We take safety seriously! Safety has always been a serious issue we considered from the design of the project all the way to lab work. We believe it is of vital importance in iGEM competition, and even more important for us, as we chose Therapeutic Track this year. In fact, our project aims to tackle the safety concerns of CAR T therapy, trying to provide an alternative solution to the severe adverse effects. We set strict rules regarding lab safety, and discussed some potential problems may come into existence in the future clinical application of our project.
We carefully assessed the risk of our project, including the organism we used, experiment conditions, training and waste management.
All information are summarized in our Safety Form.
We used E. Coli DH5alpha strain for molecular cloning, Jurkat T cell leukemia cell line and HEK293T cell line as chassis, HEK293T
cell line for lentivirus packaging, and lentivirus vectors for gene transduction.
We have level 1 lab space (open bench) for molecular experiments (cloning and western blots, etc.) and level 2 lab space for any
mammalian cell related experiment.
To protect lab personnel from infection and other hazards, our team members have received bio-safety and bio-security education and
have been trained with lab procedures and always wear laboratory gowns, gloves and masks during operation. Experiments involving
mammalian cell lines are conducted in BSL-2 laboratory equipped with sink, eyewash and emergency shower. Level 2 Biosafety Cabinet
are used for any handling of mammalian cell lines and lentivirus vectors.
As for the lentivirus vectors, the vectors are modified to prevent the lentivirus from being contagious. Team members have received
traing before conducting the experiments and conduct the experiments under the supervision of trained personnels (lab technicians and
senior graduate students).
To avoid gene modified organisms being released into the environment, all materials, including used medium, agar plates, pipette
tips, will be sterilized before disposed.
For the lentivirus vectors, to avoid potential contamination to the environment, all the materials and equipment used will be
processed with Benzyldodecyldimethylammonium Bromide (C21H38BrN).
Since our project involves a viral protein, U24, from level 2 organism human herpesvirus 6A, it is of safety concern that the protein may have pathogenic effect on lab members handling it. We therefore conducted comprehensive literature search and found no evidence of this protein alone can be pathogenic. We further reported our situation to the Safety Committee with a check-in form, and was later granted permission to use this protein in our project.
Since our device serves as an add-on for CAR-T, our project share the risks of CAR-T therapy. The main purpose of the project is to
mitigate some of the major risks associated with CAR-T therapy. We have conducted exhaustive literature search to identify risks
associated with CAR T therapy. We also consulted immunology professors in our university on the safety issue of CAR T therapy
(see our integrated HP)
The CAR-T cells may have toxic effects towards the patients, due to overactive immune response (cytokines release syndrome, tumor
lysis syndrome, etc.), on-target/off-tumor recognition, unexpected off-target cross-reactivity. These are the aspects our projects
focus on. For a detailed version.(see our description)
The lentivirus used for the gene transduction may result in insertional mutagenesis, which could be oncogenic.
The foreign proteins (CARs, rtTA, U24) may be recognized by the endogenous immune system and provoke an immune response against them.
A unexpected risk was reported recently that CAR was mistakenly transduced into cancer cells, binding surface antigen in cis and
confer resistance to CAR T therapy (Ruella et al., 2018).
