1. Researcher, public and environmental safety issues

The iGEM team 2008 Slovenia addressed the issues of biological safety concerning this year’s project at all phases from the design of the project, through experimental work and further impact of our results on future research activities. Furthermore, the public and the environmental safety were considered.

In the first instance, we performed a self-evaluation of the proposed project and predicted potential safety risks to the researchers, public health and environmental. Based on this self-evaluation, the project team was divided into several sub-groups of students whose work was closely supervised by a senior mentor with required license for particular procedure. Such organization of work proved to be more efficient regarding biosafety. Before the onset of the laboratory work, all team members participated in a course on Occupational Health and Fire safety and pass the nationally recognized exam. Additionally, a basic training on reacting in emergency situations for students was organized.

2. Local and national biosafety committees and novel potential safety issues associated with Biobricks

The project proposal and the self-evaluation of the biosafety issues were reviewed by the Department’s biological safety officer. Mainly, the officer’s task is to asses if the mentors are qualified to supervise the proposed research and that the laboratories hold the nationally required licenses to host the research work proposed. National Institute of Chemistry holds a license to work at the biosafety level II with some microorganisms and cell lines. In addition, the laboratory holds the license to work with the genetically modified organisms and is in the process of obtaining GMO level II licence. The local biosafety officer also evaluated new risk assessment forms for all the new constructs and BioBrick parts and decided whether they present an increased safety risk beyond the current license level of the laboratory.

Slovenia, as the European Union member state, has adopted and harmonized legislation concerning biosafety with EU directives. The proposed research was evaluated by the national committee for biosafety. Furthermore, the experiments involving laboratory mice had to be approved by the Veterinary Administration of the Republic Slovenia and the Ethical committee for the use of laboratory animals in research.

During the experimental work, the laboratory health and safety monitoring was in place and found no incidences of safety risks associated with the project. During animal experiment, animals were inspected on a daily basis for behavioral or visual abnormalities and none were detected. All mice were weighed regularly and none were found to have lost weight suggesting that their health status was not compromised. Groups of animals subjected to vaccination with DNA constructs were closely monitored and no local infection or inflammation was detected at the area of DNA vaccine application.

3. Potential applicability of the project and potential safety issues if applied in humans

Safety risks were also considered a top priority when evaluating potential applications of the project outcome in the future, i.e., developing efficient vaccines to treat H. pylori or other infections in the human using our synthetic biology approach.

For this reason, we chose delivery routes of our immunobrick-vaccines that have potentially the least safety risk associated with it. For example instead of using invasive strains of E. coli to deliver our recombinant vaccines we used a non-invasive strain with mutated flagellin that is risk-wise similar to commensal gut bacteria. In addition, we also employed so-called bacterial ghosts – plasmids in bacteria that are activated at the temperature of 42 C to kill bacteria while the antigens expressed on the surface of the bacteria are still available to elicit host's immune response. For the delivery of DNA vaccines in the mouse, we applied a procedure by electroporation and injection into the muscle or subdermis, which has been previously evaluated in humans and many animals and showed no serious adverse effects. Some adverse effects could include muscle contraction and local pain, which is usually not severe.

We have addressed also an important issue of possible integration of DNA into the recipient host genome leading to mutagenesis and possibly carcinogenesis. Numerous animal studies have now shown that such integration events are extremely infrequent, about 3000 times lower than the spontaneous mutation rate for mammalian genomes. Our GFP monitoring of the construct expression in the muscle of live mice by non-invasive optical imaging showed that expression of our constructs was indeed transient resulting in barely detectable GFP expression already after 7 days of injection. We therefore regard our DNA designer vaccine delivered by electroporation as a safe procedure that might in future be safely used for human trials.

In conclusion, the self-evaluation of risk assessment of the proposed project, the local biosafety officer’s review of the proposal and the relevant national biosafety committees did not recognize potential hazards or increased biosafety risk to researchers, animals, humans and environment.