Overview

Safety has always been a paramount concern in all projects and experiments conducted at Huazhong Agricultural University. This year, our team deeply recognizes the critical importance of safety measures for protecting researchers and the environment. We have implemented a series of measures, which not only prevent potential harm to individuals but also mitigate the risk of accidental pathogen release.

We strictly adhere to iGEM's official safety rules and policies, conducting thorough risk assessments for every aspect of our project. As the final product is intended for real-world application through collaboration with industry and government partners, each step has been meticulously planned with corresponding solutions to preempt uncertainties. In developing HemorrEaser, which focuses on inhibiting hemorrhoidal angiogenesis and creating a treatment suitable for specific populations like pregnant women, we paid particular attention to its potential effects on normal angiogenesis and fetal development. A series of measures were implemented to mitigate these risks and enhance overall safety. Furthermore, we maintained stringent safety standards throughout the project via comprehensive safety training and obtaining ethical approvals, ensuring full compliance with laboratory safety and ethical requirements.

Safety in Design

1. Global Regulation Circuit: To address the potential risks of engineered bacterial over-proliferation in vivo and associated issues such as therapeutic module expression fluctuations and toxicity, we designed an intelligent regulatory system based on a LuxR/TetR dual-factor cascade. This system employs a quorum-sensing mechanism to monitor bacterial density and utilizes a negative feedback loop to regulate the expression of downstream genes, thereby achieving global control.
2. Suicide and Medicine-Food Collaboration Circuit: HemorrEaser utilizes a riboswitch that negatively regulates downstream gene expression in response to a specific compound. In the absence of the ligand, the engineered bacteria initiate expression of the CcdB toxin protein, leading to self-elimination and cessation of therapy. To meet specific requirements during the treatment cycle, we incorporated an "expiry-date" circuit that delays bacterial lysis, allowing the engineered bacteria to remain active for extended periods without frequent dosing. In the topical application mode, the engineered bacteria contain only a simple negatively regulated riboswitch, enabling a rapid response to inflammation and ensuring quick elimination after the task is completed.
3. Implementation Design: Informed by our Human Practices research, the final product is designed as a bacterial powder formulated into enteric-coated capsules and ointments. This approach facilitates practical production and use, while ensuring effective self-elimination of the engineered bacteria and minimizing environmental release risks. For pregnant women, for whom the bioactive anti-angiogenesis components pose a potential risk to fetal development, only the topical ointment with anti-inflammatory effects is a safe and effective option.

Read details in Design

1. BioPROTAC: Our designed bioPROTAC, VHH-VHL, specifically mediates HIF-1α degradation. Off-target effects could potentially inhibit the hypoxia response in normal human tissues. To address this, we employed OMVs displaying the membrane-anchored OmpA-CAR fusion for targeted delivery. The CAR wound-homing peptide targets neovascular sites, and no literature currently indicates direct risks to fetal development from CAR itself. However, we acknowledge that CAR's targeting is not absolutely specific. Due to the general lack of highly specific targeting methods for hemorrhoidal tissue, we incorporated the global regulation module to maintain a constant per-cell release rate of the therapeutic agent, thereby reducing off-target risks.
2. Anti-VEGF Nanobody: Off-target effects of a conventional anti-VEGF nanobody could be harmful, particularly to fetal development. We engineered a probody by adding a masking peptide, ensuring it is only activated at the hemorrhoid site where MMP3 is highly expressed, significantly reducing its off-target potential.
3. Melittin: The engineered melittin (NKU-China, 2024) functions as the active drug in the topical formulation. Its targeting is ensured by a triple-layered safety strategy: the inherent inflammatory tropism of E. coli Nissle 1917 (EcN), regulation by the quorum-sensing-based global control circuit, and activation by an ROS-responsive promoter.

You can read details in Design

Safety in Operation

Safety remains the core principle of our project. We have completed and submitted the required safety forms detailing the project. The laboratory work utilized E. coli DH5α, BL21(DE3), and Nissle 1917 strains for gene cloning and expression. E. coli Nissle 1917 is a widely recognized probiotic with no evidence suggesting risks to human health or the environment. Other strains are handled according to strict laboratory protocols to ensure safety for personnel and the environment. All parts, reagents, and cells were obtained from commercial suppliers via safe channels.

To understand the real-life experiences, treatment perceptions, and acceptance of innovative therapies such as medicine-food collaboration and engineered probiotics among patients with hemorrhoids and related intestinal conditions, we conducted anonymous interviews and questionnaire surveys. All participants provided informed consent, with sensitive questions being optional. Data analysis was performed solely in aggregated form, without involving individual information. This study was approved by the Scientific Ethics Committee of Huazhong Agricultural University and involved only patients who voluntarily signed informed consent forms, ensuring full compliance with ethical standards. You can read the materials below.

In our analysis of public comments on Sina Weibo related to hemorrhoids and intestinal health using machine learning and deep learning models, we prioritized ethical compliance. To avoid potential violations, we obtained data support from Dr. Tingshao Zhu's group at the Institute of Psychology, Chinese Academy of Sciences, rather than collecting data independently. Their database was approved by the Institutional Review Board of the Institute. Participant consent was not obtained as it is not required for analyzing publicly available data. We only analyzed publicly accessible Weibo posts. Users are informed that their posts are public, and those preferring privacy can set their accounts private. Furthermore, all personally identifiable information was excluded from research outputs to protect user privacy.

At the project's outset, Researcher Wu Shanyu provided comprehensive laboratory safety training, covering potential hazards and correct procedures. Professor Qi Yingchun, the biological laboratory safety officer, conducts annual in-person safety training for the iGEM team and requires passing an online safety exam. Experienced members also guide newcomers. A dedicated team member oversees daily laboratory safety. We have always adhered to the principle that safety education is integral throughout our project.

Safety Policies & Compliance

This project does not involve work with animal or human subjects, gene drives, coronaviruses, GMOs, or any sequences from the WHO list of antimicrobial-resistant pathogens. Our Human Practices work involved surveys, interviews, and educational activities, all pre-approved by our university's review committee and supervised by the PI and instructors. We strictly follow the "No-Release Policy" to prevent engineered bacteria from escaping the lab.

We strictly follow iGEM safety competition rules, having submitted all required forms before the deadline. As stated, the project involves no animal testing. All laboratory work adheres to the highest safety standards. To ensure experimental safety, mycoplasma testing is routinely performed to verify the safety of both cell materials and the laboratory environment.(See in Protocol.)