On the Way from SARS-Cov-Sensitive Mice to Murine COVID-19 Model
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Research Results in Pharmacology 6(2): 1–7 UDC: 616-092.9 578.76 DOI 10.3897/rrpharmacology.6.53633 Editorial On the way from SARS-CoV-sensitive mice to murine COVID-19 model Vladislav O. Soldatov1,2, Marina V. Kubekina1, Yulia Yu. Silaeva1, Alexandra V. Bruter1, Alexey V. Deykin1,3,4 1 Core Facility “Genome editing”, Institute of Gene Biology of the Russian Academy of Sciences, 34/5 Vavilova St., Moscow 119334, Russia 2 Research Institute of Living Systems Pharmacology, Belgorod State University, 85 Pobedy St., Belgorod 308015, Russia 3 Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology of the Russian Academy of Sciences, 34/5 Vavilova St., Moscow 119334, Russia 4 Laboratory of Pathogenomics and Transcriptomics, Institute of General Pathology and Pathophysiology, 8 Baltiyskaya St., Moscow, 125315, Russia Corresponding author: Alexey V. Deykin ([email protected]) Academic editor: Mikhail Pokrovskii ♦ Received 25 April 2020 ♦ Accepted 28 April 2020 ♦ Published 4 May 2020 Citation: Soldatov VO, Kubekina MV, Silaeva YuYu, Bruter AV, Deykin AV (2020) On the way from SARS-CoV-sensitive mice to murine COVID-19 model. Research Results in Pharmacology 6(2): 1–7. https://doi.org/10.3897/rrpharmacology.6.53633 Abstract The coronavirus disease 2019 (COVID-19) is a master killer which appeared suddenly and which has already claimed more than 200,000 human lives. In this situation, laboratories are in urgent need for a COVID-19 murine model to search for effective antiviral compounds. Here we propose a novel strategy for the development of mice that can be inoculated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 causative agent. In humans, two proteins – ACE2 and TMPRSS2 – are involved in SARS-CoV-2 cells entry and, thus, we decided to intro- duce their genes into a murine genome. These genes will be placed with LoxP sites under the murine Tmprss2 promoter. Such an approach can provide a representative model with the opportunity to control the viral sensitivity of an animal population and tissue specificity of hACE2 and hTMPRSS2 expression. Graphical abstract Copyright Soldatov VO et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Soldatov VO et al.: On the way from SARS-CoV-sensitive mice to murine COVID-19 model The new COVID-19 model should be based on inducible co-expression of the human ACE2 and TMPRSS2 genes. Acti- vation of ACE2 and TMPRSS2 genes will occur only in the virological laboratory, after crossbreeding with Cre-mice. Before activation, mice will be resistant to SARS-CoV-2 for their biological safety during the pandemic. Keywords COVID-19, SARS-CoV-2, ACE2, TMPRSS2, mice, CRISPR/Cas9. Background shown as one of the up-regulators of coronavirus infec- tion pathways (Iwata-Yoshikawa et al. 2019). Interac- The COVID-19 outbreak is a dramatic, rapidly evol- tion of ACE2 and TMPRSS2 enhanced the cell entry of ving situation. The search for effective approaches to SARS-CoV and it correlated with TMPRSS2-mediated SARS-CoV-2 infection therapy and prevention has be- proteolysis of both S and ACE2 proteins (Shulla et al. come one of the most important tasks for medicine now 2011). Recently, Hoffmann et al. (2020) have discovered and in the foreseeable future. Every day, doctors and that serine protease TMPRSS2 is employed by SARS- scientists receive more and more information about the CoV-2 for S protein priming and the TMPRSS2 inhibitor effectiveness of classic antiviral medications and some prevents virus entry. off-label-used drugs. However, these data should also be quickly supple- mented by the results of pre-clinical studies that can Homology between human and provide much useful and even crucial information about mouse ACE2 proteins the most effective drugs. Unfortunately, as most labora- tories do not have an accessible SARS-CoV-2-sensitive animal model, this is the main stumbling block for quick Human and mouse ACE2 enzymes consist of 805 amino in vivo screening. The difficulty in obtaining such mod- acids with 81.86% interspecies homology. Both hACE els in the pandemic condition directed us to develop our and mACE have Collectrin (75.97%) and Peptidase M2 own SARS-CoV-2-sensitive mouse model as discussed (interdomain similarity = 84.62%) domains and the latter in this paper. directly interacts with S-protein. It has only recently been identified that amino acids Asp30, His34, Tyr41, Gln42, Lys353, Arg357, Gln24 and Met82 of human ACE2 play Virus invasion pathway a key role in binding with viral S-protein (Yan et al. 2020). Alignment of mouse and human protein ACE2 sequences To enter the target cells, SARS-CoV and SARS-CoV-2 showed that five of these eight residues differ in human use their “corona”, which is represented by numerous spi- and mice. The similarity between TMPRSS2 cleavage ke (S) proteins. It was shown that the S-protein engages sites (amino acids 697–716) of hACE2 and mACE2 pro- angiotensin-converting enzyme 2 (ACE2) as the entry re- teins is 78.95% (Fig. 1). ceptor (Li et al. 2003). During viral infection, the trimeric Three regions of SARS-CoV-2 binding site are shown. S protein is cleaved into S1 and S2 subunits (Belouzard Five of eight key residues differ between mouse and hu- et al. 2009; Simmons et al. 2013). Further, the S1 subunit, man aligned sequences (highlighted in red) when three containing the receptor binding domain, directly binds to amino acids coincide (highlighted in green). In addition, the peptidase domain of angiotensin-converting enzyme alignment of mouse and human ACE2 TMPRSS2 cleav- 2 (ACE2) (Li et al. 2005), whereas S2 is responsible for age sites is presented below. membrane fusion. Although ACE2 is present in many types of tissues (Hamming et al. 2004), SARS-CoV is highly pathogenic Homology between human and only in the lungs (To and Lo 2004). Furthermore, type mouse TMPRSS2 proteins 1 pneumocytes, which poorly express ACE2, are more sensitive to viral invasion in comparison with ACE2- rich type 2 pneumocytes (Matsuyama et al. 2010). The Both human and mouse TMPRSS2 proteins consist of 4 selective nature of tissue damage was explained by the domains: Transmembrane; LDL receptor class A; Sca- existence of a second molecule, which also contributes venger receptor cysteine‐rich; and Serine protease. Muri- to cell contagion. Thus, ACE2 immunoprecipitation ne TMPRSS2 protein contains 492 amino acids and sha- captured the transmembrane protease/serine subfamily res 81.4% similarity and 77.3% identity with the human member 2 (TMPRSS2), a known human airway and al- one. The details of comparison were presented in Vaarala veolar protease (Vaarala et al. 2001). This enzyme was et al. (2001). Research Results in Pharmacology 6(2): 1–7 3 Figure 1. Alignment of human and mouse ACE2 protein sequences. Mouse models of SARS-CoV mice with ACE2 gene under murine Ace2 promoter have infection sensitivity to SARS-CoV-2, but it is lower than that of SARS-CoV. However, these models are not widespread in laboratories and we have not found any publication where Naturally, mice are low-sensitive to SARS-CoV infecti- ACE2-humanised mice were used for preclinical studies on, but can be poorly inoculated by the virus. To improve of anti-SARS-CoV-2-therapy. Whereas, since a few mod- the virus inoculation, a few transgenic lines of mice were els have already been developed, we can take the availa- created with humanized ACE2 gene. In the first line, the ble experience and try to propose our own strategy for the hACE2 gene was introduced under the CAG promoter genetic edition. with CMV-IE enhancer (Tseng et al. 2007). This modifi- cation led to a sharp clinical manifestation with an acute wasting syndrome and deaths of the mice within 4 to 8 Novel approach to the creation of days after SARS-CoV inoculation. A second line was de- SARS-CoV-2-sensitive mice veloped by introducing the hACE2 gene regulated by the human cytokeratin 18 (K18) promoter. After the SARS- CoV contagion, these mice demonstrated a clinical picture As can be seen, there is not only ACE2 involved in of encephalitis and pneumonia, resulting in death after 3–5 SARS-CoV-2 invasion. Therefore, first of all, we consi- days of the post-inoculation period (McCray et al. 2007). der that mice with two humanized ACE2 and TMPRSS2 The transgenic line, created by the Yang et al. (2007), genes will be more sensitive to viral invasion. This ap- has the most natural tissue expression profile of ACE be- proach will not only make ACE2 more accessible for cle- cause hACE gene was introduced under the mouse’s own avage (which is important for a viral entry), but will also Ace2-promotor. In this line, SARS-CoV replicated more open up additional possibilities for drugs testing, such as efficiently in the lungs of transgenic mice than in those inhibitors of TMPRSS2. Moreover, a high expression of of wild-type mice. After the SARS-CoV inoculation, the TMPRSS2 in the epithelial cells makes it reasonable to mice had severe pulmonary lesions, including interstitial introduce both hACE2 and hTMPRSS2 under the murine hyperaemia and haemorrhage, monocytic and lymphocyt- Tmprss2 promoter. We believe that the co-expression of ic infiltration, protein exudation and alveolar epithelial two virus-inviting molecules in the lung epithelium will cell proliferation and desquamation. imitate the events that happen in the human body during As SARS-CoV and SARS-CoV-2 have a similar man- SARS-CoV-2 invasion.