Proposed Implementation

The best project is the one that has been implemented. An idea is useless if it’s only on paper. Therefore, in our project, we want it to be not only a proof of concept but also a product which can impact people’s lives.

Our proof of concept consists of (1) the dose-dependent detection system using naringenin responsive promotor and mKATE expression as our read-out (Shoot) and (2) a temperature-based killswitch mechanism which will kill the bacteria when it is removed from our body (Leave). In the first concept, the naringenin responsive promotor and fluorescent protein mKATE will be replaced by a promotor acting upon the biomarkers and therapeutic protein, respectively. Therefore, the development of this tunable biosensor is applicable for other diseases too. However, because of the current need for the early diagnosis of colorectal cancer (CRC) in Belgium and also how we design our biosensor to be a gut bacteria, our primary envisioned application is the treatment of CRC.

More specifically, according to World Cancer Research Fund International, colorectal cancer (CRC) is one of the 3rd most prevalent diseases in the world with 89.9% five-year relative survival rate for stage I and II (World Cancer Research Fund International, 2022; Fight Colorectal Cancer, 2022). However, when diagnosed in stage III and IV, the 5-year relative survival rate are 74.3% and only 14% respectively (World Cancer Research Fund International, 2022). Since earlier stages of CRC often come without any symtpoms, the need for a non-invasive early detection tool is high. About more than 1.9 millions of people are diagnosed with CRC and over 935,000 deaths are CRC-related in all over the world in 2020 (Fight Colorectal Cancer, 2022). Rates of CRC are expected to grow, primarily due to increased screening and an aging population. However, current treatment methods are invasive and have slow recovery. Aware of this need, in this project, we developed a therapeutic biosensor which has the capacity to treat CRC without harming the environment hereafter.

End Users and Implementation

The end-users who will directly benefit from our product are colorectal cancer patients.

Currently, the primary treatment methods are surgery, radiation and chemotherapy, all of which are either invasive or non-specific, resulting in a reduction in quality of life. Biosensors, on the other hand, are an emerging alternative that can overcome these disadvantages of the current treatments. The biosensor we want to develop must satisfy three main objectives:

It must be less invasive than excisting therapy

It does not cause any major harm to the human body

It does not cause harm to the environment when removed from the body

First, there are many formulations that can be choosen for our biosensor such as implants, injections or pills. After discussions with Luc Colemont from the organization Stop Darmkanker vzw, Mathias Vissers of Netic Health and other experts on biology and pharmaceutical fields (see Human Practice for more detail), we decided to envision an oral pill for the delivery of our biosensor, a genetically engineered bacteria (GEO), to the intestine. At its destination, it will detect specific biomarkers above a certain treshold if CRC is present. This detection will result in the expression of a therapeutic protein. In application, naringenin promotor will be replaced by colorectal cancer biomarker-driven promotors in an AND gate system. Furthermore, mKATE will be replaced by a therapeutic protein allowing a targeted therapy delivery. Some of the constructs have been cloned into the E. coli K12 MG1665 but our final product will be implemented in Lactobacillus spp. This species is a natural inhabitant of our gut. Thus, the oral pill has achieved two first goals.

To complete our product and to ensure the safety of the environment (requirement three), a kill switch mechanism is included for the biocontainment of the biosensor. At the human body temperature of 37°C, the toxin is neutralized by an antitoxin. However, outside of the body, at a lower temperature, the antitoxin is inactivated, and the toxin will kill the bacteria.

In a sentence, our bacterial biosensor in form of an oral pill is a non-invasive, easy, specific, and localized treatment option with a built-in biosafety mechanism. Once developed, it can be mass produced and enter the market.

Other Applications

As stated in the previous section, our proof of concept consists of two concepts which can be used for a variety of applications.

Expansion of the treatable illnesses

Our project can act as a platform to assist in other ailments. Adapting our Shoot genes to target other ailments within the gastrointestinal system such as irritable bowel syndrome can widen the scope of our products. Moreover, replacement of the biomarker-driven promotors and the therapeutic protein can expand to the treatment of many other diseases in other organisms. Stomach cancer, for example, can possibly be treated in an analogous way to CRC, with the correct adjustments to our GEO. Another example is that it may be possible to facilitate healing of ulcers within the stomach with adjustments to our platform.

Expansion of the application fields

The genetic circuit can also be applied to other industries as well. On a basic level, our sensor detects a signal, activates gene expression, detects an environmental temperature change, then kills itself. Any one of these nodes can be altered and adapted to fit various needs. Such a fundamental platform allows for a wide variety of applications. If the plasmid is adjusted placed in an appropriate bacteria, our plasmid may for example be useful in water treatment.

Surveillance sensor

According to Massimiliano Simons, an assistant Professor in Philosophy of Technology at Maastricht University, the construct of our project could be used to act as a possible surveillance sensor. If an older patient becomes senile or gets Alzheimer’s disease, the sensor’s concentration dependency can be used to check up on patients and whether they have taken the proper medication. For this to happen, however, the sensor’s construct and the recognition molecule would need to be altered with the concentration dependency.

Safety

Safety of our users

The biosensor enters the patients’ body with the oral pill, detects the target, activates the therapeutic protein. Thus, the process does not require an invasive methods which reduces the risk of infection or slow recovery for patients. Moreover, it is specified for the targeted gene. Finally, it leaves the body through digestive system and is killed when it is in the environment.

Safety of our environment

As stated before, we have implemented a temperature dependent riboswitch that will kill the cell when it is out of the body environment. Hence, it prevents the spread of the bacteria out of control in the environment and hence, prevents contamination.

Challenges

There are many challenges that we have to take into consideration if we want to implement our biosensor. However, they are also our opportunities to improve the product.

The first challenge is the technical and biological issues which should be addressed in the future that in the scope of our project was not done; for example, the determination of the threshold of naringenin concentration to produce sufficient concentration of wildtype NarX to surpass NarX mutant (see modelling or engineering part for more detail) so that the therapeutical protein can be expressed. It is necessary for tuning the biosensor, but because of the limit of time, we couldn't obtain the exact amount. In the end, the pill needs to be developped which takes another type of research. After this, the pharmacodynamics, pharmacokinetics, efficacy and safety of the pill in human bodies should be investigated thoroughly to ensure the safety of the user. A discussion with Lien Lybaert of Persomed, who is developing a personalized vaccine against CRC raised a question: “How would our biosensor react in the body, when it is already injected with the vaccine?”

Secondly, the kill switch mechanism is dependent on temperature. In the regions in the world where the temperature reaches above this temperature threshold, it would therefore limit the effectiveness of our biocontainment method. Therefore, it may be wise to improve biosafety and biocontainment by developing a more robust kill switch. Aside from developing a more binary activation of our kill switch, a genetic circuit could be designed such that we create an upper threshold for temperatures above human body temperatures. This would therefore create a narrow band in which the organism can survive, helping to prevent spread into the environment as well as unlocking the applicability in hotter climates and seasons.

Thirdly, the biggest challenges regard regulations. Within various countries and political sectors, strict regulations (including bans) have been placed on GEO. From legal perspective, regulations vary regionally and it also takes a long time to receive government approval for a new drug or treatment. They not only cover the technical issues but also the clinical trials, production, marketing, administration, logistics, distribution and storage of the products containing these organisms. In general, the engineered products must meet biosafety and biocontainment standards.

The final challenge is how to change the negative/mixed response/attitude of the general public towards genetically altered organisms. Convincing people on the safety and efficacy of those is difficult and can take a long time. To address this problem, we conducted a survey on public awareness on the use of a bacterial biosensor to treat CRC with the targeted subjects namely cancer patients, their families and health workers. We also hold many discussions with experts in bioethical fields for clarification of the issue. A discussion with Massimiliano Simons gave us an ethical point of view on our product. According to him, cancer patients and governmental constitutions will have the most participation in the legalization and integration of the product into our lifestyle. If the biosensor is ready to use, trust has to be built with the government on one side, since it is able to stop the project at any point, and the patients on the other side, who have to take in the product. It therefore combines both of our biggest challenges and to gain trust on the both sides, we should focus on the safety and our legal compliance. This aspect is also raised in the discussion with Markus Schmidt, doctor and cofounder of the organization for international dialogue and conflict management. He suggested us to pay attention on the standardization of biosafety in synthetic biology. Taking his advice, in every step of the project, we always put biosafety and biocontainment above what we could achieve.