Safe Project Design
In Boomerang project, we used bacteria, mammalian cells and virions.
The mammalian cells and bacteria used in all experiments are classified as biosafety level 1 organisms; all are harmless and were used under well-established protocols and with proper guidance and safety equipment.
Since mammalian cells are very sensitive, they will not survive in case of accidental release. In order to minimize the same risk in bacteria , we used only 2 types of antibiotics in the entire work (chloramphenicol for the backbone plasmid and ampicillin for all the rest), so the bacteria remain vulnerable to other antibiotic types. If bacteria from our experiments will accidently get released, they most likely get outcompeted by the naturally occurring and better adapted bacteria around. Moreover, we used bacteria in our experiments only as an amplification tool (DH5α) for our plasmids, which are competent cells. The promoters we used are human promoters, so it is not likely that translation will occur in other bacteria. But even if the plasmids will get transformed to other bacteria and translated, the proteins encoded in our plasmids are probably harmless, because there are few components needed to create the Boomerang system.
We used AAV (Serotype 2) as our vector to transduce the three plasmids into the cells. Replication-defective recombinant AAV vectors, as the one we used in our experiments, are non-infectious and non-hazardous materials as defined by OSHA 1919.1200. Even tough, Production of Adeno-associated virus does fall within biosafety Level 2 criteria due to the use of HEK293 cells in the production process. Since routine quality control on preclinical vectors does not include testing for cellular contaminants or for replication competent AAV, we should handle AAV with caution as potentially infectious material.
Despite this disadvantage, our goal was to try and use a potentially in vivo vector, which has very high transduction percentage. Since AAV mediated gene therapy has already been approved for marketing at 2012 by the European Commission (Glybera - for treatment of LPLD - lipoprotein lipase deficiency. More information available here), and it has some major advantages in comparison to other transduction methods (its ability to infect both nondividing and dividing cells, its ability to deliver the plasmids into the host cell's nuclear without integrating into the genome and the low pathogeny even in the WT replication-competent AAV)(1) we decided to use it with caution in Agilent kit (#240071 - AAV Helper-Free System). In this kit, the AAV ITR sequences and rep/cap genes are present on separate plasmids that lack homology and thus preventing production of recombinant wild-type virus.
Yet, in order to minimize the work with viruses, it was decided that we will perform some of the experiments using calcium phosphate (CaP) as our transfection method instead of the viral vector.
Safe Lab Work
All lab work and experiments were done according to laboratory safety policies at Ben -Gurion University. The lab team was trained and escorted by lab members and instructors of Prof. Smadar Cohen lab for the entire time of our work to ensure the safety and health of team members and lab workers alike.
Since not all team members had experience in genetic engineering lab before, all lab work was done under the supervision and guidance of at least one of the senior team members, who already had experience in that field.
The main safety aspects of our daily routine in the lab included 3 parts:
- Personal safety: all working in the lab was done with gloves, safety glasses, closed leather shoes and lab coats.
- Keeping our benches clean and organized: work benches were cleaned and sterilized with ethanol at the end of every work day. Waste was separated to viral waste, Biologic waste and regular waste. The viral waste first went through two rounds of UV light inside the biosafety cabinets, and afterwards it was autoclaved, along with the biologic waste.
- Special areas for different types of work - our lab was divided into 3 parts:
Working with the virions was done inside biosafety cabinets. The virions were placed in designated tools, and we used filtered pipet tips to prevent sample carry-over contamination. After each work with virions (preparation or infection), UV light was turned on in the cabinets for two rounds (approx. 40 min).
The first part of the lab was designated to the regular lab work (plasmid purification, restriction analysis and etc.).The second part (fume hood and a nearby bench) was designated to work with ethidium bromide and gel-electrophoresis only. The third part was for working with mammalian cells and virions (biosafety cabinets, separated culture room, bench in other laboratory).
We didn't face any special safety problems in sending our DNA parts.
References(1) Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Federico Mingozzi, Katherine A. High. Nature Reviews Genetics. 2011 May;12:341-355. doi: 10.1038/nrg2988.