with viral hepatitis B worldwide. The high precision MAD7 we provide as a therapeutic tool will also be modified to enhance the safety of treatment. This ideal of healing embodies DUT-China's hope to maintain social equality. We will ensure that all details of the project are transparent and open during the project and future development process. At the same time, we will strive to protect the privacy of all stakeholders. In the process of putting into real-world application, we will also adopt mature technologies and strive to overcome the uncertain risks of advanced technologies. These ideas and measures show DUT-China's responsibility in the face of global public health problems.
The laboratory is a concentrated area where security risks occur. In the iGEM project, DUT-China considers laboratory safety a top priority.
Before the experiment, all wet experimental group members underwent strict laboratory access safety training under the supervision of PI. After training, the team members who got full marks in the safety exam can got the laboratory access permit. Then, the wet experimental group also carried out detailed experimental operation training. This can not only ensure that the team members can obtain rigorous and accurate experimental data through good operating habits, but also avoid safety risks caused by irregular operation. After the completion of the task division, we determined the person in charge of each module in the laboratory according to the region and responsibility, and controlled the laboratory safety hazards point-to-point.
The School of Bioengineering at Dalian University of Technology provides a safe and advanced laboratory environment for the team (figure). Our laboratory rating is bio-safety level 1 - standard microbiological lab. And we ensure that experiments are conducted using microbes and manipulation techniques appropriate to the laboratory rating. The microorganisms used in this project are E.coli K-12 and E.coli DH5-alpha, which are basically non-pathogenic. In order to prevent possible infection and exposure to chemical reagents, members of the wet experimental group should take personal protection throughout the experiment, including but not limited to wearing gloves, masks, lab coats, and goggles. We strictly comply with the laboratory chemicals and reagents management regulations, and all chemical reagents are stored in chemical safety cabinets. The use of dangerous chemicals such as strong acids and alkalis requires additional records. The laboratory has also established a standard and standardized waste treatment process to ensure that various chemical and biological materials are not discharged into the environment. All waste containing biological material is autoclaved and then treated by a professional treatment company.
In addition, DUT-China members have also established a smooth communication mechanism on security issues in the project. We set up an innovative work log recording mode to record the operation of the equipment while recording the experimental results. We require the members of the wet experimental group to specify the operation details in the work log, record the reagents and equipment used in detail, and count the problems in it, so as to reduce the safety risks of different members during the handover of work as much as possible.
As an advanced in vivo gene editing therapy for hepatitis B, DUT-China's products are still a long way from being brought to market. The hepatitis B gene editing therapy we provide mainly relies on the composition of high-precision MAD7 mutants, gRNA that recognize specific targets of HBV DNA, and delivery vectors. Due to the technical characteristics of in vivo gene editing and in vivo delivery, a thorough understanding of the risks of product application is necessary for responsible iGEMer.
Before our high-precision hepatitis B gene editing therapy enters the market, it will definitely undergo human cell experiments, live animal experiments and clinical trials. First of all, these experiments are bound to involve a lot of exposure to hepatitis B virus, and controlling the risk of infection in these experiments is especially critical. Second, the technical risks and therapeutic side effects of gene-editing technology could threaten the health of subjects and product customers. During the experiment, there may also be ethical safety risks such as animal welfare and subjects' rights mentioned above.
Technically, although we are working on different aspects of the project to reduce the possibility of off-target effects of MAD7, the potential off-target risk of in vivo gene editing therapy for hepatitis B is still a major threat to application safety. On the other hand, the result of DNA repair that occurs in the host after the DNA double strand break is uncertain, which may lead to risks such as gene mutation. When delivering using LNP or other ways, an improper dosage in the delivery system can cause cytotoxicity and immune reactions, posing a threat to the patient's health.
We use engineered bacteria in the production process. The strains used in the production process will be tested and controlled in strict accordance with the requirements of laboratory and production-related regulations. Moreover, our therapeutic systems will eventually degrade into harmless small molecules once they enter the body. Therefore, the engineered organisms or components we use do not spread autonomously in the environment.
Once our gene-editing therapies are used, their efficacy will be continuously monitored and the safety of the product examined over a long period of time. At the same time, the mutation and screening strategy we used is expected to be popularized in the field of modification of CRISPR-Cas gene editing system, and the legitimacy of the high-precision Cas enzyme obtained by this method should also be one of the risks we consider.
Curing hepatitis B can give patients more rights to fair employment and social equality. We will fully respect patients' privacy and equal rights in our treatment. This will help us maintain the ethical safety of technology for use in society. In addition, we are prepared to patent the results of our research and fully respect the copyright of others in our work. Safeguarding the copyright security of all parties will also be one of the priorities to ensure security.
In the iGEM project, our team not only focuses on the current technological breakthroughs, but also focuses on the future application security of the project results. We understand that the widespread use of anyone technology can have a profound impact, so we have fully considered the sustainability, environmental friendliness and social acceptability of the technology at the beginning of the design of the project. We are committed to applying the project results to the cause of improving human life and promoting social progress. At the same time, we are always concerned about the potential risks of technology, and ensure the safety and reliability of technology application through continuous research and monitoring. We firmly believe that only under the premise of ensuring the safety of future applications, scientific and technological innovation can truly become a powerful driving force for the development of human society.
The power of synthetic biology is a double-edged sword, with both promise and risk. The innovation of science and technology is ultimately for the benefit of mankind. DUT-China team always puts safety awareness in the first place and is responsible for the legitimate interests of themselves and others. At the same time, safety is also our bottom line in the iGEM project. Through the comprehensive construction of the three modules of ethical and technical safety of project design, laboratory safety and future application safety of project results, we aim to ensure that every part of the project meets the safety requirements, and contribute our strength to the healthy development of science and technology and the harmonious progress of society.