Introduction

Nanobuddy is a preventative therapeutic, administered by a genetically modified organism (GMO), that can be seeded into the lungs and possibly gastrointestinal tracts of poultry with the goal of protecting them against avian influenza, and subsequently preventing the spread of disease. Naturally this involved significant consideration of safety regulations in the design of our constructs, laboratory handling thereof, and in the future application of Nanobuddy. Early on in our project we were helped on the right path towards a project which takes all the aforementioned considerations in proper context thanks to the RIVM. They met with us to discuss various aspects such as safe-by-design and the possibility of dual-use. From that point on we have taken the safety considerations and our Key values in laboratory safety, biocontainment, conducting a safe microbiome analysis, choice of organism and therapeutic, delivery strategy, the sustainability of our solution, informed consent, data safety and construct design.

Laboratory Safety

Introduction

The ultimate goal of Nanobuddy is to have developed a genetically modified organism (GMO) that could be implemented safely into the poultry industry. For safe implementation, the safety regulations are required to be thoroughly taken into account. Our project has aimed to honour safety regulations in several different aspects in the scope of our laboratory handlings and future implementation of Nanobuddy.

General laboratory safety

While working in a microbiology laboratory, one should be aware of the biological risks accompanied with performing certain experiments. In our project, we have experimented with various organisms and chemicals. Hence, all our team members who partake in experiments were required to obtain the Safe Microbial Techniques (SMT) certificate to ensure lab safety throughout the iGEM project. A part of our team members did not yet have the SMT certificate. As a consequence, we organized a SMT course in which our team members and others could participate. The SMT course was supervised by SMT certified team members. All participants received the SMT certificate which was issued by the biological safety officer from the University of Groningen.

Figure 1. Ronald, Shree, Sander, Bindert, Harry, and the supervisors all working together on the VMT course!

Besides the fact that all our laboratory members have a SMT certificate, our lab is up-to-date to every requirement of a ML-1 type lab. A ML-1 classification is assigned to a laboratory if several rules regarding the containment of GMOs are ensured. These rules include, among other things, proper handwashing upon entering and leaving the laboratory, daily containment of work benches or areas where GMOs are handled, and autoclaving all waste or equipment in case of GMO contact. The ML-1 lab code corresponds to the biological safety level 1 (BSL1). Any organisms classified as BSL1 implies that their exposure results in no direct danger to the environment and people. During our project, we worked with different microorganisms: E.coli strains BL-21, DH5α, JH101, and NEB C3040 and the Limosilactobacillus reuteri DSM 20016 strain. These organisms are categorized as BSL-1. Therefore, all required wet lab experiments of our project could be performed in a BSL-1 laboratory provided by the Institute of Groningen Biomolecular Science and Biotechnology of the University of Groningen.

Microbiome analysis safety

For our microbiome analysis we wanted to use animal-by-products. First, we asked for permission from the iGEM safety committee. Once we had received permission from them, we approached a large scale butcher to see whether we could receive about 20 chicken lungs as an animal-by-product. Luckily, we quickly found a butcher that was happy to cooperate with us, and had the relevant registration number needed at the Netherlands Food and Consumer Product Safety Authority (NVWA). The relevant registration numbers are, from the University of Groningen, Nijenborgh 7: 35955 (Permission for production by-products: use of Category 1 material for research or diagnosis (Cat 1 UDER)); and from W. van der Meer en Zonen B.V: 5050 (Feedban registration: company of origin of animal by-products, exclusively from non-ruminants (GEN ABP - Section (C) REG)). The applicable regulations on export by-products are the European Regulation on the Implementation of By-Products (Regulation (EC) No. 1069/2009) and the Implementing Regulation (EU) No. 142/2011). In addition, there are national laws and regulations on transportation and documentation which can all be found in the Dutch Animal-By-Product policy (bill BWBR0032335).

Once we knew the registrations were in order, we devised a plan to safely conduct this research. For starters, we had to safely transport our lungs to our lab. We had contact with the biosecurity officer who provided us with all the necessary documentation which we needed to transport the lung samples from point A (the butcher) to point B (our lab). For this, no special courier was needed but we did need to fill in a special transport-form (which is basically a formality and is only used when something goes wrong during transport.

Figure 2. Mink posing before putting the box with our 20 chicken lungs into the trunk.

We transported our own lungs, using the car from Ronald (figure 2). Before leaving, we had to make sure that we were abiding by the Cat 1 risk materials transportation requirements. These were all stated by the document provided to us by the biosafety officer. After creating a suitable containment location for our lungs, we went to visit the butcher, who already had the lungs at the ready and offered to assist us in putting them in. When putting in the lungs, we wore gloves and thoroughly disinfected our hands afterwards. The lungs were then packed into 50ml Falcon tubes, which were subsequently packed in leak proof sealable bags filled with paper towels corresponding to the ‘leakage weight’ of our sample (figure 3). These bags were then put on ice in a large box. The box was marked with the relevant documentation and logos as stated by the internal university documents.

Figure 3. The safe way we transported our lung samples to the lab, as stated by the UG internal document, as well as governmental policy.

For lab safety, we decided to carry all of our steps out in a biosafety cabinet with gloves on (figure 4). This would not only prevent us from getting into contact with the chicken lungs, but this also better prevents the microbes in the air from entering our lungs. We have to minimize this as there is not a lot of bacteria living there. This type of pollution of the sample might lead to a lot of noise when working with samples that do not have that many microbes living on them.

Figure 4. Our workstation (the biosafety cabinet) for the duration of the microbiome project.

Safe-by-Design Considerations Throughout the Project

Choice of Organism:

In the search for an appropriate carrier organism, many considerations had to be taken into account. Most importantly, to prevent disruption of the local microbiome a suitable organism has to be selected from native bacteria. Literature indicates that Influenza A mainly reproduces in the respiratory and gastrointestinal tract, therefore our search started there (insert reference).

was even shown in literature to be most commonly found when compared to other bacteria present. Therefore this became one of our first choices. To solidify the findings and bring more support to the claim that L.reuteri is present in Dutch poultry, we performed a microbiome analysis which can be found on our style="color: #ffc30b;">results page. In tandem with presence other considerations must be taken into account such as: precedents of previous successful genetic engineering, effectiveness for nanobody secretion, effects on chicken health, survivability, escape frequency, amongst others.

Biocontainment:

Due to the possibility of bacteria to incorporate foreign DNA into its own genome, DNA of our engineered bacteria could enter the environment and have unforeseen consequences. To prevent this, a killing mechanism of the bacteria was designed to be repressed by two environmental factors: high temperature and the absence of light. Both of these are characteristic of the poultry lung environment (41°C and devoid of light). In order to survive, the carriers must remain in an environment fitting these conditions, very much unlike the exterior of lung poultry. Additionally, our kill-switch can be activated using a non-toxic chemical as an extra fail-safe measure.

Choice of Therapeutic:

The classical approach towards tackling viral epidemics has been with the use of vaccines ever since Edward Jenner inoculated a boy with cowpox to confer immunity. However the development of vaccines is a costly process and carries with it certain downsides such as intensive campaigns and for poultry the limitations of trade. We initially set out to confer immunity by use of an engineered lettuce, but due to the trade regulations we quickly went for another approach.

We heard about the advantages of using nanobodies due to their small size; they are easier to manufacture than antibodies, while still retaining stability and functionality. This moved us to look into the process of nanobody grafting, where a structural skeleton is used unto which amino acids are placed to create specific nanobodies. However, one of our team members quickly found a paper that wrote about a broadly neutralizing nanobody against the HPAI. Since we wanted to focus on a product that could be market ready sooner rather than later due to the increasing severity of the avian influenza pandemic. We contacted the original authors of the paper and got permission to start working with their proprietary nanobody, and the rest is history.

Delivery Strategy:

An important aspect for our project is its scalability. In order to administer our prophylactic therapy to millions of chickens, a method of “spray” dispensation was envisioned. The engineered bacteria would be delivered through a spray vaccination machine which produces small droplets that’ll enter the respiratory or intestinal track of the poultry. There, the bacteria can produce the protective nanobodies. This strategy eases the logistical challenge because the farmers only need to walk through the farm with the spray. The possibility of having a kill-switch activated when a suppressor chemical is used up or diffused enough was also looked into. We believe that this provides enough protection for the environment against the spread of our probiotic approach. Even when the users of the product inhale the aerosols due to faulty personal protective equipment or the lack thereof.

Sustainable Solution:

We envision Nanobuddy as more than just a current solution for the avian influenza epidemic. Together with our modeling project we see Nanobuddy as a platform from which multiple viral agents can be blocked from entering life stock. By making use of interchangeable parts which encode for the specific nanobody, our project could become a broader approach towards tackling viral spreads.

Once developed, the nanotherapeutic is able to copy and reproduce itself under conditions which do not trigger the biocontainment module, thereby providing a cheap and fast method of producing more product. For the generation of sequences of good nanobody binders to new viral epitopes, we’ve developed the in silico work flow. The modeling therefore would provide the project with future perspective towards utilizing it in a broader context. Of Course this all does require the current laws and legislations to become more accommodating towards the usage of GMO’s in the livestock industry. But what our survey from the human practises has shown, is that while the approach was frowned upon at the initial introduction of GMO’s, the current climate is becoming more accommodating towards novel solutions which might provide gains in comparison to old strategies.

Informed Consent:

When working safely on an iGEM project, initially lab safety comes to mind. Especially when you are working with GMOs, this usually gets the lion's share of the attention. However, in this day and age, we are convinced that data protection is equally as important. And we extensively researched and prepared data safety and informed consent to ensure the safety of all our stakeholders.

The AREA framework from past teams allowed us to responsibly gather data to go the extra mile while considering data protection and informed consent. In particular, the contribution the iGEM team of Groningen 2021 made regarding a template based on their informed consent sheet. This template is fitting for our Human Practices approach in which notes and recordings are made of the interview, a summary is written afterwards and all of the data, except for the anonymous summary, is deleted after the iGEM season.

Additionally, we checked our informed consent sheet with your supervisors, the ethics committee and the data-security office of your university.

For more information on the informed consent implementation, please visit the stakeholder engagement part of our human practices page.

Construct design

Biocontainment

In our project we aim to develop a genetically modified Lactobacillus reuteri to produce nanobodies. For eventual implementation of such a GMO various risks and regulations are needed to be taken into account. The highest concern of GMO implementation is the probability of the GMO escaping outside its confined environment. Once environmental escape has occurred, the GMO could affect ecosystems by impacting population dynamics among microbes, propagation, or gene flow [1]. As a consequence, existing micro-organisms could be suppressed by the GMO or the availability of modified genetic material could enhance other microorganisms. This would result in an alternation in biodiversity and disruption of the food chain. Disturbances in ecosystems due to GMO exposure should therefore be minimized. Furthermore, the general public expresses concern about the unknown (longterm) effects on human health GMO contact might have [2]. To address these concerns and prevent the spread of GMOs, biocontainment strategies have been researched and developed. They primarily rely on: 1) metabolic auxotrophy, in which an essential gene for a nutrient is deleted, 2) inducible control of essential genes in which deactivation has detrimental effects on cell health, 3) rewriting the genetic code of an microbe by using synthetic or xenobiological nucleotides [1]. We have developed a suitable biocontainment strategy to prevent our genetically modified Lactobacillus reuteri from escaping into the environment or affecting human health.

References

[1] Arnolds, K.L. et al. (2021) “Biotechnology for secure biocontainment designs in an emerging bioeconomy,” Current Opinion in Biotechnology, 71, pp. 25–31. Available at: https://doi.org/10.1016/j.copbio.2021.05.004.

[2] Varzakas, T. et al. (2018) “Innovative and fortified food: Probiotics, prebiotics, gmos, and Superfood,” Preparation and Processing of Religious and Cultural Foods, pp. 67–129. Available at: https://doi.org/10.1016/b978-0-08-101892-7.00006-7.