Safety and Security

Role in iGEM

In iGEM, safety is not just a requirement but a responsibility [1]. As synthetic biologists, we are creating tools that have the potential to improve human health and society - but if handled carelessly, they could also pose risks. For this reason, our team approaches every stage of our project with careful attention to biosafety (protecting people and the environment from accidental harm), biosecurity (preventing the intentional misuse of biological knowledge or materials), and bioethics (ensuring our work respects human dignity, values, and responsibility toward future generations). We believe that developing new therapies should always go hand in hand with preventing risks, building public trust, and ensuring that our innovations truly serve to make the world a better and safer place. In the following sections, we therefore present a detailed overview of how we applied these principles to each biological system and material we worked with.

Check-ins

Human cell lines

Working with human-derived cell lines is central to our project. These models are valuable tools for testing the uptake and efficacy of antisense oligonucleotides (ASOs) in a controlled and reproducible environment. At the same time, they come with important responsibilities: safeguarding researchers from exposure, preventing contamination or release, ensuring materials are used responsibly, and reflecting on the ethical implications of using human biological resources.

Biosafety

All work with human cell lines was carried out under Biosafety Level 2 (BSL-2) conditions, in compliance with institutional and international guidelines [2].
This included:

• Use of biosafety cabinets for all manipulations to prevent aerosol exposure and contamination.
• Personal protective equipment: lab coats, gloves, protective eyewear.
• Proper waste disposal procedures (autoclaving and chemical disinfection) for cultures, media, and consumables.
• Training and supervision: All team members working with human cell cultures completed certified biosafety training and obtained the required permits for handling human cell lines. Their work was consistently supervised by experienced researchers.
• Mycoplasma and contamination control measures to ensure safe and reliable use of the cells.

Human Embryonic Kidney 293 cells (HEK293):

HEK293 (Human Embryonic Kidney 293) cells are a widely used immortalized human cell line that provides a reliable mammalian model system for molecular biology and gene expression studies [3]. In our project, HEK293 cells are transduced with a lentiviral vector carrying a GFP reporter gene, enabling fluorescent readouts of antisense oligonucleotide (ASO) activity [4], [5]. This makes them an ideal platform for proof-of-concept experiments in RNA-targeted gene regulation.

A549:

We used A549 (human lung adenocarcinoma, epithelial, ATCC CCL-185)[6] as an in-vitro model of NSCLC to quantify the activity of our antisense oligonucleotides (ASOs). Their origin from human lung cancer tissue makes them directly relevant to our therapeutic strategy.

A549-GFP Cells:

For fluorescence-based assays, we also used A549 cells engineered to express green fluorescent protein (GFP). The GFP transgene was introduced using a replication-incompetent lentiviral vector (Addgene #185473) [7]. These cells enable quantitative and visual readouts of ASO-mediated knockdown in a cancer-relevant model.

A375 Human Epithelial Melanoma Cells (ATCC CRL-1619):

We use the A375 human epithelial melanoma cell line [8] to evaluate our antibody-epitope conjugate. A375 cells are EGFR-positive and HLA-A2 positive, making them suitable for evaluating our antibody-epitope conjugate. They are used to assess whether the conjugate is internalized, processed, and presented on MHC-I molecules, enabling activation of MART-1–specific T cells.

Primary Human T Cells Expressing MART-1–Specific TCR:

We use engineered primary human T cells expressing a MART-1–specific TCR, provided by our collaborators [9] at Bar-Ilan university and described in [10]. These cells allow us to evaluate whether our antibody–epitope conjugate can be internalized by A375 epithelial cancer cells, processed, and presented on MHC-I molecules to activate MART-1–specific T cells. Because no immortalized or commercially available cell line carries this specific TCR, the use of engineered primary T cells is essential for testing whether our conjugate enables antigen presentation and immune recognition in vitro.

Biosecurity

While human cell lines themselves are not typically associated with dual-use concerns, biosecurity measures ensured that materials could not be misused:

• Restricted lab access to authorized, trained personnel only.
• Secure storage of frozen cell stocks in designated freezers with inventory tracking.
• No distribution or sharing of cell lines outside approved facilities.
• ASO sequence review to ensure no designs could be repurposed for harmful applications.

Bioethics

Our use of human cell lines was guided by ethical considerations:

• Source transparency: HEK293 and A549 are established cell lines that are widely available and accepted in research. We did not generate or collect new human-derived materials.
• Respect for human dignity: We recognize that these cells originate from human tissues, and we use them only in the context of advancing therapeutic knowledge for human benefit.
• Responsible translation: All experiments were restricted to in vitro models, with no attempt at animal or human application. Our work remains in the research stage, where it poses no direct risks to patients or the public.
• Commitment to societal benefit: Our goal in using these cell lines is to design therapies that may one day contribute to safer and more effective treatments for lung cancer.

Antisense Oligonucleotides (ASO)

Antisense oligonucleotides (ASOs) are short, synthetic single-stranded DNA molecules designed to bind complementary RNA sequences and promote their degradation or block their function [11]. In our project, ASOs serve as molecular tools to evaluate knockdown efficiency, specificity, and cell viability in human cell lines. We use:

• MALAT1-targeting ASO as a positive control for transfection and knockdown efficiency [12].
• GFP-targeting ASO for reporter-based proof-of-concept experiments in HEK293-GFP and A549-GFP cells.
• Gapmer ASOs targeting RIOK1, PRMT5, and MAT2A in A549 lung cancer cells to assess gene-specific knockdown and effects on cell viability. Gapmers are chemically modified ASOs that increase stability and RNase H–mediated cleavage efficiency, allowing potent and specific targeting of mRNA transcripts in vitro [13].

Our ASO experiments are conceptually related to Registry parts BBa_K5401005 and BBa_K2429068, which also employ antisense strategies to modulate gene expression, although the target genes and cellular systems differ [14].

Biosafety

All ASOs are handled under Biosafety Level 2 (BSL-2) conditions in compliance with institutional and iGEM safety policies [2]. Specific measures include:

• Fume hood use: All ASO solutions are prepared and handled exclusively in a fume hood to prevent accidental inhalation or aerosol exposure.
• Containment: Experiments are performed in biosafety cabinets using gloves, lab coats, and protective eyewear.
• Waste disposal: All ASO-containing waste is decontaminated using chemical disinfectants or autoclaving before disposal, preventing any environmental release.
• Cell line restrictions: ASOs are tested only in commercial human-derived cell lines (HEK293, HEK293-GFP, A549, and A549-GFP). No animal or environmental applications are performed.

Because ASOs are non-replicating, non-infectious molecules, they pose no hazard of propagation or transmission. The main potential risk is off-target modulation of unintended genes, but this risk is confined to in vitro cell culture experiments and mitigated by using ASOs only in controlled, small-scale laboratory settings.

Biosecurity

ASOs do not encode proteins or toxins and cannot be weaponized in their current form. They are synthesized at research-grade purity and used exclusively for laboratory testing of knockdown efficacy. There is no foreseeable dual-use risk or potential for misuse.

Bioethics

The use of antisense oligonucleotides (ASOs) targeting human transcripts requires ethical consideration due to their potential biological effects. In our project, all ASOs are strictly employed as in vitro research tools, never in living organisms or clinical settings. Their purpose is to validate our system and assess knockdown efficiency while minimizing unnecessary risks. We also align with iGEM’s policy that nucleic acid parts outside the White List, including non-coding RNA targets, require a Check-In, which we have acknowledged and completed.

Lentivirus

(Replication-Deficient, VSV-G Pseudotyped, VectorBuilder VB900088-2243bzq)

We use a replication-deficient, VSV-G–pseudotyped lentiviral vector to deliver a GFP-expressing construct (VectorBuilder VB900088-2243bzq) into HEK293 cells [15]. This system allows stable genomic integration of the GFP transgene, enabling long-term expression for proof-of-concept experiments testing the effectiveness and specificity of our antisense oligonucleotides (ASOs) [4] [16]. The lentiviral system is engineered to be replication-incompetent and lacks all packaging genes, meaning no additional viral particles are produced after transduction [17]. Using this approach provides consistent GFP expression, which is critical for reproducible assessment of ASO-mediated knockdown over time.

Biosafety

Although the lentivirus is replication-deficient, it carries potential risks standard for integrating viral vectors, including:

• Accidental exposure through skin puncture, mucosal contact, or aerosol generation, which could result in unintended transduction of human cells.
• Environmental release, which could theoretically allow stable integration of the vector in non-target cells.
• Insertional mutagenesis, a rare but theoretical risk due to genomic integration [18].

To mitigate these risks, all procedures are conducted under Biosafety Level 2 (BSL-2) conditions with strict adherence to institutional protocols [2]:

• Class II biosafety cabinet (BSC): All lentiviral manipulations, including production, concentration, and transduction, are performed in a certified BSC.
• Personal protective equipment (PPE): Lab coats, gloves, and protective eyewear are worn at all times, and hand hygiene is strictly enforced.
• Waste decontamination: Liquid waste is treated with bleach or appropriate disinfectants; solid waste is autoclaved prior to disposal.
• Designated workspace and equipment: Dedicated pipettes and consumables prevent cross-contamination.
• Training: All personnel handling lentivirus have completed institutional biosafety training and spill response procedures.
• No amplification in cells: Transduced HEK293 cells lack packaging elements, ensuring no new viral particles are generated.

Biosecurity

The lentiviral vector is replication-deficient, non-pathogenic, and restricted to in vitro use. It cannot be used to generate infectious viruses in the laboratory context, and all materials are secured and handled only by trained personnel. There is no foreseeable dual-use risk in the context of our experiments.

Bioethics

The lentiviral system is used solely to create a stable GFP-expressing HEK293 and A549 cell lines for in vitro proof-of-concept testing of ASO therapeutics. No human or animal subjects are directly exposed, and the system is applied only in contained BSL-2 laboratories. This approach maximizes scientific benefit while minimizing ethical and biosafety concerns.

MART‑1 Peptide Epitope

We incorporate a short peptide epitope derived from MART‑1 (Melan‑A, amino acids 26–35, ELAGIGILTV) into our engineered antibody chain (discussed below). MART‑1 is a melanocyte differentiation protein expressed in normal melanocytes and melanoma cells. Its peptide epitopes can be presented on MHC-I and recognized by T cells. In our experiments, the MART‑1 epitope is delivered to A375 human melanoma cells (ATCC CRL-1619) [8], which are then co-cultured with human T cells engineered to express a MART‑1-specific TCR [10]. T cell proliferation and activation serve as readouts for successful epitope presentation, providing a controlled system to study antigen delivery and immune response.

Biosafety

The MART-1 sequence is a short, non-infectious peptide motif embedded in a recombinant antibody. It cannot replicate, encode functional proteins beyond its antigenic role, or cause disease. All experiments are performed under Biosafety Level 2 (BSL-2) conditions using standard cell culture practices [2]:

• Biological safety cabinet (BSC): All peptide manipulations and cell culture work are performed in Class II BSCs.
• Personal protective equipment (PPE): Lab coats, gloves, and eye protection are worn at all times.
• Waste disposal: All materials are decontaminated following institutional guidelines.
• Controlled access and training: Only trained personnel access the lab, and all experiments are documented and submitted via Check-In forms to the iGEM Safety Committee.

Biosecurity

The MART-1 epitope is a non-replicating, non-pathogenic antigen fragment used solely as part of an engineered antibody in vitro. There are no dual-use concerns.

Bioethics

The MART‑1 epitope allows the use of immortalized human cell lines (A375 and engineered T cells) [8], [10] rather than pathogenic organisms or primary patient samples, minimizing ethical concerns. It is widely used in immunology research and represents a minimal-risk model system to test antigen delivery and T cell activation, fully consistent with iGEM safety guidelines [1]. Alternative epitopes would not further reduce risk, as MART‑1 already provides a standardized, safe, and well-characterized option.

Antibodies

We express Cetuximab (anti-EGFR) [19] in Chinese Hamster Ovary (CHO) cells [20] to explore antibody-based delivery strategies for our ASO therapeutic platform. The antibody sequence is codon-optimized for CHO cells to improve expression efficiency, and we evaluate expression levels experimentally. This setup allows us to study ASO conjugation strategies, demonstrate effects of codon optimization, and establish a modular in vitro platform for targeted antisense delivery. Computational tools such as ESO [21] and MNDL Bio [22] are used to predict and optimize antibody expression in CHO cells.

To enable targeted delivery of antisense oligonucleotides (ASOs), we plan to conjugate the expressed antibody to ASOs using an N-hydroxysuccinimide (NHS) ester coupling strategy [23], which covalently links the ASO to lysine residues on the antibody. This approach provides a stable, reproducible means of generating antibody–ASO conjugates, allowing us to test targeted knockdown efficiency and delivery in vitro.

Cetuximab is a well-characterized, clinically approved therapeutic antibody [24]. It is not derived from a pathogen or toxin and has no natural function in the host organism beyond its designed anti-EGFR activity. Expression in CHO cells is performed solely for research purposes, including expression testing, codon optimization, and ASO conjugation studies.

Biosafety

Cetuximab expression in CHO cells poses no known biosafety hazards beyond standard mammalian cell culture procedures [2]. CHO cells are non-pathogenic and commonly used in research and biomanufacturing. Safety measures include:

• Biosafety cabinet (Class II BSC): All manipulations are conducted in certified cabinets.
• Personal protective equipment (PPE): Lab coats, gloves, and eye protection are worn at all times.
• Good laboratory practices: Sterilization of materials, proper waste disposal via autoclaving or chemical disinfection, and careful labeling of recombinant materials.
• Controlled workspace: All CHO cells and recombinant materials are stored in designated areas to prevent cross-contamination.
• Conjugation reagents: NHS esters and related chemicals should be handled in accordance with institutional chemical safety protocols [25].

No part of the antibody sequence encodes toxins, virulence factors, or other hazardous elements.

Biosecurity

The antibody sequence is synthetic and non-replicating, and CHO cells are non-pathogenic. Materials are restricted to trained personnel in a controlled laboratory environment. There are no dual-use concerns associated with this work.

Bioethics

This work uses immortalized CHO cells and synthetic antibody sequences, avoiding any pathogenic organisms or patient-derived materials. Expression of Cetuximab and its conjugation to ASOs provides a safe, reproducible platform to test targeted antisense delivery, fully consistent with iGEM safety and ethical guidelines.

Yeast - Saccharomyces cerevisiae W303

We engineered Saccharomyces cerevisiae W303 to express GFP as a model organism to test the effectiveness and specificity of our antisense oligonucleotides (ASOs). This serves as a proof-of-concept system for RNA-targeted gene regulation, providing a simple, controllable eukaryotic context before moving to mammalian cell lines.

Biosafety

Saccharomyces cerevisiae is classified as SBiosafety Level 1 (BSL-1)S and is generally regarded as safe. The main risks involve accidental exposure to laboratory personnel or unintended environmental release. These risks are minimal because S. cerevisiae is non-pathogenic and poses negligible threat to healthy individuals and the environment [2], [26].

All work is conducted under BSL-1 conditions, including:

• Personal protective equipment (PPE): Lab coats and gloves are worn at all times.
• Surface decontamination and waste disposal: Work surfaces are disinfected, and solid/liquid waste is autoclaved prior to disposal.
• Designated work areas: Experiments are conducted in clearly assigned areas to prevent cross-contamination.
• Training and supervision: Team members are trained in biosafety and aseptic techniques, and experimental design is guided by Dr. Daniel Dovrat, an expert in S. cerevisiae handling.

Biosecurity

The GFP-expressing yeast strain is non-pathogenic, non-replicating outside the lab environment, and poses no dual-use concerns [26]. All materials are stored securely and used only by trained personnel.

Bioethics

Using S. cerevisiae allows safe in vitro experimentation without involving human or animal subjects. This choice balances low-risk handling with sufficient biological complexity to test antisense oligonucleotide activity, consistent with iGEM safety and ethical guidelines.

White List Organisms

Chinese Hamster Ovary cells (CHO)

Chinese Hamster Ovary (CHO) cells [20] are widely used mammalian cell lines in biotechnology, particularly for recombinant protein and antibody production. In our project, CHO cells are employed as a safe and reliable expression system to produce cetuximab antibodies from plasmid DNA.

CHO cells are non-human, non-pathogenic, and considered low-risk (White List, BSL-1/2 practices) [2]. All work is conducted following standard laboratory safety protocols, including use of personal protective equipment (PPE), sterile techniques in Class II biosafety cabinets, and proper decontamination of waste and surfaces. Their well-characterized nature and long history of safe use make them ideal for controlled antibody expression experiments.

Escherichia coli

We use Escherichia coli primarily as a cloning and plasmid amplification host in our project, performing standard molecular biology procedures such as plasmid preparation and vector construction [27]. Only well-characterized laboratory strains (e.g., DH5α) are used, which are non-pathogenic, incapable of surviving outside controlled laboratory conditions, and considered low-risk (White List, BSL-1 practices) [2]. All work is carried out using standard microbiological safety measures, including sterile techniques, appropriate personal protective equipment (lab coats, gloves, eye protection), and proper decontamination of liquid and solid waste by autoclaving. No pathogenic or clinical strains are employed, and the procedures are consistent with routine laboratory handling of E. coli, ensuring safe and responsible use.

Our Project

1. Antibody-mediated targeting reduces off-target exposure
   Conjugating the ASO to cetuximab concentrates the oligonucleotide payload on EGFR-expressing cells [28]. Because EGFR is substantially overexpressed in many NSCLC subtypes while being low in healthy lung tissue [29], antibody targeting increases the local concentration of ASO in tumor cells and reduces systemic exposure and uptake into non-target tissues. Antibody–oligonucleotide conjugates (AOCs) are an active area of translational research precisely for this reason: they combine the targeting specificity of monoclonal antibodies with the gene-modulating action of oligonucleotides to improve therapeutic index [30], [31].
   Practical safety measures we apply: antibody selection based on tumor vs normal expression (Prof. Benhar’s consultation), small laboratory scales, BSL-2 containment for all handling, secure storage, and inventory control of conjugates.
2. “Brother” (blocking) strand reduces off-target binding and toxicity
   To lower unintended hybridization, each ASO is initially hybridized to a partially complementary blocking strand that sterically and thermodynamically limits the ASO’s ability to bind non-intended RNAs [32]. Only after cell-specific uptake and intracellular processing does the ASO become available to engage its intended target sequence. This approach — pairing an ASO (or gapmer) with a complementary blocker or using protective oligonucleotide architectures — has been demonstrated to reduce off-target interactions and improve safety of therapeutic ASOs in preclinical studies. Recent engineered “blocking”/nano-architectures (e.g., BROTHERS / BRO designs) and related strategies show clear reductions in off-target effects compared with unmasked ASOs [33].
   Practical safety measures we apply: all conjugation and unmasking chemistry is validated in vitro; concentrations are kept at research scale; all work is carried out in fume hoods/BSC and wastes are fully inactivated and disposed of.
3. Targeting a synthetic-lethality partner spares normal cells
   Our ASO targets the synthetic-lethality (SL) partner gene of a mutated gene found in lung cancer cells [34]. The therapeutic idea is that cancer cells bearing the mutation become dependent on the SL partner; knocking down that partner kills mutant cancer cells but not wild-type (healthy) cells. This is an accepted and translationally pursued therapeutic strategy (analogs include PARP inhibitors exploiting BRCA deficiency) [35]. ASOs have been used to induce synthetic-lethality in preclinical models (proof-of-concept ASOs achieving selective cytotoxicity in mutant tumors), demonstrating feasibility and a favorable selectivity profile in vitro and in vivo. Because normal cells lack the initiating driver mutation, they are expected to tolerate the transient knockdown of the SL partner, greatly reducing the risk of systemic toxicity [36].
   Practical safety measures we apply: rigorous target validation in cell lines (mutant vs wild-type controls), dose-response testing to define therapeutic window, and strict in-vitro confinement (no animal or human administration during iGEM work).
4. Epitope release + MHC-I presentation recruits immune clearance in a controlled way
   The antibody molecule in our construct includes a peptide epitope designed to be released during intracellular degradation of the antibody after receptor-mediated internalization. Antigenic peptides derived from internalized proteins can be processed and presented on MHC-I (either by the target cell itself or by professional antigen-presenting cells via cross-presentation), thereby flagging the tumor cell for cytotoxic T-cell recognition [37]. Cross-presentation and engineered vaccine strategies have been widely studied; delivering antigenic peptides in targeted formats to improve MHC-I presentation and T-cell recruitment is a recognized immunotherapy approach [38].
   Practical safety measures we apply: the epitope is a tumor-associated epitope chosen to minimize cross-reactivity with healthy tissues; experiments testing immune recruitment are conceptual / in vitro only in the iGEM context; any future translational work would require full preclinical immunotoxicity and HLA-coverage testing.
5. Overall safety context — non-replicating components, small scale, and precedent
   Non-replicating nature: ASOs are synthetic, non-replicating nucleic acids; antibodies are non-infectious proteins. Neither component can propagate biological risk in the way live engineered organisms can. ASO therapeutics have multiple FDA-approved precedents [39] (e.g., nusinersen [40], inotersen [41]) that demonstrate the clinical tractability of ASOs when safety risks are managed. AOCs are an active clinical/industry area (several companies — e.g., Avidity [42], Dyne [43] — are advancing AOC platforms), but to date there are no FDA-approved AOC products; research and trials are ongoing and subject to rigorous safety assessment. This context supports our cautious, in-vitro-only approach during iGEM.

Our Labs

Our Molecular Biology Lab

In this laboratory, we perform experiments with Saccharomyces cerevisiae (yeast) and Escherichia coli. These organisms are used for cloning, plasmid preparation, and other molecular biology processes that support our main project. The workspace is organized into specific areas (e.g., PCR station, gel electrophoresis station, transformation area, and measurement bench), which ensures that all steps are carried out under reproducible and reliable conditions.

Biosafety note: Although both E. coli and S. cerevisiae are classified as BSL-1 organisms and are safe to handle, they are considered contaminants in mammalian cell culture. For this reason, all experiments with yeast and E. coli are strictly confined to this lab, with designated equipment and waste streams.

Our Cell Culture Lab

Our tissue culture laboratory is exclusively dedicated to mammalian cell work under sterile conditions. Here we maintain human cell lines, perform transfections, and assess gene regulation using our antisense oligonucleotides (ASOs). This environment requires strict aseptic protocols to ensure the reproducibility of experiments and the safety of both personnel and cell cultures.

Separation principle: To prevent cross-contamination, we never bring materials, organisms, or consumables from the yeast/E. coli lab into the tissue culture space. Even small traces of “outsider” microorganisms would pose a major risk to mammalian cultures, which are highly sensitive to contamination. Therefore, each lab is used only for its intended purpose, and all equipment, reagents, and waste handling procedures remain completely separate.

Lab Safety Facilities

Our Lab Safety Guidelines