Personal safety comes first. That is why, well before any of us entered the lab to conduct our experiments, the team attended a rigorous laboratory safety seminar held by our PI, Dr. Michalis Aivaliotis. During the seminar, we were educated on basic laboratory etiquette in fine detail, learning about all the “do”s and “don’t”s. We were also informed about what we should do in case of accidents, errors, or any suspicious noise, smell or activity: the standard protocol, the contact details of the lab’s safety experts, and the whereabouts plus use instructions of safety equipment like fire extinguishers, emergency showers, and first-aid kits.

Seeking to further our biosafety training and expand it, we invited experts Dr. Victoria Siarkou, Professor of Microbiology-Infectious Diseases, and Dr. Anastasia Tsingotzidou, Associate Professor of Macroscopic, Microscopic Anatomy and Embryology and of Domestic Animals, both at the Department of Veterinary Medicine of AUTH, to hold a private seminar on the proper disposal of biological waste (see our Integrated Human Practices Page). The professors had implemented a rigorous biosafety training system for the students and academic staff of the Veterinary Medicine Department, with very positive results. What we learned during the seminar about the disposal of different categories of waste is invaluable for the well-being of all the staff of our lab, the university itself, and for minimizing the ecological burden of science.

All members of the wet lab team, regardless of whether they would later take part in the experiments or not, were tutored in small groups by our instructors. These tutoring sessions focused on certain lab techniques like pipetting, PCR, DNA electrophoresis, and cell cultures, with two goals: to teach reliable handling and to hammer in and demonstrate the importance of lab safety practices. The overarching aim was to make safety habitual, something which is crucial to prevent mistakes in the lab, not just in the context of iGEM but throughout the members’ scientific careers.

Of course, the culmination of all the aforementioned safety education was our work ethic during our experiments. Diligently following procedures and protocols, using PPE such as gloves and masks, only handling dangerous chemicals under a fume hood, and communicating every single thing happening in the lab (such as who is using the space and at what time) with the entire team and instructors.

In Thessaloniki, especially during the summer months, temperatures soar high. When our team entered the lab, the air conditioning unit was malfunctioning, resulting in a dangerously elevated temperature in the lab space: intense heat that, if left unchecked, would compromise the scientists’ judgement and fine motor skills, as well as endanger the cell cultures and chemical agents. Seeing as those conditions threatened both the personal safety of our wet lab members and the integrity of our experiments, we took the necessary steps in our university to have the AC unit replaced. We saw a hazardous issue in our workspace and resolved it with the help of our instructors, ensuring optimal conditions for both the cells and the researchers, and therefore optimal handling for safer experiments, bettering the lab itself for future researchers.

At the early brainstorming stages of our project design, we were confronted with the following dilemma: should we silence messenger RNA or microRNA to induce apoptosis? Having found significant evidence supporting both options, we could only turn to stakeholders for guidance. With their input, we were able to reach a decision ultimately dictated by safety concerns: siRNA silencing is a far more documented process with approved pharmaceutical products in humans, whereas anti-miRs are of a more unpredictable nature, due to the multitude of direct targets a microRNA impacts and the lack of relevant advanced trials to this day. Ultimately, we opted for a molecule which has already been shown to be safe for therapeutic use in human patients, thinking of the well-being of our CLL patients receiving our treatment down the line.

Patient safety has been at the core of our project design (see our Design Page) as an inextricable part of it. The central molecules of our project, the siRNAs, were picked or designed after a meticulous process combining bibliographic and in silico research, with the sole purpose of minimizing possible off-target binding, while maintaining silencing efficiency. All that to minimize side effects and maximize safety and precision.

The exact same goals extended to our delivery system (see our Design Page). After an extensive search, we chose to base our LNPs on a formulation that has already been approved for human use and is currently considered the “gold standard” for siRNA therapies ¹, exhibiting efficiency as a carrier, as well as minimal toxicity, immunogenicity, bioaccumulation, or ecotoxicity ². Most importantly, we implemented a dual targeting strategy to those LNPs, ensuring first the organ-specificity with SORT molecule 18PA ³ for splenic uptake or intramuscular administration for lymphatic uptake; then, ensuring cell type-selectivity by conjugating Fab’ against ROR1: a receptor present on the surface of CLL B cells but mostly absent in normal tissues. It is well established that without those modifications, the LNPs tend to accumulate in organs such as the liver ⁴, with the potential to cause hepatotoxicity, and to require higher therapeutic doses, which lead to more significant off-target effects and mediated toxicity ⁵. Those decisions were made after meticulous bibliographic research to limit siRNA uptake by healthy organs and cells, and to keep the side effects of the drug to a minimum: improving its safety and effectiveness in lower doses.

Knowing how crucial it is for a research project to be in harmony with bioethical guidelines, we reached out to the Bioethics Committee of our university, detailing exactly what we sought to do, so we could begin realizing our project only after receiving their endorsement.

That was not enough for us, however. We wanted to learn more about bioethics, so we could be better equipped to shape every aspect of our work and our project in an ethical manner. To answer our questions about the use of siRNAs, LNPs, human cell lines and biomolecules, about data integrity, off-target effects, and how to balance benefit-danger even in the preclinical experimental stages, we consulted members of the Hellenic National Commission for Bioethics and Technoethics. On two separate occasions, we spoke with Dr. Nicholas Moschonas, Professor Emeritus of Biology and Medical Molecular Genetics at the Medical School at the University of Patras, and with Dr. Evangelos Protopapadakis (see our Integrated Human Practices Page), Professor of Applied Ethics at the School of Philosophy at the National and Kapodistrian University of Athens and President of the Research Ethics and Deontology Committee at the National Center for Scientific Research “Demokritos”: both members of the Commission. The professors reviewed our project and gave us invaluable insight, each from a new perspective born from their respective fields of Medical Biology and of Philosophy: providing us with a truly well-rounded view of bioethics.

Inspired by all this, our team wrote a Patient Communication Guide (see our Inclusivity Page). Our goal was to promote bioethics principles such as informed consent, patient-centric healthcare, and overall good practices in patient-medical professional communication. It is imperative that patients are informed fully and in a way they can deeply understand about their illness and the treatments they may receive, in our case RNAi therapeutics, in order to have knowledge and control of their health management: a fundamental aim of medical ethics.

Following a recommendation from bioethics expert Dr. Evangelos Protopapadakis, our team analysed the known side effects of currently approved siRNA therapies, of drugs making use of nanocarriers with similar compositions to our own, and of various drugs targeting the same molecular agents as our proposed therapy in CLL. In this way, we created a “Worst Case Scenario” (see our Implementation Page) for our therapeutic approach, which we could then use for comparison with existing CLL therapies and for the prediction of estimated safety concerns for human use. By examining the components of our project in the context of this side-effect list, we were emboldened by the fact that our meticulous siRNA and LNP design is mechanistically set to limit some of these adverse side effects by eliminating their root cause, making for a potentially safer and ethically justified new therapeutic.

A significant contributor to safety is always doing things “by the book”. For that reason, we reached out to Dr. Angeliki Rompoti, a member of the Scientific Board of Appeals of the National Organization for Medicines. She provided us with answers to our questions about the standard process of drug design at the preclinical and later stages, any special regulatory approaches to our parts and proposed drug formulation, as well as the procedure for drug approval and cost assessment. Through that communication, we could ensure that we're following guidelines and procedures every step of the way, while also being able to make informed decisions about our project implementation.

Thinking ahead, our team communicated with Dr. Anastasia Tsingotzidou (see our Integrated Human Practices Page), an expert in laboratory animals, to seek knowledge, answers, and guidance regarding the next experimental steps in our project: namely, in vivo preclinical research. Thanks to this meeting, we were extensively informed about the processes and protocols, the rules of bioethics regarding laboratory animal well-being, safe handling for both humans and animals, and ensuring that a drug’s safety is adequately determined before transitioning to clinical trials. Finally, we examined in great detail the European Union’s legislation for the safe and ethical use of laboratory animals, preparing ourselves for conducting in vivo experiments in the future in a safe and ethical manner.

Taking our considerations one step further, we consulted Dr. Georgios Papazisis (see our Integrated Human Practices Page), Professor of Basic and Clinical Pharmacology at the Department of Medicine, Aristotle University of Thessaloniki, and the Medical Director of the Clinical Research Unit, at the Papageorgiou Hospital in Thessaloniki. During this meeting, we shared our queries regarding clinical trial staging and protocols, with an emphasis on ensuring participant safety and privacy, and maintaining data integrity without personal bias: going one more step forward into imagining our project implementation according to safety and ethics regulations.

Finally, we took steps to ensure our project is aligned with sustainability goals and environmentally conscious, as we carry responsibility for the pharmaceutical products that will be distributed, consumed, and eventually disposed of. These steps included meetings with stakeholders like Dr. Dimitra Manou and Dr. Grigoris Zarotiadis (see our Integrated Human Practices Page) and careful consideration of our project design. For the latter, it is imperative to mention that we consulted the ERA for Patisiran-LNP, on the design of which we decided to base our own LNP carriers, which has concluded that this formulation does not pose a risk to the environment, in terms of bioaccumulation, persistence and eco-toxicity; in accordance with the Guideline on the Environmental Risk Assessment of Medicinal Products for Human Use. Therefore, the foundation of our own pharmaceutical has been proven to be environmentally friendly and emboldens us to assume that our proposed drug formulation will show similarly positive results after an analogous ERA assessment ⁶.