Corresponding author: Vladislav O. Soldatov ( 1263220@bsu.edu.ru ) Academic editor: Mikhail Pokrovskii
© 2020 Vladislav O. Soldatov, Marina V. Kubekina, Yulia Yu. Silaeva, Alexandra V. Bruter, Alexey V. Deykin.
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.
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
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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 introduce 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.
The new COVID-19 model should be based on inducible co-expression of the human ACE2 and TMPRSS2 genes. Activation 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.
COVID-19, SARS-CoV-2, ACE2, TMPRSS2, mice, CRISPR/Cas9
The COVID-19 outbreak is a dramatic, rapidly evolving situation. The search for effective approaches to SARS-CoV-2 infection therapy and prevention has become one of the most important tasks for medicine now and in the foreseeable future. Every day, doctors and scientists receive more and more information about the effectiveness of classic antiviral medications and some off-label-used drugs.
However, these data should also be quickly supplemented by the results of pre-clinical studies that can provide much useful and even crucial information about the most effective drugs. Unfortunately, as most laboratories do not have an accessible SARS-CoV-2-sensitive animal model, this is the main stumbling block for quick in vivo screening. The difficulty in obtaining such models in the pandemic condition directed us to develop our own SARS-CoV-2-sensitive mouse model as discussed in this paper.
To enter the target cells, SARS-CoV and SARS-CoV-2 use their “corona”, which is represented by numerous spike (S) proteins. It was shown that the S-protein engages angiotensin-converting enzyme 2 (ACE2) as the entry receptor (
Although ACE2 is present in many types of tissues (
Human and mouse ACE2 enzymes consist of 805 amino acids with 81.86% interspecies homology. Both hACE and mACE have Collectrin (75.97%) and Peptidase M2 (interdomain similarity = 84.62%) domains and the latter 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 a key role in binding with viral S-protein (
Three regions of SARS-CoV-2 binding site are shown. Five of eight key residues differ between mouse and human aligned sequences (highlighted in red) when three amino acids coincide (highlighted in green). In addition, alignment of mouse and human ACE2 TMPRSS2 cleavage sites is presented below.
Both human and mouse TMPRSS2 proteins consist of 4 domains: Transmembrane; LDL receptor class A; Scavenger receptor cysteine‐rich; and Serine protease. Murine TMPRSS2 protein contains 492 amino acids and shares 81.4% similarity and 77.3% identity with the human one. The details of comparison were presented in
Naturally, mice are low-sensitive to SARS-CoV infection, but can be poorly inoculated by the virus. To improve the virus inoculation, a few transgenic lines of mice were created with humanized ACE2 gene. In the first line, the hACE2 gene was introduced under the CAG promoter with CMV-IE enhancer (
The transgenic line, created by the
As SARS-CoV and SARS-CoV-2 have a similar manner of cell contagion, it is believed that the already-created models are also sensitive to COVID-19.
As can be seen, there is not only ACE2 involved in SARS-CoV-2 invasion. Therefore, first of all, we consider that mice with two humanized ACE2 and TMPRSS2 genes will be more sensitive to viral invasion. This approach will not only make ACE2 more accessible for cleavage (which is important for a viral entry), but will also open up additional possibilities for drugs testing, such as inhibitors of TMPRSS2. Moreover, a high expression of TMPRSS2 in the epithelial cells makes it reasonable to introduce both hACE2 and hTMPRSS2 under the murine Tmprss2 promoter. We believe that the co-expression of two virus-inviting molecules in the lung epithelium will imitate the events that happen in the human body during SARS-CoV-2 invasion.
In brief, to create this model, we are going to clone hTMPRSS2 and hACE sequences and introduce it into the mouse genome with the use of CRISPR/Cas9 technology. hTMPRSS2 and hACE2 will be divided by the IRES element for their equivalent expression. Additionally, to create an inducible hACE2/hTMPRSS2 expression, we wish to place LoxP sites in front of the hTMPRSS2 sequence (Fig.
Brief description of the existing SARS-CoV-sensitive transgenic mice.
Promoter | Clinical pattern and pathomorphology | Lethality level | References | ||
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SARS-CoV | SARS-CoV-2 | SARS-CoV | SARS-CoV-2 | ||
CAG promoter+CMV-IE enhancer | Acute wasting syndrome. Death within 4 to 8 days of post-inoculation period as result of inflammatory response in the brain (more intensive than in the lungs) Damaged tissues: lungs, kidneys, liver, heart, skeletal muscle, spleen, lymphatic nodes, pancreas, gastrointestinal smooth muscle and ganglia, vascular endothelium, adrenal and central nervous system (CNS) | no data | 100% (in mice with high hACE2 expression) 0% (in mice with low hACE2 expression) | no data |
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human cytokeratin 18+ alfalfa mosaic virus enhancer | Pneumonia, neuro-inflammation. Infection of the CNS is a major factor contributing to the fatal outcome observed in SARS-CoV-infected mice. Damaged tissues: Lungs, colon, small intestine, kidneys, liver, spleen, heart | no data | 100% | no data |
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mouse ACE2 promoter | Gross pulmonary edema, focal haemorrhage, consolidation and lung bullae with no significant histopathological lesions or viral antigens in myocardium, liver, spleen, kidney, cerebrum, intestine and testis. Damaged tissues: Viral antigens were observed in the bronchial epithelial cells, alveolar macrophages and alveolar epithelia. | Interstitial pneumonia with lymphocytes and monocytes infiltration. Accumulation of macrophages in alveolar cavities | 0% | 0% |
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Construction consists of homology arms, hTMPRSS2 sequence, IRES element, hACE2 sequence and an effective terminator of expression in 3’ regions. Homology arms have to direct it next to the murine Tmprss2 promoter with use of CRISPR/Cas9. Transgenic mice can be crossbred with Cre-ERT2 mice for the excision of the stop-cassette between LoxP-sites and activation of hACE2 and hTMPRSS2 transcription.
A previously-unknown coronavirus called SARS-CoV-2 has crossed the species barrier and caused a human infection outbreak, first in Wuhan and then around the world. The rapidly developing epidemic generated interest in mice expressing the human receptors for SARS-CoV-2 entry for further pharmacological screening (
In the growing avalanche of SARS-CoV-2 pathogenesis details, it is very difficult to focus on specific targets. For example, recent insight into the CD147 as a novel target of SARS-CoV-2 opens up new possibilities for explaining the disease (
We have formulated the basic requirements for the COVID-19 animal model. First of all, transgenic animals should not be a “laboratory” reservoir of the virus before the start of the pharmacological experiments. Although the SARS and MERS were successfully inoculated in mice, infection did not spread in animals after intranasal administration. In the case of a new SARS-CoV-2 virus, it is impossible to predict its degree of contagion from mice to mice and human population. For the safety of the laboratory staff, we decided to use LoxP-induced expression of humanized genes, as described in (
As shown, the hTMPRSS2 and hACE2 proteins are jointly involved in the pathogenesis of the SARS-CoV-2 invasion. Moreover, the expression profiles of these enzymes in mice and human intersect at lungs, intestine and the male reproductive system epithelium (
It is very important not only for the COVID-19 murine model to be able to be infected by the SARS-CoV-2, but also for the infection process to be as human-like as possible. The previous models showed effective infection only in high viral load conditions. At the same time, in the models based on the K18 promoter, systemic damage, neuroinflammation and lethargy developed, which is not quite representative of the human clinical picture. In the model, created by
Activation of expression is possible after crossbreeding with mice constitutively or inducibly expressing Cre-recombinase (for example, Cre-ERT2 line). We expect these mice to be a safe and representative COVID-19 model.
The authors declare no conflict of interest.
This work was performed using the equipment of the Institute of Gene Biology of Russian Academy of Sciences facilities and supported by the Ministry of Science and Higher Education of the Russian Federation. This work was supported by the Russian Science Foundation (Grant #17-75-20249). We are grateful to Academician Pavel Georgiev and Professor Mikhail Pokrovskiy for productive discussions, Vladislav Maslov for design of the figures and Olesya Serkina for the grammar review.
Vladislav O. Soldatov, junior researcher of the Core Facility “Genome editing”, Institute of Gene Biology of the Russian Academy of Sciences; junior researcher of the Research Institute of Living Systems Pharmacology , Belgorod State University, e-mail: pharmsoldatov@gmail.com; ORCID ID http://orcid.org/0000-0001-9706-0699. The author was engaged in literature analysis and paper writing.
Marina V. Kubekina, junior researcher of the Core Facility “Genome editing”, Institute of Gene Biology of the Russian Academy of Sciences, e-mail: kubekina@genebiology.ru; ORCID ID http://orcid.org/0000-0002-8834-1111. The author was engaged in paper writing and preparing graphical materials.
Yuliya Yu. Silaeva, PhD in Biological Sciences, researcher of the Core Facility “Genome editing”, Institute of Gene Biology of the Russian Academy of Sciences, e-mail: silaeva@genebiology.ru; ORCID ID http://orcid.org/0000-0003-2070-9001. The author was engaged in structuring the article and arranging references.
Alexandra V. Bruter, PhD in Biological Sciences, researcher of the Core Facility “Genome editing”, Institute of Gene Biology of the Russian Academy of Sciences, e-mail: aleabruter@gmail.com; ORCID ID http://orcid.org/0000-0002-2090-2488. The author was engaged in developing the concept and literature analysis.
Alexey V. Deykin, PhD in Biological Sciences, Head of the Core Facility and senior researcher of the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology of the Russian Academy of Sciences, leading researcher of the Laboratory of Pathogenomics and Transcriptomics, Institute of General Pathology and Pathophysiology. e-mail: aleabruter@gmail.com; ORCID ID http://orcid.org/0000-0001-9960-0863. The author generated the idea of research and was engaged in developing the concept, analysing literature and writing the paper.