NMU-China iGEM understands the potential risks and possible crises in a lab facility. We try to avoid some security problems and make sure that no personal or environmental harm occurs.
Our circuit design of switching from Mγ（CARγ macrophage） to Ms（CARMERTK macrophage） upon detection of high cytokine levels is safety feature that is unique to Toggle Macrophage. （more details in Design） Toggle Macrophages can balance the immune system via phenotypic transformation. Mγ has pro-inflammatory ability, while Ms has anti-inflammatory ability. As we don’t want the immune system to be suppressed during early stages of infection, Toggle Macrophages transform into Mγ so that the immune system can mount the necessary response against the virus. However, if our circuit detects high levels of IL-6 (such as during a cytokine storm), Mγ transformation will be halted and Ms will be induced. The Ms will then help to reduce inflammation. This switching behavior between Mγ and Ms ensures that maintaining a safe level of inflammatory response. We established a model to prove it.
The human monocytic leukemia cell (THP-1) are hard to survive without a suitable environment. There is no reported evidence for the presence of infectious viruses or toxic products in THP-1 cells, making this cell line relatively easy and safe to use. (Chanput et al., 2014)
We can assure you that our engineering cells will not be out of the laboratory or discharge into the natural environment. If there is any accident, the engineered cells were released into the environment is also don't need to worry about. Because in vitro, macrophages don't proliferate and hard to survive without a suitable environment.
Even though CAR-macrophages were accidentally injected, we can induce apoptosis of CAR-macrophages with AP1903.
The SARS-CoV2 pseudovirus was constructed based on the spike genes of the strain Wuhan-Hu-1(GenBank:MN908947) using published methods. (Nie et al., 2020)
We constructed SARS-CoV-2 pseudovirus to substitute SARS-CoV-2. Firstly, synthesize and codon-optimize the S gene for human cells and clone it into eukaryotic expression plasmid pcDNA3.1 to generate the envelope recombinant plasmids. Then, transfect the plasmids into 293T cells. After that, infect the cells with VSV G pseudotyped virus (delta-G-VSV), of which the VSV-G gene is substituted with luciferase expression cassettes. Its precautions are consistent with those of Delta-G-VSV.
Because the delta-G-VSV is non-replicating , the system is safe and could be handled in biosafety level 2 facilities. However, exposure to delta-G-VSV could also cause risks to the operators. Usually, infections of wild-type VSV in humans are asymptomatic or result in a mild febrile symptom. (Lichty et al., 2004) Besides, VSV could also pose threat to species other than human. (van den et al., 2009)
To ensure safety, any experiment engaging VSV will be performed in a BSL-2 facility in a separated area to avoid. All the experiment materials contaminated will be sterilized to avoid leakage. The operators must wear protective suit, gloves, and goggle. The operations must follow safety guidelines. The pseudoviruses will be prepared and provided by our supervisors to minimize the risks.
5. lentivirus vectors
The cDNA sequences containing the various fusion constructs were cloned into a third-generation lentiviral vector. Lentiviral infection was used to stably express CAR constructs in THP-1 cells.
The potential risks from lentiviral vectors are dependent upon the nature of the exposure. Aerosol exposures through droplet transmission are another potential route of lentiviral vector exposure. One concern of lentiviral vectors is the possibility that recombination unintentionally reconstitutes a replication-competent and pathogenic virus. (Schlimgen et al., 2016)
When operating lentiviral vector, we will use commercial Lentiviral vector production kit in a biosafety class 2 cabinet, strictly following it's protocols and procedures. Lentivirus operations that do not involve animal testing in a biosafety cabinet (BSL-2 level) are considered sufficient by the UK ACGM guidelines.
Kill Switch Design
To ensure that our probiotic is safe within the body, we incorporate a chemically-inducible kill switch. This way, the Toggle Macrophage can rapidly be eliminated in vivo. An excellent candidate for this inducible kill switch is the AP1903 described by （Clackson, et al., 1998）, as AP1903 has no other biologic effects in vivo. (Straathof, K. C., et al.,2016) Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP. (Clackson, et al., 1998）
Caspases are very important regulators of apoptosis induced by apoptosis stimuli (Stennicke and Salvesen, 1998). iCasp9 can be activated by AP1903 that has proven safe at the required dose for optimum deletional effect. (Iuliucci, J. D., et al.,2001) Caspase9 will subsequently activate downstream effector caspases, such as caspase3, and ultimately induce apoptosis.
Safe Lab Work
We deeply understand how important biotechnology safety is to our community and are informed of iGEM's safety rules for every student. Our advisor, Zetong Ma, conducted safety education for all team members when they officially entered the lab.
This year, our experiments were mainly focused on in vitro cell experiments. These experiments were of low risks, and we required the students to strictly follow safety rules in our lab and college. We handled all the biological materials, wastes and equipment strictly following the BSL-2 requirements.
Chanput W, Mes J J, Wichers H J. THP-1 cell line: an in vitro cell model for immune modulation approach[J]. International immunopharmacology, 2014, 23(1): 37-45.
Schlimgen R, Howard J, Wooley D, et al. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies. J Occup Environ Med. 2016;58(12):1159-1166. doi:10.1097/JOM.0000000000000879
van den Pol, Anthony N et al. “Viral strategies for studying the brain, including a replication-restricted self-amplifying delta-G vesicular stomatis virus that rapidly expresses transgenes in brain and can generate a multicolor golgi-like expression.” The Journal of comparative neurology vol. 516,6 (2009): 456-81. doi:10.1002/cne.22131
Nie, Jianhui , et al. "Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2." Emerging Microbes and Infections 9.1(2020):680-686.
Clackson, et al. "Redesigning an FKBP--ligand interface to generate chemical dimerizers with novel specificity. " Proceedings of the National Academy of Sciences of the United States of America (1998).
Iuliucci, J. D. , et al. "Intravenous Safety and Pharmacokinetics of a Novel Dimerizer Drug, AP1903, in Healthy Volunteers." The Journal of Clinical Pharmacology 41.8(2001).
Straathof, K. C. , et al. "An inducible caspase 9 safety switch for T-cell therapy." Blood 105.11(2005):4247-4254.
Stennicke, H.R., Salvesen, G.S., 1998. Properties of the caspases. Biochim. Biophys. Acta 1387, 17–31.