Team:UNILausanne/IntegratedHP

Integrated Human Practices

Biosafety

Context

The iGEM entity tries to ensure biosafety by closely regulating teams with multiple layers of security: from a Team Safety Form at the beginning as well as towards at the end of the project, to Animal Use Form that would need more oversight, and any problem is screened-out by a Security Committee. Rules on biosafety are not the same in all the countries, so iGEM has to impose some rules to make the playing field of the competition as equal as possible for all teams.

Working in the field of synthetic biology, a question that needs to take priority is how biosafety will be ensured throughout the entirety of the project. It is a domain that, if unregulated, could pose severe risks to the environment and human health. As our project consists on the introduction of a modified organism into the human body to secrete a drug; the possibility of unwanted adverse effects on the health of the patient raises some big biosafety issues that need to be addressed. A strict control to ensure the safety of the patient needs to be put in place to prevent any problems, noting however, that we have chosen a non-pathogenic probiotic bacteria that is known to harmlessly live in the human gut, therefore we would not be introducing a dangerous microorganism into the body or the environment.

How it influenced our project

Public opinion on GMOs

When doing the GMO survey (see GMO survey) some concerns that arose between the participants were on how GMO containment to prevent them from escaping into the environment. Here are some of the questions regarding containment and safety in the survey:

Figure 1. Answers to question 5 of the GMO survey

The participants are very divided on their opinions on how medical GMOs could impact the environment. The majority of replies (20%) selected the answer "5", so the participants did not have a clear-cut inclination. However later on in the survey, when asked why some people might have some caution when dealing with GMO's, the aspect of propagation into the environment was often brought up. So this got us thinking into how we could ensure that our bacteria wouldn't propagate once it got out of the body.

To address these concerns we created a safety mechanism called kill switch that could solve the containment issue that inherently comes with a project such as ours.

A self-destruct emergency button: kill switch

To prevent the propagation of our GMO into the environment or into areas of the body that are not intended for our bacteria, we have designed a kill switch for our vector, to assure the death of the organism in any environment that is not the tumoural area of the colon (see kill switch).

Table 1: Conditions of survival of bacteria with kill switch circuit
 

Colon

Blood circulation

Phosphate pill to colon

Outside body

Toxin Concentration

Low

Low

Low

High

Antitoxin Concentration

High

Low

Low

Low

B.O.T

ALIVE

DEAD

DEAD

DEAD

The kill switch is a biosafety mechanism that assures that the bacteria will not propagate outside the areas of intended use (like in the blood or even outside of the body). It is based on the balance of toxin and antitoxin production inside of the bacteria. If the environment properties are different than the ones in the expected area of action, there is a stop to the anti-toxin and the bacteria effectively dies. It also ensures a plan B, in case the system gets deregulated inside the colon: if the patient takes a phosphate pill, the kill switch system will be activated effectively killing the bacteria. This mechanism gives an extra layer of security containment that would make our product even safer for human use.

Expert Advice

Tobias Vornholt is a PhD student, experienced in working in the synthetic biology field and was the iGEM advisor for ETH Zurich 2018 and 2019 as well as a iGEM member of Team Bielefeld-CeBiTec in 2015. We interviewed him to get some feedback on our safety system. He explained that using bacteria as a therapeutic vector comes inherently with multiple difficulties in its efficacy and safety, as working with a living organism can be unpredictable. We need to ensure first off that the bacteria are able to maintain a normal activity for an extended period of time in a variable environment (such as the digestive track). There is always the risk of adverse effect in the patient through unpredicted modifications to the native microbiome or potentials of horizontal gene transfers (creating antibiotic resistances for example). And finally we also need to ensure that the organisms does not proliferate outside the human body. However, as we are using a probiotic, the escape of such an organism should be of little concern depending on how we engineer it.

He advised the use of a combination of multiple independent safety organisms to be able to limit proliferation inside and outside the body; we could inactivate the pathogenic characteristics of the bacteria, give it an evolutionary disadvantage, and even physically contain it (like in a capsule for example). In regards to the kill switch, he asserted that it was a good starting point for the containment, but that it wouldn't be enough. As this system depends on only two parameters (temperature and phosphate) it is quite risky. The kill switch should then be tested for the frequency of escape mutations. It would be better if these mechanisms depended on sensing several variables to reduce the chance of the bacteria finding a favourable environment.

Legal Safety Considerations

Our product it's at its core a genetically modified organism, and therefore quite controversial in the public eye. In the European Union, clinical trials using GMOs require three levels of review, done by independent national agencies[1] , Our bacteria would have to adhere to the Cartagena Protocol on Biosafety, "an internationalagreement which aims to ensure the safe handling, transport and use of living modified organisms (LMOs)[2] ". We are creating a product that would befall under the class "Deliberate Release" (in opposition to the "Contained Use" class) of the EU GMO framework, as the bacteria after have gone through the digestive track it would then be released upon the environment (through the sewage system). We would need to prove with convincing evidence that this release would not be harmful to the environment to obtain permission to go forward with our clinical trials.

In conclusion, a GMO combined with a chronotherapy approach comes with a lot more challenges, a security consideration that we thought at the beginning of this project. However, scientific rigor and a good team can help us go far and develop B.O.T to its maximum potential.

Gender Disparities in Scientific Data

Context

Historically, women have been excluded from research studies when studying diseases that are prevalent in both sexes [3]. In recent decades, it has been acknowledged that clinical and animal trials have not correctly analyzed sex-differences in data for drug interactions in the body. These information gaps put women in unnecessary danger when getting treatment for diseases that have only been studied in men.

How it influenced our project

In our project, we are studying a treatment that is based on timing azurin release with the circadian cycle for treating CRC; a system based on chronotherapy, a topic that is still heavily controversial in the medical field. If we obtain promising results, it could reinforce the use of chronotherapy as an effective tool against pathologies and a step into the direction of more effective medicine. As of now, the number of chronotherapy studies have been few and far in between, with the results being heavily debated.

Most of the data that exist on the circadian cycle comes from studies on men, and in recent years, it has been shown that the circadian cycle characteristics differ on men and women [4]. To obtain this data, we would need to search in the literature or [5] conduct the experiments ourselves, as it is an essential piece of information needed to give adequate treatment. We could further develop this chronotherapy aspect with more tests, to assess if giving drugs at certain time points of the circadian cycle helps fight diseases in a more efficient manner, and establish a more robust fundamental knowledge on the subject.

Modeling

After obtaining different data when the optimal time for the drug release would be we ideal for each sex, we could use our in silico modeling to know what variables could  be changed in the lab to obtain different oscillation periods adapted to each sex.

Figure 1. Simulations showing how to change the period of the oscillations. β is the mRNA lifetime divided by protein lifetime. Higher values of β give shorter periods.

With an ODE model of the repressilator, we showed that the ratio of mRNA lifetime to protein life-time (referred as β in our model) is a key parameter to modulate the amplitude of the oscillations. As we see in figure 1, β is inversely proportional to the period of the oscillations. For instance, if we focus on the protein lifetime, the longer it is, the greater the period of the oscillations. Hence to modify the period, we could take advantage of the synthetic protein degradation system in E.coli recently developed [5]. Indeed, previous iGEM teams have already made use of protein degradation tags [6]. By coupling these degradation tags to the proteins in the repressilator we could potentially reduce the period of the oscillations.

Our system does not naturally have any protein degradation tag that we could remove, so increasing the period of the oscillations by extending protein lifetime would be challenging. We attempted to develop a completely different approach based on a recent publication about the effect of space within the cell in genetic circuits [7]. We hypothesized that cloning the three genes in three separated spots in the chromosome of E.coli could increase the period of the oscillations, as the time of a transcription factor needs to diffuse within the cell to find the next gene would increase, hence delaying the system. However, a video call with the first author of the publication (Ruud Stoof) made us realize that would not be efficient, as the time we could gain would be in the range of minutes and could introduce some noise.

Our individual-based model (see Modeling) finally helped us find the solution on how to increase the period of the oscillations: by reducing cell growth rate. For example, by reducing the probability of cell division at each time step (which is equivalent to reducing the growth rate) we can increase the period of the oscillations (Fig 2). The growth rate could be changed due to the environment (e.g. recommend a diet to the patient that promotes slower bacterial growth in the gut) or genetic modifications in our bacteria.

Figure 2.Simulations with our individual based model of the repressilator in one dimension. Reducing the division probability and thus the growth rate, would increase the period of the oscillations.

Women in STEM

Finally, women are not only under-represented on scientific data, but in the scientific domain as well. The problem has to be solved from the inside-out, with more women directing the studies, perhaps the sex-bias will be decreased. To see how different people feel about this issue and maybe how it could be solved we conducted some interviews. Moreover, we talked a lot with Christine Sempoux, President of the Commission for the Promotion of Women of the University of Lausanne. She explained to us how important it was to include the female gender in scientific studies and gave us several examples of problems related to this under-representation. (see Women in STEM).

References (click to see)

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