Revision as of 00:55, 14 October 2017

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 on the research being conducted in the institute. For this reason, an internal department of Safety, Security, Health and Environment exists providing all the necessary information to the ETH students and employees, regarding their actions in case of an emergency. Safety brochures, lectures and podcasts are also used in order to make the personnel aware of the safety rules, enhancing 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 contained 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 blanket and a fire-extinguisher, 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

Finally, a description of our iGEM project was also 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.

Safety of our current project design

In our iGEM 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. There were multiple bacteria suitable for tumor colonization according to the literature. Escherichia coli Nissle 1917 was the bacterial strain equipped 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. Other E. coli strains, such as E. coli DH5-α, E. coli TOP 10, were used in cloning experiments. All these organisms are commonly 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 zero risk. For this reason, we stick to all safety regulations for biosafety level 1 laboratory. During our experiments, there was no intravenous administration of the engineered bacteria to any living organism.

Furthermore, in order to see the effect of our anti-cancer treatment (either produced or purified azurin), several cancer mammalian cell lines were also used, namely HeLa, HEK, 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 proper 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. This strain should be handled in a BSL-2 lab, however in our work we did not work with this strain, but we ordered the gene sequence of azurin as g-Block in order to clone it to our 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. However, all the experiments conducted with this anti-cancer peptide took place in a BSL-2 laboratory, for increased safety.

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. According to Dr. Simon J. Ittig, 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. Interestingly, the administered azurin to the tumor proliferates within the system, as long as we end up with more bacteria than we injected initially. This is a big safety advantage over the classical anti-cancer drugs, where high drug dosages lead to toxic off-target effects. Finally, there are bacterial treatments already approved and administered in Switzerland, such as Bacillus Calmette-Guérin (BCG) acting against bladder cancer. 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.

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 survive from 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.

Risk of off-target colonization

After administration, the bacteria will take advantage of their inherent ability to specifically colonize the patient’s tumors, as they provide the immunosuppressed and nutrient-rich microenvironment necessary for bacterial growth. However, bacterial colonization on 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. In order to address this potential safety risk, intracellular accumulation of the cytotoxin azurin and the MRI contrast agent bacterioferritin is activated only once a bacterial population threshold is reached and lactate, a tumor marker, is present. Additionally, azurin is being released only if the physician confirms the colocalization of the bacteria (via MRI) and the tumor. This release is taking place via a thermo-inducible cell lysis, activated by focused ultrasounds during MRI imaging. Finally, administration of antibiotics would immediately clear out the treatment out of the patient’s system, in the case of any adverse effects (e.g. fever etc.).

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 treatment (heat induced cell lysis), 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. The release of such bacteria to the environment can pose a risk, as the E. coli Nissle 1917, due to its probiotic nature, can be transferred to other species exposed to it. In that way, both the horizontal gene transfer risk and the risk of E. coli Nissle 1917 spreading amongst species increases. For this reason, a knock-out strain should be created which would rely on a metabolite provided externally during the treatment but cannot be found freely in the environment.

References

Stritzker, Jochen, et al. Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. International journal of medical microbiology 297.3 (2007): 151-162.
Stritzker, Jochen, et al. Enterobacterial tumor colonization in mice depends on bacterial metabolism and macrophages but is independent of chemotaxis and motility. International Journal of Medical Microbiology 300.7 (2010): 449-456.
Fuge, Oliver et al. Immunotherapy for Bladder Cancer. Research and Reports in Urology 7 (2015): 65–79. PMC. Web. 15 Sept. 2017.