Safety and Ethical Risks

Our gene integration strategy was designed for use in existing ex vivo gene transfer processes to mediate targeted integration. The immediate use case is constrained to research projects in lab environments, however the strategy could hypothetically also be used eventually in gene therapies to correct recessive genetic diseases by integrating DNA encoding for the wild type, functional protein. The system could, with little modification, be used to integrate arbitrarily large DNA constructs into virtually any genome. The risks of this enablement are extensive, but do not greatly exceed existing gene transfer technologies. The off-target activity of CRISPR/Cas9 is well established, and may result in the integration of the transgenic construct outside of the targeted genomic safe harbour. However, such risk is negligible compared to the pseudorandom integration typical of virus-mediated integration. Although the strategy may be appropriate for eventual use in human gene therapy, that potential is beyond the scope of our work. Further evaluations and investigations into the ethical risks of our project can be seen on our Integrated Human Practices page.

Safety in the Lab

Before beginning work in the lab, all team members, mentors, and Principal Investigators completed lab safety courses developed by the University of Calgary Environment Health and Safety (EHS) services. The courses covered a wide range of topics such as occupational health and safety, laboratory safety, hazard assessment, incident reporting and investigation, spill response, biosafety, bloodborne pathogens, and WHMIS. The completion of these courses ensured that everyone on the team worked safely in the lab and could respond to incidents or hazards appropriately. Furthermore, the University of Calgary Biosafety Committee outlines guidelines for safe biological laboratory practices. These guidelines were observed and adhered to during all of our experimental work.

Our project used a common lab strain of Escherichia coli, DH5-alpha. This strain is non-pathogenic and is classified as Biosafety Level 1 (BSL-1), therefore its use posed low risk to researchers. We also used an immortalized human cell line, HEK293T cells, which are Risk Group 1 organisms. HEK293T cells have adenovirus genetic contents and Simian Virus 40 large T antigen (SV40T), however, these cells do not contain the complete viral genomes, therefore the risk of viral particle generation by these cells is low (ATCC, 2016). The cells are not themselves infectious, are not known to carry any disease-causing agents, and are virtually unable to survive outside of the lab. All experiments with HEK293T cells was done in a BSL-2 Biosafety Cabinet to ensure the safety of researchers and to work within University of Calgary safety guidelines. Our lab is a certified BSL-2 lab, therefore it was equipped for safe work with both E. coli DH5-alpha and HEK293T cells.