Safety

Before Starting the Lab Work

During the initial planning stages of our project, the whole team received a detailed safety briefing from our PI, Nicole Gensch, according to our university standards. To ensure safe working conditions, we were introduced to general safety precautions in our laboratory as well as the necessary safety measures when working with genetically modified organisms in a Biosafety Level 1 laboratory.

As our team is made up of undergraduate and graduate students with varying levels of laboratory experience, we also spend a weekend with our supervisors learning good microbial techniques, general laboratory methods such as cloning, sterile handling of bacteria and the use of standard laboratory equipment. We decided that a few selected students should be responsible for safety in the lab, which consisted of being responsible for the use of the autoclave and waste. These students are also part of the safety team to ensure that everyone knows how and where to dispose of their waste and that the lab staff follow the rules.

Lab Work

We established early on various containment measures, administrative controls, and personal protective equipment (PPE) for handling our S1 organisms, including all E. coli strains, and P. fluorescens all classified as risk group 1. The safety team ensured that all materials and waste were autoclaved, and that the access to the laboratory was possible for authorized personnel only.

For administrative controls, protocols were written before the experiments for handling, culturing, and disposal. The handling of these different bacteria was restricted to given areas, each with their own materials, such as medium, pipettes etc. to avoid cross contamination and ensure good microbial practice. Personal protective equipment, such as lab coats, safety goggles, gloves and closed-toed shoes were provided and had to be worn at all times in our facility.

To minimize the risk of spreading hazardous substances, restricted areas for working exclusively with dangerous substances, such as Acrylamide were defined [1]. For each new machine (e.g., Cabinet safety II) and each new technique (e.g., Western Blot) planned for our experiment, we were given specific instructions on how to handle the machines and materials properly.

Safety Precautions in Our Project

Bacterial Strains

In our project, we exclusively use model strains of E. coli derivatives (Top10, DH5α, BL21(DE3), Omp8) and P. fluorescens (DSM 50090), which are specifically designed for laboratory work. These strains are on the whitelist and did not require specific approval from an ethics committee or special containment. In our experiments we use P. fluorescens as a model organism, which serves as a proof of concept for our target pathogen P. aeruginosa. We chose this approach because P. aeruginosa is classified as a Risk Group 2 biological agent, which can cause disease in humans and may pose a risk to laboratory workers. By working with the safer P. fluorescens, we can develop and test our concepts without the added risks associated with P. aeruginosa. We have taken great care to ensure that none of our genetically modified organisms ever leave the laboratory. Even if this were to happen, given that all strains were developed for laboratory use, they would not survive in a natural environment.

Antimicrobial Peptides (AMPs)

In our project, we started with Sushi S1, an antimicrobial peptide (AMP) originally found in the horseshoe crab. In its native organism it functions as part of the innate immune response against bacterial invasion. The mechanism of action of Sushi S1 involves binding to bacterial membranes, causing disruption and leakage of cytosolic contents, ultimately leading to bacterial cell death. While Sushi S1 is dangerous to bacteria in its parent organism (horseshoe crab), it is not inherently dangerous to other organisms. In our project, we express Sushi S1 in bacteria and P. fluorescens. It is important to note that the peptide’s potency may differ when expressed in these organisms compared to its activity in the horseshoe crab.

We have expanded our AMP repertoire to include D-CONGA-Q7, a synthetic peptide developed by Prof. Dr. Wimley’s laboratory at Tulane University School of Medicine. This novel AMP’s function is still under investigation.

Safety Features

To ensure a specific targeting mechanism on our delivery system, we used phage tail proteins on outer membrane vesicles (OMVs) and Gb3 on LNPs and Liposomes. The use of a Pseudomonas-specific promoter prevents AMP synthesis in non-targeted organisms, such as other bacteria or mammalian cells. In addition, the short sequence of the AMPs ensures low stability once outside the cell. Indeed, studies have shown that engineered versions of Sushi S1 have low haemolytic and cytotoxic activity against mammalian cells [2]; the same is true for our second AMP, D-CONGA-Q7 [3].

Cytotoxicity Testing

We assume that both AMPs encoded on a plasmid pose only minimal risk to human lung tissue due to their specificity for target pathogens. However, we recognize the importance of rigorous safety testing. To validate this assumption and assess potential cytotoxic effects in our experimental settings, we implemented two complementary assays using A549 mammalian lung cells:

1. SYTOX Green Assay: This fluorescent probe specifically stains cells with compromised membrane integrity. By quantifying the fluorescence, we can assess the extent of membrane damage caused by our AMPs.
2. MTT Assay: This colorimetric test measures cellular metabolic activity, serving as a reliable indicator of cell viability, proliferation, and potential cytotoxicity. The assay relies on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by viable cells to form a purple formazan product.

By employing these two distinct methodologies, we aim to comprehensively evaluate the safety profile of our AMP-encoded plasmids. The SYTOX Green assay provides insight into direct membrane effects, while the MTT assay offers a broader view of cellular health and metabolism. Together, these tests allow us to rigorously assess any potential cytotoxic effects of our engineered system on human lung cells, ensuring the safety of our approach before further development.

Potential Risks and Mitigation Strategies

While our product is designed for inhalation to treat P. aeruginosa or other harmful bacteria in infected human lungs, we recognized potential risks that must be addressed.

• Promoter mutation leading to product inefficacy
• Unintended spread of plasmid components
• Immune reactions to bacterial endotoxins

1. Addressing Promoter Mutation: There is the possibility of promoter mutation rendering our product ineffective. To avoid this, we could express a restriction enzyme that cuts and degrades the plasmid ori. Additionally, implementing multiple promoters and AMPs could reduce the likelihood of complete failure due to mutations.

2. Preventing Plasmid Spread: Our liposome-based delivery system is designed to be safe and have limited potential for widespread dissemination. The primary concern for potential spread lies with the plasmid component. In the final application, the antibiotic resistance genes will be deleted fromthe plasmid in order to prevent spreading of antibiotic resistances.

3. Reducing Immunogenicity: Another potential side effect we have considered is the risk of an immune reaction. This concern arises because our OMVs may contain bacterial endotoxins, specifically lipopolysaccharide (LPS). To address this, we propose using an E. coli strain with inactivated genes encoding lipid A acyltransferase and other lipid components. This approach is based on research by Watkins et al. [4] and could significantly reduce the immunogenicity of our product.

Future Safety Considerations

Before finalizing our product, we recognized the importance of completing all clinical stages of drug development. As well as conducting double-blind tests on both patients and healthy volunteers. These steps are crucial for ensuring the safety and efficacy of our treatment in humans.

References

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