. Safety .

1.Overview

Every team member is well-informed and highly vigilant in ensuring safety throughout the competition, acknowledging the inherent risks associated with our work. We dedicate significant efforts to preventive measures, firmly believing that maintaining a safe and secure environment for research and collaboration is paramount to responsible innovation and effective teamwork. Our team has implemented comprehensive safety protocols encompassing preventive actions, operational safety, and post-execution guarantees. By continuously identifying and mitigating potential risks, we minimize harm to the iGEM team, stakeholders, project participants, the broader society, as well as the environment. Thus, we have adopted a holistic approach to safety that encompasses physical and laboratory hazards while also addressing privacy concerns and data security of participants. This page provides an overview of the safety measures we have implemented across all facets of our work.

Experimental Safety

2.1 Laboratory Safety

The HUBU-4-CHN team strictly adheres to the safety policies and regulations of iGEM 2024. The majority of organisms and biological parts we use are included in the iGEM 2024 whitelist. For parts not included, we submitted the necessary check-in forms and obtained approval prior to their use. Throughout the project, we rigorously followed all national and institutional safety regulations.

2.2 Safety Preparation Before Laboratory Work

The team operated in ML-1 and ML-2 (equivalent to Biosafety Levels 1 and 2) classification laboratories this year, although only ML-1 work was conducted. All team members were required to attend multiple safety orientation sessions provided by laboratory technicians. These sessions encompassed laboratory-specific safety policies and procedures and included a demonstration of the location and utilization of safety equipment (Fig.1).

Fig.1 Safety supplies in the laboratory. The left shows emergency shower equipment; the upper right indicates usage precautions, and the lower right shows a fire extinguisher

Fig.2 A team member performing experiments in compliance with laboratory safety standards

2.3 Safety Preparations Before Laboratory Work

3.Project Safety

3.1 Choose EcN as the Chassis Cell

We selected Escherichia coli Nissle 1917 (EcN), a non-pathogenic, BSL-1 probiotic found in the human gut, as our chassis organism. EcN is widely used in clinical settings to treat gastrointestinal disorders such as Crohn's disease and ulcerative colitis. It colonizes the gut, preventing pathogenic bacteria from establishing infections, while also strengthening the intestinal mucosal barrier and modulating immune responses to reduce inflammation. Recent studies suggest EcN has tumor-targeting potential, and in combination with chemotherapy, it enhances anti-tumor effects. All work involving genetically modified bacteria was recorded in our transgenic notebook.

3.2 Use of Outer Membrane Vesicles (OMVs) for Delivery

We employed artifical bacterial outer membrane vesicles (OMVs) produced by engineered EcN as a delivery system for Cas9 ribonucleoproteins (RNPs). OMVs are nanoscale liposomes, typically 20–200 nm in size, produced by Gram-negative bacteria. Since OMVs are not live bacteria, they do not cause bacteremia when used in vivo, significantly reducing the risk of bacterial infection or disease transmission, as well as immune responses. OMVs offer advantages over traditional delivery systems, as they avoid the risk of antibiotic resistance gene transfer and exhibit lower cytotoxicity compared to adenoviral vectors and cationic liposomes.

3.3 Proof of Concept Experiment

For safety reasons, we opted not to conduct animal experiments. Instead, we tested the effectiveness of the OMV-based targeted CRISPR/Cas9 delivery system in commercially available HeLa and A549 cell lines.

3.4 gRNA Registration for Human Genome Targets

Our project strictly follows iGEM safety protocols. For components not included in the whitelist, we submitted the necessary Check-in Forms to ensure proper risk management. Our OMV delivery system targets the human PRDX4 gene in the HeLa cell line. Since gRNAs targeting human genes are not pre-approved, we emphasize the importance of safety risk management in synthetic biology and adhere to iGEM’s safety rules to ensure the project progresses safely.

3.5 Potential risk assessment

In the future, the OMV-Cas9 RNP delivery system could potentially treat human genetic diseases. The system is designed for intravenous injection to deliver gene-editing components to target sites, maintaining normal cellular functions while correcting genetic errors. However, discussions with professionals revealed potential risks, such as the possibility of CRISPR gRNAs mistakenly recognizing non-target DNA sequences, which could damage healthy cells or cause cell death. Rigorous testing of gRNA off-target effects is essential before proceeding with further experiments.

3.6 Reducing Cas9 Immunogenicity via Gene Mining and Synthetic Biology

To minimize the immunogenic side effects of Cas9 RNPs, our future work will involve the introduction of a two-plasmid system co-expressing Cas9 RNP and alkaline phosphatase (ALP). ALP catalyzes the removal of the 5′-PPP modification on gRNA, converting it to 5′-OH, which reduces innate immune activation via the RIG-I pathway. Additionally, since over 70% of humans have pre-existing antibodies against SpCas9 and SaCas9 proteins, we will explore gene mining and synthetic biology techniques to develop Cas9 variants with reduced immunogenicity. This approach aims to improve the safety and efficiency of gene editing therapies.

4.Human Practice Safety

     As a responsible iGEM team, we prioritize the privacy and security of project participants. We have studied relevant laws, including the Constitution and Civil Code of the People's Republic of China, and consulted the Legal Aid Center to ensure our human practices comply with these laws. We prepared a privacy protection notice with legal assistance to inform participants about the data we collect, how it is processed, and how it is stored securely.

    We always believe that building a good relationship of trust with our project participants is beneficial to the development of both parties. We infected the participants with warm and sincere words and shared our project with them. In the process, we not only build a friendly relationship, but also make them feel like an integral part of our project.

    Here, you can swipe below to see the privacy protection notice for project participants as an example. This includes information we collect about program participants, how we process the information, and how we securely store and use the information to ensure the privacy and security of program participants.

Attached: iGEM HuBu-4-CHN 2024 Project Information Form and Personal Privacy and Security Notice

5.Safety Activaties

5.1 The 1st International Symposium on the Application and Safety of EcN

Objective:
The first International Symposium on the Application and Safety of EcN was organized by the School of Biological and Pharmaceutical Engineering of Nanjing University of Technology and Peking University Health Science Center.
Activities:
1. Discuss the potential application and safety issues of EcN in the field of synthetic biology with the participating iGEM team in the online Tencent meeting
2. After the meeting, the division of labor and cooperation will complete the writing of the EcN white paper

Details:
On July 26, 2024, at 1 p.m., the first International Symposium on the Application and Safety of Escherichia coli Nissle 1917 online conference began. All members of our team participated in this seminar, which aimed to bring together organizations, teams, and individuals from all over the world who have in-depth research and practical applications of EcN. They discussed the latest advances, challenges, and future directions of its scientific research and practical applications and shared valuable experience and insights in EcN research and applications. After a brief opening ceremony, the symposium officially began. At this time, our team listened carefully to the thematic sharing of relevant experts and related companies and learned about the therapeutic potential of EcN in the origin of inflammatory bowel disease and the tumor microenvironment. In the free question and exchange session, our team actively communicated with other iGEM teams to share our team's project, namely the potential application of EcN-secreted outer membrane vesicles in targeted therapy and the safety of EcN as a chassis cell: "Although there are still a series of problems in the application of EcN in the field of biotherapy, such as immune response, the carriage of virulence genes, and drug resistance, in general, EcN, as an engineered bacterium, has promising application prospects in disease treatment and diagnostic strategies. With the continuous advancement of synthetic biology and genetic engineering, the modification and optimization of EcN will continue to improve its safety and therapeutic potential, bringing more effective and safer treatment options to patients." Other teams agreed with our team's presentations and made suggestions for our project. For example, one team said: "The use of EcN's outer membrane vesicles as a delivery platform for gene-editing complexes is very innovative, but the safety of the toxicity of the outer membrane vesicles secreted by EcN is also a matter that needs to be considered." Our team was greatly inspired. After the workshop, our team carefully searched for relevant literature and integrated it into the "EcN White Paper" in collaboration with other teams. This white paper included a section on "Side Effects and Biosafety Perspectives for EcN Involved Therapies" (attached). After participating in the first seminar, our team gained a lot, actively reflected and adjusted, and helped our team's project to use EcN more safely and efficiently.

Attached: EcN White Paper

5.2 Compilation of the "Ethics Manual of Synthetic Biology"

Objective:
By jointly writing the "Ethics Manual of Synthetic Biology" with various universities and based on our team's project, we aim to supplement relevant safety issues, establish the ethical framework of synthetic biology, promote the healthy development of synthetic biology technology, ensure that it adheres to ethical principles in the application process, and facilitate the improvement and development of the research and practice of the team project in the directions of safety, responsibility, and in-depth education.

Implementation Plan:
Multiple iGEM teams, such as CJUH-JLU-China and our team, collaborate to jointly write the "Ethics of Synthetic Biology" manual.

Activities:
Hosted by the relevant iGEM team of Jilin University, through online communication, discussion, and division of labor to write the ethics manual of synthetic biology and share relevant track experiences.

Details:
On August 11, 2024, our team's HP began working on the initial draft of the manual. CJUH-JLU-China serves as the chief editor, completing the writing of the preface, integration and typesetting, text review, and other contents. Each team writes the responsible part separately and submits the completed draft to the CJUH-JLU-China team. After each team makes phased progress, an online meeting will be held for summary.

   Based on the projects of each team, ethical topics of synthetic biology are explored to provide an ethical guidance framework for synthetic biology researchers, policymakers, ethics review institutions, and the general public in China and even globally, guiding synthetic biology research and practice towards a more responsible and sustainable direction.

Our team (HUBU-4-CHN) is mainly responsible for writing the first draft of the ethics manual of synthetic biology. Based on our team's iGEM project, we are mainly responsible for writing the manual in the medical aspect.

Attached: Ethics of Synthetic Biology