Our team upholds iGEM values and our university’s high standards for the safety of our experiments. Our project complies with all relevant safety rules and policies of the iGEM competition, our university and other authorities. For a comprehensive overview of the safety measures implemented in our project, please refer to the Safety Form.
The Implementation page also contains a comprehensive description of all safety aspects that need to be considered for the future potential execution of our project.
Microbial and Cell Safety
Microorganisms safety
The basic microorganism used in our experiments is Escherichia coli, a lab friendly, harmless microorganism, very helpful at transformations. It is compatible with the safety of the laboratory we are using (level 1). The strains we used [E. coli (NEB® Stable Competent E. coli High Efficiency-C3040H) K12 strain background] belong to the white list of iGEM safety.
Cell safety
We have specific cell lines that undergo transfection using a lentiviral-based system. The transfection is intended to express a Chimeric Autoantibody Receptor (CAAR) construct. HuT 78 (TIB-161) is a cutaneous T-lymphocyte cell line derived from a 53-year-old, white, male patient with Sezary Syndrome. This cell line is classified under Biosafety Level 1 (BSL-1) according to ATCC guidelines. Its classification reflects the low risk associated with this cell line, allowing it to be handled under basic laboratory safety practices without requiring special containment.
Part Safety
Chimeric CAAR protein
During our project, 8 of the human protein domains in our part collection were selected and cloned into C1 PGK plasmids for lentiviral production and subsequent transfection into HuT 78 (TIB-161) cells.
We attempted to classify them in three groups, based on immunogenicity and potential off-target effects. Consequently, before designing our chimeric protein, the risk of the original peptides was ranked in the table below (Table 1).
Part
Risk
Function
MOG peptide
High
Immunogenic
MBP peptide
High
Immunogenic
CD28
Moderate
Signalling
4-1BB
Moderate
Signalling
CD3ζ
Moderate
Signalling
Igκ signal peptide
Low
Structural
CD8a Hinge
Low
Structural
CD8a Transmembrane
Low
Structural
Table 1: Genes of origin of the epitopes we selected for ARC.
MOG Peptide, MBP Peptide – Immunogenic- can induce autoimmune responses. Their use is quoted in many scientific works aiming to simulate Multiple Sclerosis in the lab. Our use differs, as we do not aim to induce an immune response. We selected the particular epitope (of approx. 20 amino acids) that binds to the respective anti-MOG/MBP antibodies, harnessing them to our cell.
CD28, 4-1BB, CD3ζ – Involved in T cell activation. They carry risks of overactivation and immune-related side effects, particularly in vivo. Our project design focused heavily on mitigating those risks and repurposing the signaling towards beneficial results. We are drafting a scientific paper towards this aim, with hopes of publishing it and contributing to further insights into this repurposing.
Igk Signal Peptide, CD8α Hinge and Transmembrane – Primarily structural components. Risks depended on our chimeric construct design, which we reviewed for unwanted sequence creation.
Parts for CRISPR/Cas9-VPR
Our project also involves guideRNAs for epigenetic induction of FOXP3. Single guideRNAs might bind to unintended genomic sites, activating genes other than FOXP3. The changes in gene expression that this might cause can potentially lead to uncontrolled proliferation, altered immune responses, or even transformation into a cancerous state, depending on the accidentally activated gene.
This does not arouse safety concerns in the lab, however, should the project continue, in vitro proliferation assays and other experiments should quantify the risk and identify ways to safely and accurately remove damaged cells.
Parts for microRNA release mechanism
The third component of our project refers to the design of single strand DNA oligonucleotide sequences that hybridize to create a sophisticated release mechanism for microRNAs 338 and 219. We designed two DNA parts that are essentially the mature microRNA-338-3p and microRNA-219-5p sequences.
Work with naked DNA or RNA oligos is considered safe. The DNA hairpins are negatively charged molecules. It is almost impossible for them to cross the physical barriers of the skin. The only likely situation in which they enter the body is via injection.
In case of an accident, the DNA hairpins do not encode any gene and the microRNAs are rapidly destroyed in the environment. The microRNAs in our mechanism hybridize normally with the hairpins, forming non-functional molecules unless in the target cell.
Laboratory Safety
The team was included and trained in the laboratories of Prof. Michalis Aivaliotis and Prof Nikoleta Psatha of the Aristotle University of Thessaloniki, as well as the laboratory of Dr. Evangelia Yannaki in the Hematology Dept. of Papanikolaou General Hospital, Thessaloniki. We attended biosafety seminars with FunPATh (Functional Proteomics and Systems Biology Research Group) where we received specialized training concerning laboratory work in a biosafety level 1 (S1) environment and lab safety working with cell cultures. We have also scheduled sessions with Prof. Lambropoulou, the Head of toxic and lab waste management in our university. Our experiments follow previously tested protocols (outlined by esteemed biotechnology companies like Sigma-Aldrich and Thermo Fisher Scientific) and were conducted after review and communication with our professors. As per university policy, the team was always supervised in the lab during experimental processes.
Greek legislation
Since 1995, Greek legislation has defined a number of conditions and limitations which scientists working with GMOs have to abide by. This includes specific frameworks for their management and release as well as penalties for misconduct. The legislation is constantly updated (most recently in 2019) with new information that corresponds to the rapid changes in the scientific landscape 1.
EU legislation
There are extensive texts outlining the laws, regulations and recommendations of European legislative bodies, including the European Commission, on the deliberate release into the environment of genetically modified organisms 2.
The relevant page of the Leiden 2021 iGEM team contributed significantly to our understanding of the legal framework 3.
Lab security
Before starting our training and experiments, we wanted to establish some ground rules for lab conduct that we would all follow at all times, even in the simplest of experiments. We met with our PIs and instructors and made a simple guide with instructions that, although basic, are often overlooked even by experienced scientists. We always kept those in mind in the lab to ensure we were behaving safely and responsibly without putting ourselves or those around us at risk.
Figure. Our guide for lab safety.
Human Practices Safety
As Cicero once said, the safety of people shall be the highest law.
In iGEM Thessaloniki, we place a high significance on ethical and responsible research involving human participants. We conducted our research diligently and in accordance with the Code of Conduct of the Aristotle University of Thessaloniki Bioethics Committee.
Privacy and safety of the patients are our utmost concerns so we took several measures to secure it. As a first step, we consulted a psychologist and board member of the Greek MS Society which highlighted the necessity of compliance with the GDPR (General Data Protection Regulation) of the European Parliament which dictates that patient data collection should be done anonymously 4.
Then, to be able to undertake Human Practices research with human participants it was crucial to acquire official institutional authorisation. The Bioethics Committee at Aristotle University of Thessaloniki assisted us in preparing informed consent forms to ensure that participation in our research complied with EU and Greek law and protected the participants.
Here is an example of informed consent forms for patients regarding qualitative studies (questionnaires, interviews, focus groups) as well as sample management . It offers detailed information on data collection, processing and implementation 5.
If the following PDF does not render, you can view it here.
References
Hellenic Institute for Occupational Health and Safety (ELINYAE). https://www.google.com/url?q=https://www.elinyae.gr/lexeis-kleidia/genetikos-tropopoiimenoi-mikroorganismoi-gtm&sa=D&source=docs&ust=1727653101481053&usg=AOvVaw1RkVoGunZeGel3L-BhKRc-
Introduction