Safety

Safety in the lab

Safety is a prerequisite for any research project implemented either in academia or in industry. ETH Zurich values the importance of safety for the research being conducted in the institute. For this reason, an internal Department of Safety, Security, Health and Environment is organized to provide all the necessary information to the students and employees of ETH Zurich regarding safety. Brochures, lectures and podcasts are also used in order to make the personnel aware of the safety rules and to enhance accident prevention.

Specifically, our team attended a Facility and Lab Safety instruction before starting any experimental work. The lecture was held by Mr. Niels Buerckert, who is the Technical Manager and head of the D-BSSE Safety Committee. The topics covered safety practices and regulations in the lab. In a separate course, two of our team members had the opportunity to get practical experience on the usage of a fire blankets and a fire-extinguishers, putting out a real fire. As far as the biosafety measures are concerned, all iGEM team members got familiar with the safety practices for BSL- 1 and BSL-2 laboratories. Some of the topics covered were:

proper clothing (lab coat for BSL-1/2) and accessory equipment (gloves and goggles)
proper disposal of BSL-1 and BSL-2 waste
handling of liquid waste containing heat resistant antibiotics (e.g. kanamycin)
handling of hazardous chemicals
actions in case of chemical or biological contamination

Safety of our current project design

The description of our iGEM project was sent to the Swiss Department of Health (BAG) in order to assess the safety risk of the biological agents used in our project. The agency characterized our project as compatible with the Swiss regulations.

In our project, we implemented proof of concept experiments to test the functionality of our engineered bacteria in a controlled laboratory environment, simultaneously emulating the real–application conditions. According to the literature, multiple bacteria are suitable for tumor colonization. Escherichia coli Nissle 1917 was the bacterial strain we planned to equip with our synthetic circuit. Our choice was based on both the strain's safety (probiotic strain) and its ability to colonize tumors within the patient. [1][2]

Other E. coli strains, such as E. coli DH5α, E. coli TOP 10, were used in cloning experiments. All of the organisms mentioned here are used in biology research on a daily basis and are characterized as BSL-1 organisms. However, we are working with GMO strains which cannot be released into the environment and low risk does not equal no risk. For this reason, we stuck to all the safety regulations for biosafety level 1 laboratory. During our experiments, there was no intravenous administration of the engineered bacteria to any living organism, as our final application goal would suggest.

Furthermore, in order to see the effect of our Anti-Cancer Toxin (either produced or purified azurin), several cancer mammalian cell lines were also used, namely HeLa CCL-2, HEK293, HCT-116, HT-29. All these cell lines are characterized as BSL-1 strains according to the Swiss regulations. However, our laboratory is equipped with a separate BSL-2 space, where all the experiments using mammalian cells took place. We started working in this lab only after receiving a BSL-2 laboratory training.

The anti-cancer peptide, azurin, was kindly provided in a purified form by the Freiburg iGEM Team. Azurin is a non-virulence factor involved in electron transfer during denitrification in Pseudomonas aureginosa. [3] This strain should be handled in a BSL-2 lab, however in our work we did use this strain, but we ordered the gene sequence of azurin as gBlock in order to clone it into E. coli Nissle 1917 strain. As long as azurin is not a virulence factor of P. aureginosa we were allowed to use it in a BSL-1 laboratory.

Notably, there are actually already bacterial treatments approved and administered in Switzerland, such as Bacillus Calmette-Guérin (BCG), acting against bladder cancer. [4] At the same time, there are also U.S. companies that have taken approval to conduct Phase II clinical trials. All of these examples show that our designed bacterial therapy could result in a safe, effective and realistic anti-cancer treatment.

Expert’s opinion regarding the safety of our project

The opinion of experts on the field of bacterial cancer therapies was a very valuable input during the designing of our treatment. For this reason, we came in contact with T3-pharmaceuticals, a spin-off company that is currently developing novel bacterial anti-cancer treatments exploiting the powerful bacterial T3 secretion system, to deliver their anti-cancer payload to the cells. [5] According to Dr. Simon J. Ittig, CEO of T3-pharmaceuticals, people suffering from advanced stages of cancer are willing to take the risks that come along with a bacterial cancer treatment, especially because the potential side-effects are so minimal in comparison with other treatment options.

On the other hand, in our correspondence with Prof. Polšek, an expert in bioethics, we were encouraged to think about this from a different perspective and to always keep in mind that "Despair is not the viable justification for granting a judicious informed consent".

Safety of our final project design

The final goal of our project is to engineer E. coli Nissle 1917 to specifically deliver an anti-cancer peptide against malignant tumors. The administration of the treatment would be done by trained medical personnel, in a hospital environment. There are several safety risks, which we need to consider in the design of our genetic circuit and the application of the treatment.

Risk of sepsis

The treatment starts by intravenous administration of our engineered bacteria. The bacterial dose administered should be high enough to allow the bacteria to initially survive the human immune response, and allow colonization to take place. However, high concentrations of bacteria in the bloodstream can induce acute response of the immune system leading to sepsis and finally to a septic shock. For this reason, thorough investigation of the bacterial treatment administration is mandatory, in order to determine the dosage that can be administered without compromising the patient’s health. Important to mention, however, is that even in the worst case of bacteria initiating a septic response, there is the possibility to administer antibiotics at any point during the treatment, as soon as any suspicious symptoms are present (e.g. fever).

Risk of off-target colonization

After administration, the bacteria will take advantage of their inherent ability to specifically colonize tumors, as they provide the immunosuppressed and nutrient-rich microenvironment necessary for bacterial growth. However, bacterial colonization in healthy tissues (off-target) is still a possibility, even though literature data support that any off-target colonization is cleared-out by the immune system. {2} In order to address this potential safety risk, intracellular accumulation of the Anti-Cancer Toxin azurin and the production of an MRI Contrast Agentbacterioferritin is activated only once the bacterial population threshold is reached and lactate, a tumor marker, is present (see Tumor Sensor). Additionally, azurin is being released only if the physician confirms the co-localization of the bacteria (via MRI) and the tumor. This release is taking place via a thermo-inducible Cell Lysis, activated by focused ultrasounds (see Heat Sensor) during MRI imaging.

Risk of horizontal gene transfer

As long as our engineered microbes are accumulating into immunosuppressed areas (tumor environment), other bacteria might also reside in these areas. In that way, horizontal gene transfer could potentially generate antibiotic resistant bacteria that can further lead to patient infection. In order to minimize such a risk, we would have to clone our system into a chromosome to eliminate the need for antibiotic resistance. Another option would be to use metabolic complementation instead of antibiotic selection for plasmid maintenance.

Risk of the release in the environment

After the treatment, the remainings of our bacteria are going to be cleared out from the patient’s system. In that way, the patient’s secretions might still contain some DNA fragments, or even living bacteria.

One way to approach this problem is to use a procedure in the clinics similar to what is routinely done in nuclear medicine, where patients treated with radioactive substances remain in the treatment facility for an appropriate amount of time needed to completely clear out the dangerous substances from their body. During this time, all secretions, even items that the patients come in contact with, are carefully collected and disposed of according to regulations. [6]

On the other hand, a knock-out strain could be created which would rely on a metabolite that cannot be found freely in the environment, but would be supplemented during the treatment.

References