Our project, ViruGuard, will address the treatment of primary liver cancer and improve patient survival. From the very beginning, we designed our programs on the premise that they could be clinically practical, so we primarily needed to consider the risks that the oncolytic virus might pose to producers, developers, patients, and health care workers in the actual production and use of the virus. As a biological drug, our products need to fully guarantee the safety and effectiveness of use, so we have carried out a multi-level safety assurance design from the level of virus gene editing design while ensuring that our virus is sufficiently lethal, this section will be shown in our "safe design" section.

Possible risks to the human body and the environment during our use of ViruGuard will be shown in the 'Threat to the environment and human' section. In addition, in the 'Bio-security' section, we focus on the possibility of 'dual use' of our bio-bricks and the information risks of the project. Finally, in 'Lab safety', we demonstrate our laboratory rules in the course of the project, ensuring the safety of team members and the safety of the process during the development of the project.

Safety design

According to the guidelines in the Guidelines for Clinical Trial Design of Lysodyvirus Drugs (Trials)issued by the Drug Review Center (CDE) of the State Drug Administration of China in early 2021, we have selected human type5 adenovirus as the chassis organism. It is a virus widely used in current research on the immunotherapy and gene therapy. The main adverse reactions of it are fever and flu symptoms, so even in the course of use there is a virulence recovery or accidental leakage problems, will not lead to very serious consequences.

In addition, we have also carried out a variety of experimental design to increase the safety of the design. Considering the virus' broad-spectrum killing effect to cells, we have made our design based on the intracellular microenvironment differences in normal and tumor cells, setting up miRNA kill switch, tumor-specific expression promoter, extracellular specific recognition and other mechanisms to ensure the safety from the module design.

Tumor specific promoter
Cell recognition mechanism

miRNA modulated suicide switch

As for virus, the normal expression of the E1A gene is very important for its survival and function in cells. Thus, by inserting complementary sequences of miRNAs: 199a, 195 and 22, which are highly expressed in normal cells and low in tumor cells, to the 3' UTR sequence of the E1A gene, we form a "suicide switch" that is activated under a specific environment. When the level of specific miRNA in the environment is high, it binds to 3' UTR of E1A through complementary pairing, which inhibits the normal function of the E1A gene and, although it is not possible to completely kill the virus,this mechanism can still effectively affect the function and proliferation of the virus. This type of suicide switch reduces the toxicity of the virus in normal cells and ensures that when the virus leaks or causes an accidental infection, it will not have serious consequences to the normal human body.

Risk to the environment and human

Adenovirus production is usually carried out using intergenerable cell lines that cannot survive independently in the environment and do not have the ability to infect plants.
Thus, if adenovirus leakage occurs during production, transportation or use, the main problem we need to consider is:
(1) the recovery of the virulence of the oncolytic virus.
(2) The infection of the virus to normal human body or animals.
(3) Malignant mutations caused by the instability of viral genes during production and use.

Fig.1 The possible risk the Viruguard may cause. Pictures come from open source picture websites.

Virulence recovery

In the laboratory stage, there may be the problem of virulence recovery in the process of modifying and studying the virus, and therefore there is a threat to researchers. In this regard, we have chosen to use the human type 5 adenovirus with limited venom as a basal organism, so its venom recovery will not bring a very serious threat, and there are obvious signs to recognize and treat in a timely manner. In addition, the laboratories we conducted the study were safe enough to prevent adenovirus from leaking out of the lab which provides adequate protection for researchers.

In the course of use, the virulent recovery may cause harm:
(1) the oncolytic virus lost therapeutic effect.
(2) Causing additional diseases of light or heavy in patients with weak immunity.
The former will not cause more serious consequences, but the latter may cause the patient's condition to worsen. So after the use of oncolytic virus treatment, the patient needs real-time monitoring. The recovery of the virulence is usually a chance after a large amount of proliferation of the virus, so the likelihood of recovery in clinical application is extremely low.

Leaking infection

Although we have experimentally designed to reduce the virus's ability to recognize and infect normal cells, it still has the ability to infect normal cells, so there is a possibility of infecting normal humans or animals and spreading the virus to nature. This requires preventing the virus from leaking by standardizing its production, transportation, storage, and use. Because the E1A gene of ViruGuard has been modified to limit its viability, it is hard for the virus to survive in nature for long.

Virulence increases

Viral venom enhancement may occur during mass production, and this mutation is often an uncertain, random occurrence. Therefore, through a variety of ways for the production of viral products to identify, the use of phage plaque identification of virus strains, the use of TCID50, EID50, LD50, and other ways to determine the changes in viral toxicity, in order to ensure that it remains in a normal state before clinical use.


Dual use and possible danger

As mentioned previously by the iGEM2019 Team Wageningen, the threat of biological warfare has become even more severe due to advances in easy-to-use genetic engineering (GE) tools and the custom synthesis of DNA fragments. Moreover, the number of communities of biohackers who experiment without appropriate training and have little knowledge of experimental ethics is growing.
Therefore, the advancement of technology or the exploration of new bio-mechanisms may provide people with bad intentions with the tools to create hazardous organisms. We believe that the technology itself doesn't have the intention to be harmful or not, it's the way that it's used by people that leads to a positive or negative result. The abuse of technology in the wrong area will be dangerous.
Though there are possible risks of technique abuse, transparency is still the cornerstone of the scientific world. So we shouldn't be too overcautious to publications of research, so long as we make a full assessment of the positive and negative influence of the knowledge. Most of the things in the world is like a double-edged sword, so we hold the view that if the positive trumps the negative, the knowledge or the design will do more good to people. This view was also shown in the discussion on dual-use by the iGEM2019 Team Wageningen.

Fig.2 Biology hackers are a group of people who may use biology technology to realize their malicious intention. Picture comes from the stage photo of TV series < BIOHACKERS >.

In our design, we achieved immunocamouflage by inserting TLR9i sequences into the viral genome so that they could be paired with TLR production on the surface of immune cells, reducing the likelihood that the virus would be removed by the immune system early. Our design to increase the virus' resistance to the immune system may be used to enhance the ability of some deadly bacteria to become infected so that it can be used in the development of biological weapons. We assessed the likelihood of this happening and the harm it would do and weighed the potential benefits of our project.

Our design temporarily obscures the immune system and does not cause long-term and direct damage to the immune system. And the immune cells we're targeting use TLR to bind to the CpG sequence in adenovirus to initiate the process of an immune response, rather than broad-spectrum immune silencing, so the range of viruses that this mechanism can apply is very limited, greatly reducing the likelihood that they will be applied to other types of viruses to enhance infection. This mechanism of immune masking effectively enhances the effectiveness of the oncolytic virus by preventing the immune system from clearing the virus early in our project. So, in summary, the significance of this design outweighs its possible drawbacks. And because our design limits only one part of many immune processes, and there are many ways in which the immune response can be stimulated, even in the worst-case scenario, the harm caused by activating and enhancing other immune response processes can be avoided, and the risk of misuse of this design is low.

Besides, we evaluate the information risk of our project. The parts that have been flagged as potentially applicable for malicious intent are based on previously published research. The method of protein fusion to increase the specificity of combination is common in immunotherapy and the genes chosen for treatment are common targets in tumor therapy. The design of shRNA is based on Thermofisher online tools. Therefore, these facts indicate that our project doesn't have extra information risk. Moreover, we have chosen the widely used organism in gene therapy, adenovirus, rather than other types of high virulence organisms, which means we only offer information about this model organism. The adenovirus that we chose allows us to reduce the possibility of vicious use. Under the circumstances, our project will not offer additional information for possible malicious acts

Lab safety

Lab safety is the rule that all the team members must follow in order to safely conduct experiments, avoiding causing damage to the researcher or the environment. All in all, complying with lab safety rules should therefore be taken seriously, as it is the first step towards overall safety.

Therefore, before we started our business in the lab, we had a 'safety education class' delivered by the lab instructor to train us what rules to follow in the lab and how to deal with the possible emergency.

Fig.3 The management regulations of our laboratory.

After the training, we must take a test in order to confirm that we are capable to conduct the experiment by ourselves. The class and test included basic experiment skills, lab cleaning, experimental waste disposal, ethics, chemical reagents storage, lab rules, equipment safety, and emergency measures. Besides, the lab that we conducted experiments in has a professional lab instructor who takes charge of the safety of the lab, so our experiment specification was under the control of the teacher in order to avoid any possible danger.

Within the ViruGuard project, we have worked in two different types of lab, which are the BSL1 and BSL2 labs. These facilities are based upon the amount of containment needed to decrease the risk associated with microorganisms classified in a certain risk group.

To guarantee safety during our work, we divided our project into two stages. Firstly we conducted the confirmatory experiments in the BSL1 lab by taking apart our design into bricks so that we are able to assemble the parts into a whole and verify the feasibility of each part efficiently and safely.

Fig.4 A picture of our laboratory.

Before we conducted our bricks into the cells to confirm their functions, we talked to our lab instructor to discuss whether it would be infectious to ourselves or to the environment. We examined the function of our design in a safe way. Later, in the future, we would conduct the function verification of the ViruGuard in a BSL2 lab, which possesses all the functional parts that we design. The way we carried our project allowed us to conduct the experiment efficiently and reduced our exposure to virus so that the possibility of infection or leakage is low.

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