Overview
As a team participating in iGEM, it is necessary for NMU-China iGEM to follow safety regulations and prove to the world that our projects and experiments are safe. We discussed and verified the safety of genetic circuits, chassis cells, experimental procedures and operations, products, etc., and explain them.
Circuit Design & Termination Switch
The circuit is not only a functional pathway to promote the selective apoptosis of CAR-NK cells, but also a safe pathway to ensure the safety of CAR-NK cells in patients. The circuit is a chemical-induced functional switch that induces timely the apoptosis of CAR-NK cells.
We used AP1903 as an inducer, which induces the dimerization and activation of iCASP9 in vivo (Iuliucci et al., 2001), then leading to cell apoptosis. Relevant studies have shown that AP1903 has no other biological effects in vivo. Remodeled dimerization reagents 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). No obvious adverse reactions have been reported after the injection of AP1903.
Caspase-9 is an important regulator of caspase-dependent apoptosis pathway (Kuida et al., 2000). iCASP9 can cleave downstream caspase-7 and caspase-3 zymogens to induce apoptosis, which is induced by AP1903.
Gal4-KRAB is a commonly used transcription suppressor and is safe for in vivo application. A single injection of a large number of highly proliferative NK-92 cells may cause disorder in the body's immune system, while a single injection of insufficient cells may fail to achieve the therapeutic effect. To this end, when we constructed the CAR-NK library, we attempted to establish a mathematical model to find a balance between safety and efficacy, thereby determining the number of CAR NK-92 cells for a single injection.
Experimental Safety
The Biosafety Law of the People's Republic of China was adopted at the 22nd Meeting of the Standing Committee of the 13th National People's Congress on October 17, 2020. The experiments of NMU-China iGEM were carried out under the premise of complying with relevant laws
Chassis cells NK-92
NK-92MI cell line was purchased from American Type Culture Collection (ATCC) and identified by short tandem repeat sequence analysis. Clinical trials have shown that NK-92 infusion is safe even at high doses (Suck et al., 2016). Unlike T cells, they do not cause graft-versus-host disease. So far, NK-92 has been well studied. These cells showed excellent cytotoxicity against many tumor types in test.
Lentivirus carrier
Lentivirus packaging kit was purchased from Shanghai Ji kai Ji Yin Chemical Technology Co., LTD. Lentivirus vectors including pCDH-iCAS9-KRAB lentivirus vector and pCDH-scFv-CAR lentivirus vector were used to transfect NK-92 cells to get targeted role and has the suicide lines of NK cells. Studies have shown that lentivirus vector have important biological safety characteristics (Schambach et al., 2012), its application in the body is relatively safe. 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 (Schlimgen et al., 2016). And the non-specific nature of transgene integration by the viral integration machinery carries an inherent risk for genotoxicity (Schenkwein et al., 2020). When operating lentiviral vector, we will use commercial Lentiviral vector production kit in a biosafety class 2 cabinet, strictly following its 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.
CAR-NK cells
We have strict requirements for every step of the CAR-NK cell experiment to ensure that CAR-NK cells will not enter the environment, and even if they do enter the environment, they will be difficult to survive due to insufficient nutritional intake. When CAR-NK causes severe adverse reactions in vivo, we can inject AP1903 to induce apoptosis
Safe Lab Work
Strict laboratory safety training is necessary. After understanding IGEM safety rules, we invited our advisor Shi Hu to the laboratory to conduct safety education for all members. We required each member to strictly abide by the safety guidelines of the laboratory and the school.
We treat all biological materials, waste and equipment in strict accordance with BSL-2 requirements.
Pandemic Prevention & Control
It is still a special time when the COVID-19 is raging, so we stipulate that our members must wear masks when entering the laboratory and disinfect their hands before and after the experiment to avoid contracting COVID-19. We've been granted a safe, healthy environment on campus to conduct our experiments.
References
1. Iuliucci JD, Oliver SD, Morley S, Ward C, Ward J, Dalgarno D, Clackson T, Berger HJ. Intravenous safety and pharmacokinetics of a novel dimerizer drug, AP1903, in healthy volunteers. J Clin Pharmacol. 2001 Aug;41(8):870-9. doi: 10.1177/00912700122010771. PMID: 11504275.
2. Clackson T, Yang W, Rozamus LW, Hatada M, Amara JF, Rollins CT, Stevenson LF, Magari SR, Wood SA, Courage NL, Lu X, Cerasoli F Jr, Gilman M, Holt DA. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10437-42. doi: 10.1073/pnas.95.18.10437. PMID: 9724721; PMCID: PMC27912.
3. Kuida K. Caspase-9. Int J Biochem Cell Biol. 2000 Feb;32(2):121-4. doi: 10.1016/s1357-2725(99)00024-2. PMID: 10687948.
4. Suck G, Odendahl M, Nowakowska P, Seidl C, Wels WS, Klingemann HG, Tonn T. NK-92: an 'off-the-shelf therapeutic' for adoptive natural killer cell-based cancer immunotherapy. Cancer Immunol Immunother. 2016 Apr;65(4):485-92. doi: 10.1007/s00262-015-1761-x. Epub 2015 Nov 11. PMID: 26559813.
5. Schamba ch A, Zychlinski D, Ehrnstroem B, Baum C. Biosafety features of lentiviral vectors. Hum Gene Ther. 2013 Feb;24(2):132-42. doi: 10.1089/hum.2012.229. PMID: 23311447; PMCID: PMC3581032.
6. Schlimgen R, Howard J, Wooley D, Thompson M, Baden LR, Yang OO, Christiani DC, Mostoslavsky G, Diamond DV, Duane EG, Byers K, Winters T, Gelfand JA, Fujimoto G, Hudson TW, Vyas JM. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies. J Occup Environ Med. 2016 Dec;58(12):1159-1166. doi: 10.1097/JOM.0000000000000879. PMID: 27930472; PMCID: PMC5152689.
7. Schenkwein D, Afzal S, Nousiainen A, Schmidt M, Ylä-Herttuala S. Efficient Nuclease-Directed Integration of Lentivirus Vectors into the Human Ribosomal DNA Locus. Mol Ther. 2020 Aug 5;28(8):1858-1875. doi: 10.1016/j.ymthe.2020.05.019. Epub 2020 May 23. PMID: 32504545; PMCID: PMC7403359.