Team:BOKU-Vienna/Implementation
Outlook & Implementation
Friendzyme could be a daily treatment for people suffering from irritable bowel syndrome, non-celiac gluten sensitivity, or similar disorders where fructans play an essential role. These patients usually suffer from symptoms like diarrhea, bloating, and abdominal pain when eating food that contains fructans and other FODMAPs (fermentable oligo-, di-, monosaccharides, and polyols). Thus, going to restaurants or cafés is difficult as many types of food contain FODMAPs. For individuals suffering from such disorders, Friendzyme could be the solution for their food sensitivity-related problems.
Three Shades of Friendzyme
We imagine Friendzyme to be a daily treatment consisting of three layers:
First: Engineered Lactobacillus cells, which continually produce the enzymes needed for the degradation of fructans. Lactobacillus in general is classified as a GRAS (generally recognized as safe) organism and is even known to have probiotic effects. However, as we aim to use a genetically modified version, it will also feature a kill-switch as an additional biosafety measure. This kill-switch shall prevent the modified Lactobacillus from spreading inside the body or into the environment.
Second: The cells will be encapsulated in a biocontainer, which as the name suggests, contains our Lactobacillus and stops the bacteria from escaping while still allowing the flow of smaller particles, like secreted enzymes and nutrients. It also improves the pharmacokinetics of our drug as it has been shown to protect encapsulated cells from the low pH inside of the stomach.(1)
Furthermore, the thiolated surface regions of the biocontainer adheres to mucosal membranes through disulfide bonds. Thereby, we overcome one of the main problems of enzyme therapy in recent years, which is the lack of tissue specificity.(2) The small intestine is the first mucosal membrane after passing the stomach, and right where we want to degrade fructans before other microorganisms get a chance to do so.
Third: The idea is to make Friendzyme available as tablets to prevent unwanted mucoadhesion through unspecific binding to cysteine residues on the mucosa surface before entering the gut, e.g., inside the mouth or esophagus. This makes the tablet, which typically consists of a binder, antiadhesives, colorants, fillers, and a coating surrounding them, our third layer. It further protects the bacteria from gastric acid and makes the administration easier for the patient easier by providing increased and standardized size, and inhibiting unwanted odor and taste.
Friendzyme – a versatile delivery platform
The usage of our Friendzyme delivery platform is not limited to fructan degradation, nor to the digestive system. The concept of bacteria that secrete active ingredients inside a biocontainer that can attach to mucosal membranes offers a plethora of possibilities, as conversations with Prof. Andreas Bernkop-Schürch, one of the main experts in the field of thiolated polymers, confirmed. From our mouth, over our digestive system, and to the anal, vaginal, and penile cavity, all over our body, we have mucous membranes which secret protective fluids and represent the primary barrier between the external world and the interior of the body. Especially cavities lined by a mucous membrane with a slow turnover represent a promising target as they would benefit most from the prolonged API (Active pharmaceutical ingredient) release of a living cell-based system. Another criterion is accessibility. Some body cavities are more accessible than others, e.g., the anal cavity can be reached via a simple suppository, while the urethra might require more invasive methods.
One prime example is the vaginal mucosa. Its turnover is one of the slowest and it is relatively easy to access. A possible treatment here would be the use of spermicides as contraceptives. Most long-lasting forms of birth control are hormone-based, which comes with all kinds of drawbacks and side effects or the requirement of an invasive procedure. Continuously delivered spermicides via the Friendzyme delivery platform would work via a non-hormonal suppository that needs no surgery.
Bioethical and biosafety considerations
While our new approach on enzyme therapy has the potential to bring long-needed improvement compared to classical methods, it also comes with a number of aspects, that bear the potential of becoming serious biosafety issues.
First and foremost, that includes the use of live GMOs in human therapy, which is often seen critically by authorities and the general public. To our knowledge, there are no medicinal products containing genetically engineered organisms on the market so far.
To address these issues and get an idea of what our chances to actually bring Friendzyme towards implementation are, we, together with our collaboration partner iGEM TU-Eindhoven talked to Prof. Johannes Rath, an expert on bioethics and biosafety. He has a strong background in toxicology, microbiology, and law and he worked for the United Nations and the European Commission, where he was an active member of a biosafety committee. Currently, he is working in the Netherlands and shares his knowledge about research ethics in a lecture series at the University in Vienna.
He gave us valuable insights into the bioethical implications of our project setup and which steps would be needed to be completed before Friendzyme could go into the process of approval and commercialization.
He also pointed us towards the fact, that for certain somatic gene therapies, some of which are currently undergoing phase II clinical trials, also genetically engineered cells are used. This made us feel positive, that an approach like ours that uses genetically engineered bacteria, could also gain a broader acceptance and become a standard treatment one day. Classical enzyme therapy products are much simpler to get to the market due to the considerably lower number of potential safety issues compared to live cell-applications. Nevertheless, if the methods brings significant benefits and thereby shifting the risk-benefit ratio, using a set-up like ours could become an option.
However, we would need to prove that our drug has advantages compared to what is considered the current standard of care (the best currently available treatment for the condition). In our case, since no treatments for IBS are available yet, we would have to show the effect compared to a placebo instead.
The performance of a new medicinal product is not only a question of the benefits but also of the amount of risk the method involves. According to Prof. Rath it could still be the case that the sum of risks is too high, and the product does not get approved, even if the benefits are very strong and outweigh the risks.
In our case three different types of risk assessment need to be carried out:
Safety in relation to the Human Subject
This includes adverse effects and risks for the patient. We planned to take the first steps in this direction by using a human gut-on-a-chip model system and testing our capsules' effect on the human in vitro system. Unfortunately, due to time reasons, we did not manage to conduct a full screening at the end. However, Prof. Rath confirmed to us that this would be a sensible first step in also following the 3R principles of animal testing (3) that state that all possibilities of in vitro testing should be taken before going into pre-clinical in vivo animal testing.
Safety assessment in relation to possible horizontal gene transfer in the gut
The possibility of propagation of engineered genetic material must be eliminated. Since our team planned the stable genomic integration of the constructs in the final Friendzyme version, horizontal gene transfer via plasmids can be avoided.
Environmental risk assessment
Another thing that must be avoided at all costs is the uncontrolled propagation of GMOs in the environment. This would probably also be one of the main concerns of the general public regarding our project. Naturally, towards the end of the envisioned product life cycle, the GMOs will be expelled together with fecal matter. Under unfortunate circumstances, the feces could get into contact or be ingested by animals and the GMOs potentially proliferate further.
In conclusion, safety considerations need to be a key element in the development of our system. While being aware of that from the start, biosafety became an integral part of our project set-up. Hence, we included a biocontainer, an envisioned kill-switch, and natural auxotrophies of the Lactobacillus into our project. We planned these measures to ensure that the GMOs we intend to use are unable to escape into the environment and, even if they do, would not be able to survive (see wiki page Safety). Of course, it would need to be proven that all planned safety levels are indeed effective. However, the final decision upon the acceptable risks naturally lies within the respective authority.
In general, only a very low percentage of developed future drugs make it to the market. Since chances for drug approval are rather low, it is very important to carry out tests and assessments logically to avoid spending a lot of time and resources on an approach that does not have a realistic chance of ever getting implemented.
In that regard, more data regarding the scaffolds, the enzyme secretion, and the mucoadhesion is vital. Based on that, initial tests on biocompatibility and adverse effects would need to be done by using e.g., a gut-on-a-chip system, like we planned to do, and then proceed with the in vivo evaluation using small animals like mice or rats used in standard translational medicine if prior results look promising.
Only when this supporting data has been obtained and paints an overall very positive picture, it will make sense to seek approval in accordance with the responsible authorities' regulatory framework. In our case, this will be the European Medical Agency (EMA) and the Austrian Federal Office for Safety and Health Care (BASG).
However, the path to implement a product like Friendzyme would definitely be a time- and cost-intensive one and the chances of failing the process at some point are rather high.
Biohacking
We also asked Prof. Rath about the potential threat of biohacking and dual use in the context of treatments involving GMOs. He informed us that one of the main concerns in this context is the safety of patient data that sooner or later would have to be generated during the implementation process. This is a topic that should not be left out when making safety considerations regarding our project.
References
(1) Fonseca, F., Cenard, S., & Passot, S. (2015). Freeze-drying of lactic acid bacteria. Methods in molecular biology (Clifton, N.J.), 1257, 477–488. https://doi.org/10.1007/978-1-4939-2193-5_24
(2) De la Fuente, M., Lombardero, L., Gómez-González, A., Solari, C., Angulo-Barturen, I., Acera, A., Vecino, E., Astigarraga, E., and Barreda-Gómez, G. (2021). Enzyme Therapy: Current Challenges and Future Perspectives. International Journal of Molecular Sciences 22, 9181. https://doi.org/10.3390/ijms22179181
(3) Wikipedia. (2021). Directive 2010/63/EU (Wikimedia Foundation Inc.).