Safety and Risk Management

Safety and risk management are integral components of our project, aimed at ensuring the well-being of team members, the environment, and potential future users of the technology. We are committed to developing a precise mRNA-based therapeutic platform for liver cancer, which necessitates a comprehensive assessment and management of potential biological, chemical, and operational risks throughout the experimental process. The project strictly adheres to iGEM safety guidelines, relevant Chinese biosafety regulations, and our university's laboratory safety protocols. This section details the identified risks, corresponding management strategies, and specific protective measures implemented.

Risk Identification

Our project involves various biological and chemical materials, as well as the development and use of hardware equipment. Potential risks primarily include:

Biological Material Risks: The human cell lines used (e.g., HEK293, liver cancer cell lines) are mostly classified as Risk Group 1. However, handling during culture still poses potential biohazards such as cell culture contamination or aerosol generation.

Chemical Reagent Risks: The experimental process utilizes various chemical reagents, some of which are toxic or flammable. Examples include high-concentration ethanol and guanidinium thiocyanate found in RNA extraction kits, posing risks of exposure or inhalation.

Hardware Equipment Risks: The self-developed microfluidic syringe pump involves electrical components, carrying potential risks of short circuits, overheating, or liquid leakage, which could lead to equipment damage or experimental failure.

Technology Application Risks: Theoretically, off-target effects of the designed mRNA therapeutic could lead to expression of the target protein in non-target tissues or unintended immune responses. Although we have employed multiple strategies (e.g., miRNA-responsive switches, targeted delivery) to enhance specificity, the risks of off-target effects and immunogenicity remain and require vigilance.

Risk Management Measures

To effectively manage the aforementioned risks, we have implemented multi-layered safety measures:

Institutional Framework and Training: All team members received and passed systematic safety training and assessment before entering the laboratory. Training covered the correct use of Personal Protective Equipment (PPE), biosafety practices, chemical hazardous material management, instrument operating procedures, and emergency response protocols. We strictly comply with Dalian University of Technology (DUT) laboratory safety regulations and undergo regular inspections by laboratory safety officers.

Experimental Operational Protocols:

Biological Experiments: All cell culture, transfection, and RNA operations are conducted within appropriate Class II Biological Safety Cabinets or laminar flow hoods to prevent aerosol release. All biological waste is autoclaved prior to disposal by a licensed professional waste management company.

Chemical Experiments: Use of toxic or hazardous chemical reagents is performed within fume hoods with appropriate PPE (gloves, safety goggles, lab coats). Chemical waste is segregated and collected in designated containers for unified disposal by a qualified professional company.

Hardware Operation: The design and testing of the microfluidic device are conducted on well-ventilated lab benches. Electrical connections and inspections are performed by knowledgeable team members with the power disconnected, ensuring proper insulation and waterproofing. The device is monitored continuously during operation.

Environmental and Waste Management: All biohazardous waste is autoclaved before disposal. Sharps are disposed of in dedicated puncture-resistant containers. Chemical waste liquids are categorized and stored in specific, clearly labeled containers according to their properties. Solid waste generated from hardware production is sorted as electronic waste or general waste.

Risk Control Strategy - Safety by Design

We have integrated two types of cell-selective molecular switches (toehold switches and exCAG switches) into our mRNA platform, enabling translational control of the Open Reading Frame (ORF). These switches keep the ORF translationally silent in non-target cells and are activated specifically in target cells, lifting the repression to allow normal protein expression.

These switches not only demonstrate our capability for precise molecular control but also highlight the core safety-by-design principle embedded throughout our project. From the outset of the mRNA therapeutic development, we have proactively addressed potential risks, including unintended effects and immunogenicity due to off-target expression. By incorporating miRNA-responsive regulatory switches, we significantly reduce the risk of expression in non-target tissues, enhancing the controllability and safety of our therapeutic strategy. Furthermore, the Lipid Nanoparticle (LNP) delivery system has been optimized for preferential liver targeting, increasing tissue specificity.

iGEM Rules and Policy Compliance

We fully adhere to iGEM competition safety and policy requirements:

· No Risk Group 3 or 4 organisms or their parts were used.

· All experiments were confined to the laboratory; no genetically modified organisms were released into the environment.

· No human or animal experiments were conducted. All cell lines used are from commercial sources listed on the iGEM White List and possess clear provenance documentation.

· We have performed the necessary Safety Check-Ins for non-standard biological parts used in our project (e.g., siRNA targeting TGF-β, miRNA21 sequences) and reported them to the iGEM Committee with appropriate documentation.

Part and Material Safety Check-In

Key Biological Parts: Core coding sequences (e.g., human HNF4α mRNA, PROTAC elements targeting TGF-β) are sourced from public databases or published literature and lack known pathogenicity. These elements were amplified and prepared in E. coli or in vitro systems, with final products purified to remove host residue.

Cell Lines: Cell lines used (e.g., HEK293, HepG2) are classified as Risk Group 1 and were handled within standard BSL-2 laboratory facilities. All cell culture work followed aseptic techniques to ensure experimental reliability and laboratory safety.

HP - Responsible Research

In our Human Practices activities, we consistently prioritize safety, ethics, and social responsibility:

· We proactively integrated the Responsible Research and Innovation (RRI) framework from the initial project design phase, engaging with clinical experts, patient groups, and bioethicists to gather diverse feedback on public concerns.

· In communications with patient groups, clinicians, and the public, we emphasized accurate scientific communication, avoiding unrealistic promises or expectations, and clearly stating the early research and development stage of the technology.

· We actively consulted experts in biosafety and drug regulation to understand the regulatory pathway and safety requirements for therapeutic mRNA products from development to clinical application, ensuring our project design aligns with future industrialization standards.

Through this comprehensive risk management framework and responsible research practices, we have ensured biosafety, operational safety, and environmental safety throughout the project lifecycle, demonstrating the R-Home team's firm commitment to safety, ethics, and public welfare.