Our project was designed to be conducted in a biosafety level 1 laboratory. We took different precautions to ensure minimal risk and maximum safety to our team, the environment and to the general population.
Prior to starting our project in the wetlab, we received several safety briefings. Since our work was conducted in three different laboratories, we made sure that each team member received the necessary introductions.
Our Principal investigator Prof. Dr. Wölfl was responsible for the safety introduction in the BioQuant lab used for siRNA production and minor experiments of the liposome team. He took time to explain common mistakes that can occur while working in the lab as well as the correct procedures afterwards. The staff of the laboratories of the AG Fricker and the IPMB, which were used for liposome experiments and cell culture, made sure we understood the safety procedures thoroughly. Topics such as lab access and rules, disinfection and sterilization, emergency procedures, physical and personnel security as well as chemical, fire and electrical safety were discussed in detail. The staff of each laboratory was always available for safety advice and further questions. Additionally, we received several instructions and safety briefings for using special equipment, such as centrifuges and sonicators.
To strictly regulate which persons had access to the laboratories, we were given keys and key cards to the IPMB and BioQuant labs. We kept a detailed list following the exchange of the keys between different members of the cell culture and siRNA team, so everyone knew who had the keys at any given time. For access into the liposome lab, we had to announce ourselves prior to coming to the lab.
While conducting experiments we made sure to wear the appropriate protective gear which can be seen in Figure 1. While lab coats and protective gloves were worn within the lab at all necessary times, safety goggles, laboratory face shield and thermal safety gloves were donned for particular experiments. Besides latex gloves, we made sure nitrile gloves were available in case a team member had an allergy.
We paid special attention that no one was left alone in the laboratory while conducting experiments. Additionally, we established a system to record lab accidents and distributed sheets containing the contact information of safety officials in our laboratories for emergencies.
While most of our members had participated in first aid training up to this point in their life, we decided to take no chances on the wellbeing of our members. Therefore, two members of our team participated in training to become certified first responders, while one medical student already had the necessary qualifications. This brought our number of first responders up to three for a team of eighteen student members.
Due to the ongoing COVID-19 pandemic, we devised several plans to work efficiently and productively while adhering to national rules concerning the pandemic. We held team meetings primarily online to avoid unnecessary contact and risk for infection. Early on, we divided ourselves into sub-teams that tackled specific parts of our project. Our goal was to minimize physical contact between sub-teams, so that an infection occurring in one sub-team would only lead to the potential quarantine of those members and not the entire iGEM team. If one team member had infected themselves with the virus, they immediately went into quarantine and communicated this quickly to the entire team. Therefore, persons who had come into contact with that member could conduct rapid tests after receiving the news. In the laboratories we maintained a safe distance, conducted hygiene work, and made sure to wear face masks.
When performing experiments in the lab we took precautions to avoid contaminating our product. For instance we used ethanol to clean all work spaces where we performed our experiments before and after lab work. This was to avoid contaminating and therefore falsifying our results. Sterile work on the bench was performed using a bunsen burner, which creates an updraft, preventing cross-contamination and creating a sterile working environment which ensures a safe work environment.
When disposing of unneeded chemicals, we paid special attention to discard it in the appropriate way, as we were shown during our introductions to laboratory safety. All biohazard material was autoclaved before it was safely removed from the site. Therefore, there was no risk of anything escaping our lab or contamination of the environment. Furthermore, no harm came to team members due to wrong disposal of chemicals.
As many team members worked in the wetlab, good communication and documentation skills were essential to keep everyone updated and to ensure smooth and continual progress. With this goal in mind, we focused on establishing good documentation skills. By doing so, everyone could focus on giving their best in the lab without losing strength and time due to unnecessary mistakes and miscommunication. Therefore, the safety was enhanced, as every team member knew which experiments were performed in the lab at exactly what time. By conducting inventory controls, we kept track of where any materials were kept. We were able to reduce errors to a minimum, saving time and resources in the process.
When Human Practices created surveys, the first slide was dedicated to informing the participants about what data we intended to collect and how it would be used. Additionally, we provided our iGEM email address in case any potential participant had further questions. Keeping personal data from participants to a minimum was important to us. Consequently, there was not too much time needed to anonymize the surveys before processing and evaluating the data.
When designing our project, we faced the challenge that HSV-1 is normally classified as a biosafety level 2 organism, as it can be transmitted by direct contact with human epithelial or mucosal surfaces. To keep our team safe, we selected specific sequences from HSV-1. We were able to successfully implement a safer design by choosing parts of the UL-19 sequence, which encodes the HSV-1 major capsid protein.
We had it synthesized by a commercial supplier (Eurofins Genomics) and sub-cloned into the standard E.coli vector pEX-A258. The sequence was amplified and identified using PCR and restriction digest. We cloned it in a mammalian expression vector backbone taken from pRK5. Therefore, the vector expresses only one non-functional protein part of the entire HSV genome. As there is no full protein sequence no correct folding is possible. By choosing this approach, we were able to conduct our experiments in S1 laboratories instead of S2, reducing the risk to our team members significantly.
Our final product will not be able to spread in the environment autonomously, since it is not an organism but siRNA packaged in liposomes.
For further information, you can also refer to our project safety form.
When creating our tabletop device for automated LNP production we paid special attention to making the execution process as safe as possible. You can see an illustration of the device in Figure 2.
First of all, the microfluidic device will be installed securely on the bench, so that injuries and damage to the microfluidic device by accidentally bumping into it can be prevented. We reduced the risk of harm to personnel and contamination of samples significantly by ensuring that the three peristaltic pumps will only start working after the lid has been securely closed. The maintenance cabinet below the main work area contains a waste container and the cleaning solution, which are designed to be easily replaceable in a safe fashion. Additionally, the cleaning solution can be used to cleanse the chip for potential reuse or to ensure a secure disposal.
Read more on our microfluidic device here.
Dual use research of concern has increased significantly over the past two decades. The term DURC refers to research findings in life-sciences that are intended for peaceful and beneficial purposes, but can be misused to cause harm to people and/or the environment (World Health Organization, 2021). For further information about dual use in general and DURC specifically, we would like to refer you to our essay titled “Challenges for young researchers”. There we discuss appropriate definitions, the dual use dilemma and national and international laws concerning dual use.
Here, we would like to discuss whether our project itself has dual use issues.
Since our aim is to provide a therapeutic giving straightforward access to treating neuroinfections, our project has to be easy to execute and simple to reproduce. The opportunity to exchange the siRNA sequence, which is responsible for targeting specific proteins in viruses, gives the therapeutic the potential for widespread application in treating a variety of neuronal infections.
However, these requirements also bear the risk of our project being misused. If the siRNA sequence can be changed to target other viral infections, it can also be changed to target essential cell functions leading to fatal consequences. It has the potential to be used as a biological weapon of unforeseen consequence.
Additionally, the results of our project could be combined with other technologies. Our chosen lipid formulation and production method could be used to create liposomes containing not only siRNA, but also toxins or other hazardous substances.