INTRODUCTION


Our project aims to develop a new alternative for obtaining CBD using synthetic biology tools. In order to achieve this goal, it is necessary to analyze various safety aspects, because in addition to the biological risks linked to the development of a GMO, we also need to pay attention to the Brazilian regulations for the production of cannabinoids in the laboratory, since we are dealing with a controlled substance in our country.



PROJECT SAFETY


DANGERS OF CANNABIDIOL


The risk of absorption of CBD through the skin or respiratory tract is low due to its low stability and bioavailability when administered orally (around 6% bioavailability), which prevents possible poisoning through accidental contact during laboratory experiments (Millar et al., 2020). Knowing these risks, even if they are low, we used PPE such as gloves, goggles and lab coats throughout the experiments.


BUREAUCRATIC PROVISIONS ON CBD PRODUCTION IN BRAZIL


Cannabidiol (CBD) is a substance controlled by government agencies because it is a derivative of the Cannabis sativa plant, which is illegal in Brazil. Throughout the development of the project, we have researched the legal implications of producing CBD in the laboratory from a GMO, and during our research, we have not found any legal impediments to the realization of the CBDynamics project. This is possible because we are not going to use the plant in any part of our experiments, as this is the main legislative bottleneck. In addition, we are also not going to commercialize or carry out human trials with the CBD produced, as for this we would have to go through a series of processes and authorizations from our country's regulatory bodies such as the National Health Surveillance Agency (ANVISA), the Ministry of Agriculture and Livestock (MAPA) and the National Biosafety Technical Commission (CTNbio) and research ethics committees. Finally, it is also important to mention that as a safety and containment measure, we will destroy everything produced in the laboratory during the experiments.

Brazilian legislation, like that of the rest of the world, is still working on and perfecting the regulation of GMOs, as this is a relatively new technology, which is why we don't yet have specific regulations for cannabinoids that come from recombinant DNA technology, as is the case with CBDyanamics. For this reason, we at the iGEM USP-EEL-BRAZIL team have developed a bill for this specific case of CBD production through genetically modified organisms, so we take a big step forward for future companies wishing to bring in this new approach to cannabinoid production without having to worry about lengthy bureaucratic processes, and we also make it easier for Brazilian society to access this compound.



USE OF GMO CBD


LABORATORY SAFETY MEASURES


  1. General laboratory arrangements

    2. The experiments for the CBDynamics project were carried out in the Synthetic and Molecular Biology Laboratory (SyMBLab) (biosafety level NB2) (Level 2 - moderate containment). All the members present in the laboratory underwent training with our instructor Júlia Ortega in good laboratory practices, as well as using PPE and CPE during all the activities. In addition, when carrying out experiments, we always work with a flowchart to guide the activities and avoid possible risks.


    Figure 1: flowchart of laboratory experiments

    Figure 1: flowchart of laboratory experiments

    Source: Author’s (2024)

  2. GMO risk assessment in Brazil

    3. Activities with genetically modified organisms (GMOs) are widely regulated to avoid any kind of environmental and/or human risk.

    The National Biosafety Council (CNBS) and the National Technical Biosafety Commission (CTNBio) are the government bodies responsible for overseeing and regulating activities involving GMOs. In accordance with the provisions of Law No. 11.105 of March 24, 2005, this implies the presence of various pieces of equipment for biological containment, such as laminar flow cabinets, autoclaves and self-closing lockable doors. All this equipment is present in the laboratory where we work and we are always instructed on good laboratory practices to avoid the risk of contamination.


  3. Biological risk assessment in CBDyamics

    4. All our experiments were carried out with the bacterium Escherichia coli NEBturbo and the yeast Saccharomyces cerevisiae SC9721. According to the legal provisions mentioned above, the species we worked with have risk level 1, as do the parts we worked on to build the circuits and vectors used for propagation and expression. In this way, both the bacteria and the genetically modified yeast would be considered risk level 1, since we are not working with any potentially pathogenic organisms or parts that generate any toxicity.


  4. Containment strategies

    5. We are not using a killswitch in our circuits, but our expression system requires very specific conditions for our yeast to develop, and also for it to express the enzymes of the metabolic pathway and produce CBDA. This is due to its auxotrophy marks, and also to the need for hexanoic acid supplementation for functional and expressive CBD production, to do this, we developed a kinetic model. In addition, all our experiments must be carried out in a biosafety 2 laboratory, with all the appropriate PPE, biosafety protocols and cleaning processes, such as thermal inactivation and sterilization, and good microbiology and molecular biology practices. In addition, all our constructs have been optimized for yeast cells, which would not lead to possible risks for other organisms, since the translation step of protein synthesis would be greatly affected. All of these strategies together guarantee good microbial containment and so we avoid as far as possible any leakage of organisms or parts outside the laboratory environment.


  5. Biosafety and inclusion

    6. In addition to all the precautions mentioned above, our team paid special attention to the importance of biosafety in the laboratory, developing inclusive solutions for people with low vision. We created QR codes that provide descriptions of common and dangerous compounds present in the environment, to ensure that everyone has access to critical information in an accessible way. These QR codes direct you to a document in high contrast, with appropriate lettering for easy reading, and we offer the same description in audio format for each reagent. This project was designed to promote the safety and autonomy of visually impaired people, reaffirming our commitment to biosafety for everyone in the laboratory.