Policy and Practice

Interviews with Experts

In order to get a better perspective of the clinical applications of our project we interviewed Dr. Toni Winder who is a neurologist specializing in ischemic stroke. He elaborated on current stroke treatments and offered a clinical opinion on the use of genetic therapies for treating stroke and other traumatic brain injuries in patients. We also interviewed Dr. Randall Barley, an expert on cell culture therapy, to learn more about the current concerns with gene and cell culture therapy. These two interviews helped us become more aware of potential complication in our project and the clinical impact our work could have. We will be performing another interview with with a stroke victim, which allowed us to gain a better understanding of how this injury can affect a person’s daily life. The final interview will be available for viewing in at the Giant Jamboree this year in Boston.

Dr. Toni Winder Neurologist Lethbridge, Alberta

An interview with Dr. Winder discussing brain injury and his experience with treatment

Dr. Randall Barley Ph.D. Experimental Surgery Lethbridge, Alberta

An interview with Dr. Barley discussing cell and gene therapy

Concerns about Cell Therapy

With regards to human practice, because microglia can be derived directly from patient bone marrow cells, this study has the potential to provide a method of personalized, non-immunogenic neural rehabilitation [1]. In addition, we are also addressing the growing prevalence of bacterial antibiotic resistance around the globe [2].

Antibiotic Resistance Reduction

“A post-antibiotic era – in which common infections and minor injuries can kill – far from
being an apocalyptic fantasy, is instead a very real possibility for the 21st Century.”

World Health Organization, 2014

In a report released this year [2], the World Health Organization warns that the growing resistance to antibiotics threatens to compromise our ability to treat even the smallest infections. Misuse of antibacterial drugs in medical, agricultural, and laboratory applications has led to elevated environmental levels of these drugs and increases the selective pressure for microbes to develop antibacterial resistances.

We not only took this global concern into consideration when thinking about the design of our therapeutic plasmid, but also some concerns specific to our project. Plasmids with antibiotic resistance cassettes in them have the potential to transfer to cells within the patients microbiome [5], conferring antibiotic resistance to yet more organisms that live within the human body. Administering plasmids containing resistance is advised against by FDA guidelines on gene therapy for this reason [6].

These project-specific concerns, along with the growing prevalence of antibiotic resistance, has motivated our team to look elsewhere for plasmid selection mechanisms. In investigating potential methods of selecting for plasmids in culture, we came across an interesting synthetic translation riboregulation system, whereby an RNA stem-loop (RNA-OUT) acts in trans to base-pair with a stretch of mRNA (RNA-IN), obscuring the ribosome binding site and silencing expression [7]. By placing a “kill switch” gene, a T4 Holin lysis cassette or ccdB gyrase in our case, downstream of the RNA-IN sequence, the expression of this lethal gene can be prevented by blocking translation with RNA-OUT present on the plasmid. The RNA-OUT, inserted in the plasmid backbone in the place of the antibiotic resistance cassette, reduces the overall plasmid size, prevents the transfer of antibiotic resistance to human cells or microbiota, and reduces the levels of antibiotics in laboratory waste from cloning.

Animal Ethics

As the next step towards validating our genetic constructs in vivo, we plan to use a rodent model. This will involve the injection of microglia cells into the circulatory system of mice, where they will then cross the blood brain barrier [8] and migrate towards areas of inflammation. To develop the surgical protocols necessary for this procedure, we have been working in collaboration with the university veterinarian and Animal Welfare Committee. By going through these channels we can ensure the safety of our mice while still performing our experiments.

We are also cognizant of the possibility of our engineered cells containing a potential pathogen. Long-term we will investigate this by utilizing an outside party to analyze a culture sample for pathogenic genes. To minimize risk before this validation can occur, we are setting up a quarantined surgery suite to perform our procedures. This will allow us to segregate our experimental animals from the general population for surgery, recovery, and eventual tissue processing.

All members of the team involved with the animal experiments have also received formal training on animal husbandry, research ethics, and biosafety from the Canadian Council on Animal Care. Additionally, members that will be directly involved with the surgical procedures have passed a Rodent Stereotaxic Surgical Competency Exam administered by the university veterinarian. These regulations ensure that the members of the team have received the necessary training to work with animals.

Collaboration with Public Health Agency of Canada

The University of Lethbridge iGEM team collaborated with members of the Public Health Agency of Canada (PHAC) Center for Biosecurity. One of our members, Suneet Kharey, travelled to Ottawa in October and presented did two presentations. The first presentation was regarding biosafety and biosecurity issues revolving around iGEM and other synthetic biology projects. The second presentation was on our past and current iGEM project, as well as an emphasis on safety regarding misuse of synthetic biology projects. The presentation was attended by over 70 participants (in person and via WebEx) from PHAC, Health Canada, and other departments including Environment Canada, Fisheries and Oceans, Foreign Affairs Trade and Development, and the Canadian Institutes of Health Research (CIHR). During the conference, it became evident that there is a growing concern over the decreasing costs of DNA synthesis and the potential impact on public safety. In a controlled environment, the exploitation of DNA synthesis technology was demonstrated by the successful reconstruction of the Spanish Influenza Virus in 2005 [3]. It is nearly a decade since and the cost of DNA synthesis has decreased significantly and will continue to decline as technology is continually revolutionized [4].

Currently, a very hot topic is the potential of dual-use. Following the presentations by Suneet Kharey and Kathrina Yambao (Policy Analyst for the Center of Biosecurity, PHAC) a discussion occurred regarding current policies concerning synthetic biology. We discussed the risks and safety about doing synthetic biology in an institutional and/or professional environment and how this differs from do it yourself (DIY) labs. It was evident that policies and regulations need to be kept up to date with the current state and evolution of synthetic biology.

Overall, there was excellent discussion and we were able to develop the beginnings of a working relationship with PHAC. We have given very valuable feedback regarding the formation of the iGEM Safety Form as well as potentially hosting an annual Policies and Practices related iGEM conference for Canadian teams.

References

[1] Hinze, A. & Stolzing, A. (2012). Microglia differentiation using a culture system for the expansion of mice non-adherent bone marrow stem cells. Journal of Inflammation, 9, 12.

[2] World Health Organization. (2014). Microbial resistance: global report on surveillance. Retrieved from http://www.who.int/drugresistance/documents/surveillancereport/en/

[3] Tumpey, T.M. (2005). Characterization of the Reconstructed 1918 Spanish Influenza Pandemic Virus. Science, 310, 77-80.

[4] Bügl, H (2007). DNA synthesis and biological security. Nature Biotechnology, 25, 627-629.

[5] Susanne Schjørring and Karen A. Krogfelt, “Assessment of Bacterial Antibiotic Resistance Transfer in the Gut,” International Journal of Microbiology, vol. 2011, Article ID 312956, 10 pages, 2011. doi:10.1155/2011/312956

[6] U.S. Food and Drug Administration. (1998). Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy. Retrieved from http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/ucm072987.htm.

[7] Mutalik, V.K., Qi, L., Guimaraes, J.C., Lucks, J.B., & Arkin, A.P. (2012). Rationally designed families of orthogaonal RNA regulators of translation. Nature Chem. Bio., 8, 447-454

[8] Imai, F., Suzuki, H., Oda, J., Ninomiya, T., Ono, K., Sano, H., & Sawada, M. (2006). Neuroprotective effect of exogenous microglia in global brain ischemia. Journal of Cerebral Blood Flow & Metabolism, 27(3), 488-500.